U.S. patent application number 13/399031 was filed with the patent office on 2012-08-23 for tyrosine kinase receptor antagonists and methods of treatment for pancreatic cancer.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Michael Campbell, Ira GOLDFINE, John Kerner, Peter Kushner, Betty A. Maddux, Jack F. Youngren.
Application Number | 20120214758 13/399031 |
Document ID | / |
Family ID | 37997233 |
Filed Date | 2012-08-23 |
United States Patent
Application |
20120214758 |
Kind Code |
A1 |
GOLDFINE; Ira ; et
al. |
August 23, 2012 |
TYROSINE KINASE RECEPTOR ANTAGONISTS AND METHODS OF TREATMENT FOR
PANCREATIC CANCER
Abstract
A method of treatment is disclosed whereby cancer cells are
brought into contact with a formulation comprising an inhibitor of
tyrosine kinase receptors. The formulation may be comprised of an
injectable carrier and two or more tyrosine kinase receptor
inhibitors which may be nordihydrogluaiaretic acid (NDGA) and
doxyrubicine.
Inventors: |
GOLDFINE; Ira; (San
Francisco, CA) ; Kerner; John; (San Francisco,
CA) ; Maddux; Betty A.; (San Francisco, CA) ;
Campbell; Michael; (Woodside, CA) ; Youngren; Jack
F.; (San Francisco, CA) ; Kushner; Peter; (San
Francisco, CA) |
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
37997233 |
Appl. No.: |
13/399031 |
Filed: |
February 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11552686 |
Oct 25, 2006 |
8143226 |
|
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13399031 |
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60828937 |
Oct 10, 2006 |
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60825663 |
Sep 14, 2006 |
|
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60731384 |
Oct 28, 2005 |
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Current U.S.
Class: |
514/34 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/70 20130101; A61K 31/404 20130101 |
Class at
Publication: |
514/34 |
International
Class: |
A61K 31/704 20060101
A61K031/704; A61P 35/00 20060101 A61P035/00 |
Claims
1.-26. (canceled)
27. A method of treating a human pancreatic cancer patient
comprising: orally administering to the patient a therapeutically
effective amount of a formulation comprising: (1) a
pharmaceutically acceptable carrier; (2) nordihydroguaiaretic acid
or pharmaceutically acceptable salts thereof; and (3) doxorubicin,
wherein the NDGA and doxorubicin act synergistically to inhibit
growth of the pancreatic cancer cells, and wherein doxorubicin and
NDGA are administered in amounts below a therapeutically effective
amount of either doxorubicin or NDGA when doxorubicin or NDGA are
administered alone.
28. The method of claim 27, wherein the amount of doxorubicin
administered is less than 60 mg/m.sup.2 once in 21 days, or less
than 30 mg/m.sup.2 daily for 3 days every four weeks.
29. The method of claim 27, wherein the amount of NDGA administered
is less than 100 mg/kg orally three times a week, or 37.5 mg/kg
intraperitoneally three times a week.
30. The method of claim 27, wherein side effects are decreased
compared to giving either doxorubicin or NDGA at higher
dosages.
31. A method of treating a human pancreatic cancer patient, wherein
pancreatic cancer cells in the cancer patient do not express
Her2/neu receptor, comprising: orally administering to the patient
a therapeutically effective amount of a formulation comprising: (1)
a pharmaceutically acceptable carrier; (2) nordihydroguiaretic acid
(NDGA) or pharmaceutically acceptable salts thereof; (3) and
doxorubicin, wherein the NDGA and doxorubicin act synergistically
to inhibit growth of the pancreatic cancer cells, and wherein
doxorubicin and NDGA are administered in amounts below a
therapeutically effective amount of either doxorubicin or NDGA when
doxorubicin or NDGA are administered alone.
32. The method of claim 31, wherein the amount of doxorubicin
administered is less than 60 mg/m.sup.2 once in 21 days, or less
than 30 mg/m.sup.2 daily for 3 days every four weeks.
33. The method of claim 31, wherein the amount of NDGA administered
is less than 100 mg/kg orally three times a week, or 37.5 mg/kg
intraperitoneally three times a week.
34. The method of claim 31, wherein side effects are decreased
compared to giving either doxorubicin or NDGA at higher
dosages.
35. A method of treating a pancreatic cancer patient, wherein
pancreatic cancer cells in the cancer patient express Her2/neu
receptor, comprising the steps of: administering to the patient a
therapeutically effective amount of a formulation comprising: (1) a
pharmaceutically acceptable carrier; (2) nordihydroguaiaretic acid
(NDGA) or pharmaceutically acceptable salts thereof; and (3)
doxorubicin, wherein the NDGA and doxorubicin act synergistically
to inhibit growth of the pancreatic cancer cells, and wherein the
amount of doxorubicin administered is less than 60 mg/m.sup.2 once
in 21 days, or less than 30 mg/m.sup.2 daily for 3 days every four
weeks and the amount of NDGA is less than 100 mg/kg orally three
times a week, or 37.5 mg/kg intraperitoneally three times a
week.
36. A method of treating a pancreatic cancer patient, wherein
pancreatic cancer cells in the cancer patient do not express
Her2/neu receptor, comprising the steps of: administering to the
patient a therapeutically effective amount of a formulation
comprising: (1) a pharmaceutically acceptable carrier; (2)
nordihydroguaiaretic acid (NDGA) or pharmaceutically acceptable
salts thereof; and (3) doxorubicin, wherein the NDGA and
doxorubicin act synergistically to inhibit growth of the pancreatic
cancer cells, and wherein the amount of doxorubicin administered is
less than 60 mg/m.sup.2 once in 21 days, or less than 30 mg/m.sup.2
daily for 3 days every four weeks and the amount of NDGA is less
than 100 mg/kg orally three times a week, or 37.5 mg/kg
intraperitoneally three times a week.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 60/731,384 filed Oct. 28, 2005; 60/825,663 filed
Sep. 14, 2006; and 60/828,937 filed Oct. 10, 2006, which
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Many tumor cells depend on the activity of tyrosine kinases,
which act, among other functions, to depress apoptosis in the cell.
The tyrosine kinases are usually overproduced in malignant cells,
which contributes to the cell's ability to resist apoptosis.
Modulating the activity of these proteins provides an effective
means of treating cancer while not unduly damaging normal tissues.
For example, about 25% of breast tumors express unusually high
levels of the Her2 protein, a tyrosine kinase receptor that
normally plays a part in the development of the mammary epithelium.
Herceptin.RTM. (Trastuzumab) is a humanized antibody that is
currently used to treat breast cancer by targeting and blocking the
function of the Her2 protein. Other treatments focus on interfering
with the receptors to overexpressed tyrosine kinase proteins.
Receptors include HER2/neu and IGF-1R. See, Meric et al. (Apr.
2002) J. Am. Coll. Surg. 194(4):488-501.
[0003] The major lignin in chaparral, known as nordihydroguaiaretic
acid (NDGA) is a potent antioxidant and was originally used in
commercial food products as a preservative. See, U.S. Pat. No.
2,644,822. Later, it was discovered that NDGA is useful in the
treatment of diabetes. Hsu et al. (2001) Cell Transplant.
10(3):255-262. More recently, NDGA was investigated as a treatment
for cancer because it inhibits the platelet derived growth factor
receptor and the protein kinase C intracellular signalling family,
which both play an important role in proliferation and survival of
cancers. Moreover, NDGA induces apoptosis in tumor xenografts.
Although it is likely to have several targets of action, NDGA is
well tolerated in animals. However, high concentrations of NDGA are
required for efficacy and it has been suggested that more potent
analogs may be required. See, McDonald et al. (2001) Anticancer
Drug Des. 16(6):261-270.
[0004] Other cancer drugs include doxorubicin hydrochloride (DOX),
which is used alone or in combination with other drugs for
treatment of malignant lymphomas and leukemias. DOX is believed to
bind DNA and inhibit nucleic acid synthesis. Examples of tumors
amenable to treatment with DOX are acute lymphoblastic leukemia,
acute myeloblastic leukemia, Wilm's tumor, soft tissue and bone
sarcomas, breast carcinoma and ovarian carcinoma. The dosage needs
to be closely monitored because it can cause irreversible cardiac
damage. A typical dose for adults, when given intravenously is
60-75 mg/m2 once in 21 days, or 30 mg/m2 daily for 3 days every
four weeks, where the total cumulative dose should not exceed 550
mg/m2 without monitoring for cardiac function.
[0005] It is well established that breast cancer is regulated by
receptors for the female sex steroids, estrogen and progesterone.
It is now appreciated that receptor tyrosine kinases (RTKs) are
also very important for breast cancer growth (Arteaga C L, Moulder
S L, Yakes F M: HER (erbB) tyrosine kinase inhibitors in the
treatment of breast cancer. Semin Oncol 29:4-10, 2002; Averbuch S,
Kcenler M, Morris C. Wakeling A: Therapeutic potential of tyrosine
kinase inhibitors in breast cancer. Cancer Invest 21:782-791, 2003;
Baserga R: The IGF-I receptor in cancer research. Exp Cell Res
253:1-6, 1999; Dickson R B, Lippman M E: Growth factors in breast
cancer. Endocr Rev 16:559-589, 1995; Gross J M, Yee D: The type-1
insulin-like growth factor receptor tyrosine kinase and breast
cancer: biology and therapeutic relevance. Cancer Metastasis Rev
22:327-336, 2003; and Nahta R, Hortobagyi G N, Esteva F J: Growth
factor receptors in breast cancer: potential for therapeutic
intervention. Oncologist 8:5-17, 2003).
[0006] Accordingly, RTKs are targets for anti-tumor therapy. RTKs
are transmembrane proteins that typically contain an extracellular
ligand binding domain, activated by peptide hormones, and an
intracellular tyrosine kinase domain. Two RTKs of demonstrated
importance in breast and other cancers are the insulin-like growth
factor receptor (IGF-1R) (Heinemann V: Present and future treatment
of pancreatic cancer. Semin Oncol 29:23-31, 2002) and
c-erbB2/HER2/neu (HER2/neu) (Morin M J: From oncogene to drug:
development of small molecule tyrosine kinase inhibitors as
anti-tumor and anti-angiogenic agents. Oncogene 19:6574-6583,
2000). Based on their major role in regulating cancer cell growth
and survival, inhibitors of these RTKs are undergoing drug
development (Morin M J: From oncogene to drug: development of small
molecule tyrosine kinase inhibitors as anti-tumor and
anti-angiogenic agents. Oncogene 19:6574-6583, 2000; Bruns C J,
Solorzano C C, Harbison M T, Ozawa S, Tsan R, Fan D, Abbruzzese J,
Traxler P, Buchdunger E, Radinsky R, Fidler I J: Blockade of the
epidermal growth factor receptor signaling by a novel tyrosine
kinase inhibitor leads to apoptosis of endothelial cells and
therapy of human pancreatic carcinoma. Cancer Res 60:2926-2935,
2000; Bruns C J, Harbison M T, Davis D W, Portera C A, Tsan R,
McConkey D J, Evans D B, Abbruzzese J L, Hicklin D J, Radinsky R:
Epidermal growth factor receptor blockade with C225 plus
gemcitabine results in regression of human pancreatic carcinoma
growing orthotopically in nude mice by antiangiogenic mechanisms.
Clin Cancer Res 6:1936-1948, 2000; Blum G, Gazit A, Levitzki A:
Substrate competitive inhibitors of IGF-1 receptor kinase.
Biochemistry 39:15705-15712, 2000).
[0007] Signaling via the IGF-1R is important for normal cell growth
and differentiation. In addition, the IGF-1R stimulates mitogenesis
and suppresses apoptosis of cancer cells (Lowe W L: Biological
actions of the insulin-like growth factors. In LeRoith D (ed):
Insulin-like growth factors: molecular and cellular aspects. Boca
Raton, CRC Press, 1991). Following binding of the ligand to the
IGF-1R, a conformational change induces trans-autophosphorylation
of the .beta.-subunits on select tyrosine residues, and subsequent
activation of tyrosine kinase activity (Lowe W L: Biological
actions of the insulin-like growth factors. In LeRoith D (ed):
Insulin-like growth factors: molecular and cellular aspects. Boca
Raton, CRC Press, 1991). Phosphorylation of several target
substrates activates divergent signaling cascades, though the
anti-apoptotic effects of the IGF-1R are primarily mediated via the
Akt/PKB pathway (Kulik G, Klippel A, Weber M J: Antiapoptotic
signalling by the insulin-like growth factor I receptor,
phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17:1595-1606,
1997).
[0008] Tyrosine phosphorylation of the insulin receptor substrate
(IRS) family of proteins by the IGF-1R allows binding of the
regulatory subunit of phosphatidylinositol 3-kinase (PI3K) to the
IRS proteins via SH2 domains. Activated PI3K serine phosphorylates
and activates the serine kinase Akt (Alessi D R, Andjelkovic M,
Caudwell B, Cron P, Morrice N, Cohen P, Hemmings B A: Mechanism of
activation of protein kinase B by insulin and IGF-1. EMBO J
15:6541-6551, 1996). Akt can phosphorylate the protein BAD, which
prevents BAD from forming a pro-apoptotic complex with Bcl-2
proteins (Virdee K, Parone P A, Tolkovsky A M: Phosphorylation of
the pro-apoptotic protein BAD on serine 155, a novel site,
contributes to cell survival. Curr Biol 10:1151-1154. 2000).
[0009] Interruption of the IGF-1R signaling system, either by
reducing effective IGF-1 levels or targeting the receptor, can
block growth and proliferation of cancer cells (Kahan Z, Varga J L,
Schally A V, Rekasi Z, Armatis P, Chatzistamou L, Czompoly T,
Halmos G: Antagonists of growth hormone-releasing hormone arrest
the growth of MDA-MB-468 estrogen-independent human breast cancers
in nude mice. Breast Cancer Res Treat 60:71-79, 2000:
Neuenschwander S, Roberts C T, Jr., LeRoith D: Growth inhibition of
MCF-7 breast cancer cells by stable expression of an insulin-like
growth factor I receptor antisense ribonucleic acid. Endocrinology
136:4298-4303, 1995; Prager D, Li H L, Asa S, Melmed S: Dominant
negative inhibition of tumorigenesis in vivo by human insulin-like
growth factor I receptor mutant. Proc Natl Acad Sci USA
91:2181-2185, 1994; Weckbecker G, Tolcsvai L, Liu R, Bruns C:
Preclinical studies on the anticancer activity of the somatostatin
analogue octreotide (SMS 201-995). Metabolism 41:99-103, 1992; and
Yee D, Jackson J G, Kozelsky T W, Figueroa J A: Insulin-like growth
factor binding protein 1 expression inhibits insulin-like growth
factor I action in MCF-7 breast cancer cells. Cell Growth Differ
5:73-77, 1994). While overexpression of the IGF-1R can drive
transformation and mitogenesis, it is the requirement for its
constitutive presence in cancer cells (Rubin R, Baserga R:
Insulin-like growth factor-I receptor. Its role in cell
proliferation, apoptosis, and tumorigenicity. Lab Invest
73:311-331, 1995) that makes this RTK an attractive target for
anti-tumor therapies.
[0010] The HER2/neu (c-erbB-2) protooncogene encodes a 1,255 amino
acid, 185 kDa member of the class I RTK family. HER2/neu is
overexpressed in 20-30% of breast cancers, most commonly via gene
amplification, and overexpression is associated with poor prognosis
in these patients (Slamon D J, Godolphin W, Jones L A, Holt J A,
Wong S G, Keith D E, Levin W J, Stuart S G, Udove J, Ullrich A:
Studies of the HER-2/neu proto-oncogene in human breast and ovarian
cancer. Science 244:707-712, 1989; Slamon D J, Clark G M, Wong S G,
Levin W J, Ullrich A, McGuire W L: Human breast cancer: correlation
of relapse and survival with amplification of the HER-2/neu
oncogene. Science 235:177-182, 1987). Evidence from transgenic
animal studies indicates that HER2/neu overexpression directly
contributes to transformation and tumor progression (Bol D, Kiguchi
K, Beltran L, Rupp T, Moats S, Gimenez-Conti I, Jorcano J,
DiGiovanni J: Severe follicular hyperplasia and spontaneous
papilloma formation in transgenic mice expressing the neu oncogene
under the control of the bovine keratin 5 promoter. Mol Carcinog
21:2-12, 1998; Bouchard L, Lamarre L, Tremblay P J, Jolicoeur P:
Stochastic appearance of mammary tumors in transgenic mice carrying
the MMTV/c-neu oncogene. Cell 57:931-936, 1989; and Lucchini F,
Sacco M G, Hu N, Villa A, Brown J, Cesano L, Mangiarini L, Rindi G,
Kindl S, Sessa F: Early and multifocal tumors in breast, salivary,
harderian and epididymal tissues developed in MMTY-Neu transgenic
mice. Cancer Lett 64:203-209, 1992), and suggests that its
prognostic significance arises from the particularly aggressive
phenotype it confers (Hynes N E, Stern D F: The biology of
erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta
1198:165-184, 1994). The efficacy of targeting HER2/neu in
anti-cancer therapy has been demonstrated by the clinical use of an
antibody to HER2/neu to treat certain patients with breast cancer
(Albanell J, Baselga J: Trastuzumab, a humanized anti-HER2
monoclonal antibody, for the treatment of breast cancer. Drugs
Today (Bare) 35:931-946, 1999).
[0011] Nordihydroguaiaretic acid (NDGA) is a phenolic compound that
was identified as a major component of a tea made from resinous
extracts of the creosote bush Larrea divaricatta. It has been used
for centuries by Native North Americans as a remedy for diverse
illnesses, including tumors (Duisberg P C: Desert Plant
Utilization. Texas J Sci 4:269, 1952; Hawthorn P: Medicinal uses of
plants of Nevada used by Indians. Contr Flora Nevada 45:1-139,
1957). NDGA has been reported to inhibit the growth of various
tumors both in vitro and in animals (Wilson D E, DiGianfilippo A,
Ondrey F G, Anderson K M, Harris J E: Effect of
nordihydroguaiaretic acid on cultured rat and human glioma cell
proliferation. J Neurosurg 71:551-557, 1989; Avis T M, Jett M,
Boyle T, Vos M D, Moody T, Treston A M, Martinez A, Mulshine J L:
Growth control of lung cancer by interruption of
5-lipoxygenase-mediated growth factor signaling. J Clin Invest
97:806-813, 1996; Rose D P, Connolly J M: Effects of fatty acids
and inhibitors of eicosanoid synthesis on the growth of a human
breast cancer cell line in culture. Cancer Res 50:7139-7144, 1990;
and Shimakura S. Boland C R: Eicosanoid production by the human
gastric cancer cell line AGS and its relation to cell growth.
Cancer Res 52:1744-1749, 1992). NDGA also has been reported to
induce apoptosis in a variety of cell lines (Ding X Z, Kuszynski C
A, El Metwally T H, Adrian T E: Lipoxygenase inhibition induced
apoptosis, morphological changes, and carbonic anhydrase expression
in human pancreatic cancer cells. Biochem Biophys Res Commun
266:392-399, 1999; La E, Kern J C, Atarod E B, Kehrer J P: Fatty
acid release and oxidation are factors in lipoxygenase
inhibitor-induced apoptosis. Toxicol Lett 138:193-203, 2003;
Seufferlein T, Seckl M J, Schwarz E, Beil M, Wichert G, Baust H,
Luhrs H, Schmid R M, Adler G: Mechanisms of nordihydroguaiaretic
acid-induced growth inhibition and apoptosis in human cancer cells.
Br J Cancer 86:1188-1196, 2002; Tong W G, Ding X Z, Witt R C,
Adrian T E: Lipoxygenase inhibitors attenuate growth of human
pancreatic cancer xenografts and induce apoptosis through the
mitochondrial pathway. Mol Cancer Ther 1:929-935, 2002; and Tong W
G, Ding X Z, Adrian T E: The mechanisms of lipoxygenase
inhibitor-induced apoptosis in human breast cancer cells. Biochem
Biophys Res Commun 296:942-948, 2002). Still, the mechanism of this
anti-cancer effect of NDGA is not well understood. It has been
reported that NDGA inhibits the tyrosine kinase activity of the
platelet-derived growth factor receptor (PDGFR), but not the
epidermal growth factor receptor (EGFR), in cells and in vitro
(Domin J, Higgins T, Rozengurt E: Preferential inhibition of
platelet-derived growth factor-stimulated DNA synthesis and protein
tyrosine phosphorylation by nordihydroguaiaretic acid. J Biol Chem
269:8260-8267, 1994). While one report suggests that NDGA is
inactive against the IGF-1R (Seufferlein T, Seckl M J, Schwarz E,
Bell M, Wichert G, Baust H, Luhrs H, Schmid R M, Adler G:
Mechanisms of nordihydroguaiaretic acid-induced growth inhibition
and apoptosis in human cancer cells. Br J Cancer 86:1188-1196,
2002), a compound with a very high degree of structural homology to
NDGA has been described as a potent inhibitor of this receptor(Blum
G, Gazit A, Levitzki A: Substrate competitive inhibitors of IGF-1
receptor kinase. Biochemistry 39:15705-15712, 2000: Blum G, Gazit
A, Levitzki A: Development of new insulin-like growth factor-1
receptor kinase inhibitors using catechol mimics. J Biol Chem
278:40442-40454, 2003). The effects of NDGA on the HER2/neu
receptor, which also plays a critical role in breast cancer, have
not been explored. We have now found that NDGA antagonizes the
activation of both the IGF-1 and HER2/neu receptors, inhibits the
cellular anti-apoptotic signaling pathway of the IGF-1R, and
inhibits the growth of breast cancer cells both in vitro and in
vivo.
[0012] There is a need for therapeutic cancer treatments that block
the tyrosine kinase receptors with lower dosages of these powerful
drugs to reduce side effects. The present invention addresses this
and other related needs.
SUMMARY OF THE INVENTION
[0013] Treatments for breast and pancreatic cancer are disclosed.
The malignant cells are brought into contact with NDGA and
diarylurea 21834, either singly or in combination with other
compounds, such as doxorubicin and Herceptin.
[0014] Nordihydroguaiaretic acid (NDGA) is a phenolic compound
isolated from the creosote bush Larrea divaricatta that has
anti-cancer activities both in vitro and in vivo. These anti-cancer
properties in breast cancer cells are created by the ability of
NDGA to directly inhibit the function of two receptor tyrosine
kinases (RTKs), the insulin-like growth factor receptor (IGF-1R)
and the c-erbB2/HER2/neu (HER2/neu) receptor. In MCF-7 human breast
cancer cells, low micromolar concentrations of NDGA inhibited
activation of the IGF-1R, and downstream phosphorylation of both
the Akt/PKB serine kinase and the pro-apoptotic protein BAD.
[0015] In mouse MCNeuA cells, NDGA also inhibited ligand
independent phosphorylation of HER2/neu. This inhibitory effect in
cells is due to a direct action on these receptors. The
IGF-1-stimulated tyrosine kinase activity of isolated IGF-1R is
inhibited by NDGA at 10 .mu.M or less. A composition of NDGA is
also effective at inhibiting autophosphorylation of isolated
HER2/neu receptor at similar concentrations. In addition, NDGA
inhibits IGF-1 specific growth of cultured breast cancer cells with
an IC50 of approximately 30 .mu.M. Treatment with NDGA
(intraperitoneal injection 3 times per week) also decreases the
activity of the IGF-1R and the HER2/neu receptor in MCNeuA cells
implanted into mice. Inhibition of RTK activity is associated with
decreased growth rates of MCNeuA cells in vivo. Accordingly, the
anti-breast cancer properties of NDGA are related to the inhibition
of two important RTKs and as such formulations of RTK inhibitors
provide a means of treating breast cancer.
[0016] One aspect of the invention comprises methods for using NDGA
in the treatment of breast cancer.
[0017] A further aspect of the invention is methods for treating
breast cancer with a combination of NDGA and Doxorubicin. This
formulation provides an unexpected synergistic effect in
combination at low concentrations compared to individual dosages.
When given in combination, lower dosages may be used to achieve a
greater effect, which has the additional benefit of decreasing
side-effects compared to the individual drugs given at higher
dosages.
[0018] Another aspect of the invention is methods for using
Diarylurea 21834 (DAU) alone or in various combinations with other
compounds for the treatment of breast cancer.
[0019] One aspect of the invention comprises methods for using NDGA
in the treatment of pancreatic cancer.
[0020] A further aspect of the invention is methods for treating
pancreatic cancer with a combination of NDGA and Doxorubicin. This
formulation provides an unexpected synergistic effect in
combination at low concentrations compared to individual dosages.
When given in combination, lower dosages may be used to achieve a
greater effect, which has the additional benefit of decreasing
side-effects compared to the individual drugs given at higher
dosages.
[0021] Another aspect of the invention is methods for using
Diarylurea 21834 (DAU) alone or in various combinations with other
compounds for the treatment of pancreatic.
[0022] As aspect of the invention is treating a breast cancer
patient by administering tamoxifen to the patient; determining that
the patient is not sufficiently responsive to tamoxifen; and
treating the patient with a combination of tamoxifen and NDGA.
[0023] Another aspect of the invention is diagnosing a cancer
patient as having estrogen receptor (ER) positive MCF-7 cells that
overexpress HER2 (MCF-7/HER2-18) and administering to the patient a
therapeutically effective amount of a combination of tamoxifen and
NDGA.
[0024] Another aspect of the invention is a formulation
manufactured for the treatment of breast cancer specifically where
the cancer has been shown to be resistant to tamoxifen, wherein the
formulation comprises a therapeutically effective amount of a
combination of tamoxifen and NDGA.
[0025] Another aspect of the invention is a method of treatment
wherein a patient is treated with tamoxifen and found to be
insufficiently responsive which treatment is followed by
administration of both tamoxifen and NDGA.
[0026] Still yet another aspect of the invention is a kit comprised
of tamoxifen, NDGA, and instructions with respect to the treatment
of patients having estrogen receptor (ER) positive MCF-7 cells that
overexpress HER2 (MCF-7/HER2-18).
[0027] These and other aspects of the invention will become
apparent to those persons skilled in the art upon reading the
details of the formulations and methods as more fully described
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows the chemical structure of nordihydroguaiaretic
acid (NDGA).
[0029] FIG. 2 shows the chemical structure of diarylurea 21834
(DAU).
[0030] FIG. 3 is a graph showing the results of using NDGA to
inhibit the growth of pancreatic cancer cells in vitro.
[0031] FIG. 4A is a graph showing the effectiveness of NDGA in
inhibiting IGF-1 activation in pancreatic cancer cells. FIG. 4B is
a graph showing the effectiveness of NDGA in inhibiting Her2/neu
activation in pancreatic cancer cells.
[0032] FIG. 5 is a graph showing that DAU inhibits tyrosine kinase
activation in Panc 1 pancreatic cancer cells.
[0033] FIG. 6A is a schematic representation of an isobologram and
FIG. 6B is a schematic representation of an isobologram analysis
for DOX and NDGA on SKBR-3 cells.
[0034] FIG. 7 is a bar graph showing the affect of different
concentrations of NDGA on prostate cancer cells.
[0035] FIG. 8 is an image of a Western Blot obtained by incubating
different concentrations of NDGA with a peptide corresponding to
the kinase domain of IGF-1R.
[0036] FIG. 9 is a bar graph showing the results of IGF-1R
incubated with various concentrations of NDGA prior to addition of
IGF-1 wherein results were determined by ELISA employing an
anti-phosphotyrosine antibody to readout.
[0037] FIG. 10 is an image of a Western Blot wherein HER2/neu
recepetors were incubated with ATP alone or with NDGA.
[0038] FIG. 11A is a bar graph and FIG. 11B is two images of
Western Blots showing results where MCF-7 breast cancer cells were
incubated with various concentrations of NDGA.
[0039] FIG. 12 is an image of a Western Blot showing results
wherein MCNeuA breast cancer cells were incubated with varying
concentrations of NDGA.
[0040] FIG. 13A is a bar graph showing results of incubating MCF-7
cells with varying concentrations of NDGA and FIG. 13B is a bar
graph of results showing results wherein such cells were incubated
in serum or a concentration of 10 nM IGF-1.
[0041] FIG. 14 is a bar graph showing results wherein NDGA was
administered over a period of 21 days in vivo to MCNeuA tumors.
[0042] FIG. 15 is a graph showing the results of MDNeuA cells of a
tumor being treated over a period of days with NDGA.
[0043] FIGS. 16A and 16B are each bar graphs showing the affect of
DMSO and NDGA on neuroblastoma cells.
[0044] FIGS. 17A and 17B are each bar graphs showing the affect of
DMSO and four different concentrations of NDGA on neuroblastoma
cells.
[0045] FIGS. 18A and 18B are each bar graphs showing the affect of
DMSO and three different concentrations of NDGA on neuroblastoma
cells.
[0046] FIGS. 19A and 19B are each images of Western Blot analysis
showing results obtained with DMSO and three different
concentrations of NDGA.
[0047] FIG. 20A is an image of a Western Blot showing the affects
of DMSO and three different concentrations of NDGA. FIG. 20B is an
image of a Western Blot showing the affects of DMSO and NDGA. FIG.
20C is a bar graph showing the results obtained using DMSO and NDGA
in three different concentrations.
[0048] FIGS. 21A and 21B are each bar graphs showing the affects of
DMSO and NDGA. FIG. 21C shows the affects of DMSO and NDGA used to
treat mice by injection.
[0049] FIG. 22 shows gel images which show the expression of IGF-IR
and HER2 in MCF-7/neo and MCF-7/HER2-18 cells. The gels were
created using cell lysates which were separated by SDS-PAGE,
transferred to nitrocellulose membranes, and probed with antibodies
specific for IGF-1R and HER2. Two concentrations of total protein
10 .mu.g and 14 .mu.g were analyzed to confirm the linearity of the
assay.
[0050] FIGS. 23A and 23B are each graphs which show the effects of
gefitinib and NDGA on the growth of MCF-7/neo and MCF-7/HER2-18
cells. Cells were grown in the presence of various concentrations
of gefitinib (23A) or NDGA (23B) for 6 days. Cell growth was
assessed with a CyQuant cell proliferation assay. The results are
expressed as mean.+-.SEM of triplicate wells and are representative
of three separate experiments.
[0051] FIGS. 24A and 24B are each graphs which show the effects of
gefitinib and NDGA on IGF-IR and HER2 phosphorylation in
MCF-7/HER2-18 cells. Cells treated with various concentrations of
gefitinib (A) or NDGA (B) were lysed and assayed for IGF-1R and
HER2 phosphorylation by ELISA. For the IGF-1R ELISA, cells were
stimulated with 3 nM IGF-I for 10 min. For the HER2 ELISA, cells
were not stimulated. The results are expressed as mean.+-.SEM of
triplicate wells and are representative of three separate
experiments. The effects of NDGA were confirmed by Western blot
(panel B, inset) where phosphorylated IGF-IR and HER2 were detected
with phospho-specific antibodies to pIGF-IR and pHER2,
respectively.
[0052] FIG. 25 shows gel images which show the effect of NDGA on
Akt/PKB phosphorylation. MCF-7/HER2-18 cells were incubated in the
presence or absence of 3 nM IGF-1 for 10 min, with or without NDGA
treatment. Cells lysates were prepared and separated by SDS-PAGE,
transferred to nitrocellulose membranes, and probed with an
anti-pAkt antibody.
[0053] FIGS. 26A, 26B, 26C and 26D are graphs which show the
effects of combined treatment with tamoxifen and NDGA on the growth
of MCF-7/neo and MCF-7/HER2-18 cells. MCF-7/neo (panels 26A and
26C) and MCF-7/HER2-18 (panels 26B and 26D) cells were incubated
for 6 days with 100 nM tamoxifen, in the presence or absence of
various concentrations of NDGA and cell growth was assessed with a
CyQuant assay. Cell proliferation was expressed as a percentage of
untreated control cells (mean.+-.SEM) (panels 26A and 26B). Panels
26C and 26D express the results as percent growth inhibition.
[0054] FIG. 27 is a graph which shows results where human breast
cancer MCF-7 cells were grown for 5 days with medium plus 10% fetal
calf serum and treated with the indicated concentrations of
valproic acid (VAPA), nordihydroguaiaretic acid (NDGA), or
rapamycin (RAPA). At the end of 5 days the cultures (in a 96 well
plate) were assayed for total nucleic acid with a CyQuant dye based
assay. Shown is the OD in this assay.
DETAILED DESCRIPTION OF THE INVENTION
[0055] Before the present compositions for and methods of treating
cancer are described, it is to be understood that this invention is
not limited to particular compositions and methods described, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
[0056] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0057] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, potential and preferred methods and materials are now
described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited.
[0058] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cancer cell" includes a plurality of such
cancer cells and reference to "the methods of administration"
includes reference to one or more methods and equivalents thereof
known to those skilled in the art, and so forth.
[0059] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Definitions
[0060] The term "nordihydroguaiaretic acid" is also referred to as
"NDGA" and is the compound shown within the structure of FIG. 1 and
see U.S. Pat. No. 2,644,822 incorporated here to disclosed NDGA as
well as related compounds and their method of manufacture. It is
pointed out that pharmaceutically acceptable salts and amines of
the acid may be formed during use and are considered to be
encompassed by the term unless specifically indicated
otherwise.
[0061] The term tyrosine kinase receptor blocker and inhibitor of
tyrosine kinase are used interchangeably to describe compounds
which selectively and specifically bind to tyrosine kinase
receptors. The binding preferably has an antagonist effect. Such
compounds include compounds such as Her2 inhibitors, doxorubicine
and Herceptin.TM..
[0062] The terms "treatment," "treating," and the like are used
herein to generally mean obtaining a desired pharmacological and/or
physiological effect. The effect may be prophylactic in tee ins of
completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of partially or completely
curing a disease and/or adverse effect attributed to the disease.
In general, methods of the invention involve treating diseases
referred to as cancer and may be applied to a variety of different
types of cancer by utilizing combinations of compounds such as
tyrosine kinase receptor inhibitors which are known to bind to the
receptor site. "Treatment" as used herein covers any treatment of
such a disease in a mammal, particularly a human, and includes:
[0063] (a) preventing and/or diagnosing the disease in a subject
which may be predisposed to the disease which has not yet been
diagnosed as having it;
[0064] (b) inhibiting the disease, i.e. arresting its development;
and/or
[0065] (c) relieving the disease, i.e. causing regression of the
disease.
[0066] The invention is directed towards treating patients with
cancer and is particular directed towards treating particular types
of cancer which are not generally treatable with normal surgical
methods. More specifically, "treatment" is intended, in preferred
circumstances, to mean providing a therapeutically detectable and
beneficial effect on a patient suffering from cancer.
Formulations and Methods
[0067] Formulations of the invention combine compounds and
excipients to obtain desirable results with respect to the
biochemical inhibition of certain receptors. The compound such as
nordihydroguaiaretic acid (NDGA), IFG-1 inhibitors and recombinant
Her2 inhibitors as well as doxorubicine and Herceptin.TM. can be
used in a pharmaceutically acceptable excipient carrier in various
combinations. The combinations of the invention obtain a
synergistic effect. This synergistic effect is specifically defined
in connection with the present invention. Those skilled in the art
will understand that the use of compound A to inhibit receptor X
cannot be increased beyond certain points simply by adding more of
compound A. At some point the effect of compound A is not increased
by adding the amount or the increase is not practical in view of
the toxic effects. Thus, the combination of "compound A" and
"compound B" may be synergistic in blocking receptor "X" even when
the combination of "A" and "B" is not additive in terms of blocking
receptor "X". Obtaining, a modest increase in the blockage of
receptors without an increase in adverse effects or even with an
acceptable level of adverse effects may be all that is necessary to
effectively treat a given cancer.
[0068] Certain compounds may be used by themselves in particular
dosage amounts and treatment regimes. For example, NDGA may be used
in the treatment of pancreatic cancer. FIG. 3 shows the results of
NDGA inhibiting the growth of pancreatic cancer cells in vitro.
FIGS. 4A and 4B show the effectiveness of NDGA in inhibiting
certain receptors. Specifically, FIG. 4A shows NDGA in micrograms
per milliliter inhibiting Her2/NEU activation in pancreatic cancer
cells. FIG. 4B shows NDGA being used in micrograms per milliliter
inhibiting IGF-1 activation in pancreatic cancer cells.
[0069] FIG. 5 is a graph showing that DAU inhibits tyrosine kinase
activation in Panc 1 pancreatic cancer cells.
[0070] FIG. 6A is a schematic representation of an isobologram. The
envelope of additivity, surrounded by Mode I (solid line) and Mode
II (dotted lines) isobologram lines, is constructed from the
dose-response curves of two drugs (A and B). The data points Pa,
Pb, Pc and Pd, obtained from the combination dose-response curves
of A and B, would classify the two-drug interactions as
supra-additive (synergistic), additive, sub-additive and mutually
protective, respectively. FIG. 6B is an isobologram analysis for
DOX and NDGA on SKBR-3 cells. At low concentrations, the two
compounds act synergistically.
[0071] When the combinations of drugs are used in accordance with
the invention improved results of some manner are expected with
respect to inhibiting the growth of cancer cells. FIG. 6A is a
graph which schematically represents a basic concept behind an
aspect of the invention. FIG. 6A shows an envelope of additivity
for two drugs "A" and "B". As indicated in the graph the amount of
drug: "B" increases in an upward direction along the "X" axis and
the amount of drug "A" increases to the right along the "Y" axis.
Using less drug is generally more desirable when the same amount of
the drug can obtain the desired effect. Thus, the solid line and
the area below the solid line represents a supra-additive
effect.
[0072] FIG. 7 is a bar graph showing the affect of NDGA on PC3
prostate cancer cell growth wherein the cells were plated at
10.sup.5 per well in the absence and presence of NDGA for four days
with the results shown as the mean.+-.SD4 triplicate
determinations.
[0073] The present invention shows that NDGA is a direct inhibitor
of both the IGF-1R and the HER2/neu receptor in isolated receptor
preparations, cultured breast cancer cells, and tumors in vivo.
Inhibition of these RTKs was accompanied by a reduction in cell
growth both in cell culture and in vivo. The present invention
demonstrates studies indicate that inhibition of RTKs has an
important anti-tumor effect of NDGA.
[0074] The IGF-1R is an essential component of the transformation
process and an attractive target for anti-cancer agents (Gross J M,
Yee D: The type-1 insulin-like growth factor receptor tyrosine
kinase and breast cancer: biology and therapeutic relevance. Cancer
Metastasis Rev 22:327-336, 2003). Cells in tissue culture that lack
the IGF-1R can not be transformed (Baserga R: Oncogenes and the
strategy of growth factors. Cell 79:927-930, 1994). Following
transformation, overexpression of IGF-1R is observed in many cell
types (Rubin R, Baserga R: Insulin-like growth factor-I receptor.
Its role in cell proliferation, apoptosis, and tumorigenicity. Lab
Invest 73:311-331, 1995). In vivo, in primary breast tumors, the
IGF-1R is overexpressed and hyperphosphorylated (Arteaga C L,
Kitten L J, Coronado E B, Jacobs S, Kull F C, Jr., Allred D C,
Osborne C K: Blockade of the type I somatomedin receptor inhibits
growth of human breast cancer cells in athymic mice. J Clin Invest
84:1418-1423, 1989). In vitro, in a variety of breast cancer cell
lines, we have found that the IGF-1R is overexpressed (Sciacca L,
Costantino A, Pandini G, Mineo R, Frasca F, Scalia P, Sbraccia P,
Goldfine I D, Vigneri R, Belfiore A: Insulin receptor activation by
IGF-II in breast cancers: evidence for a new autocrine/paracrine
mechanism. Oncogene 18:2471-2479, 1999). In addition, there is
strong evidence linking hyperactivation of the IGF-1R with the
early stages of breast cancer (Baserga R: The IGF-I receptor in
cancer research. Exp Cell Res 253:1-6, 1999; Khandwala H M,
McCutcheon I E, Flyvbjerg A, Friend K E: The effects of
insulin-like growth factors on tumorigenesis and neoplastic growth.
Endocr Rev 21:215-244, 2000; and Suiinacz E: Function of the IGF-I
receptor in breast cancer. J Mammary Gland Biol Neoplasia 5:95-105,
2000). Anti-proliferative effects against cultured breast cancer
cells have been observed by employing antisense IGF-1R
(Neuenschwander S, Roberts C T, Jr., LeRoith D: Growth inhibition
of MCF-7 breast cancer cells by stable expression of an
insulin-like growth factor I receptor antisense ribonucleic acid.
Endocrinology 136:4298-4303, 1995), monoclonal antibodies (Sachdev
D, Li S L, Hartell J S, Fujita-Yamaguchi Y, Miller J S, Yee D: A
chimeric humanized single-chain antibody against the type I
insulin-like growth factor (IGF) receptor renders breast cancer
cells refractory to the mitogenic effects of IGF-I. Cancer Res
63:627-635, 2003; Arteaga C L, Osborne C K: Growth inhibition of
human breast cancer cells in vitro with an antibody against the
type I somatomedin receptor. Cancer Res 49:6237-6241, 1989),
transfection with a dominant negative IGF-1R (Prager D, Li H L, Asa
S, Melmed S: Dominant negative inhibition of tumorigenesis in vivo
by human insulin-like growth factor I receptor mutant. Proc Natl
Acad Sci USA 91:2181-2185, 1994), or small-molecule catechol mimics
(Blum G, Gazit A, Levitzki A: Development of new insulin-like
growth factor-1 receptor kinase inhibitors using catechol mimics. J
Biol Chem 278:40442-40454, 2003). Expressing dominant negative
IGF-1R in breast cancer cells also inhibits tumor growth in vivo
(Prager D, Li H L, Asa S, Melmed S: Dominant negative inhibition of
tumorigenesis in vivo by human insulin-like growth factor I
receptor mutant. Proc Natl Acad Sci USA 91:2181-2185, 1994).
[0075] HER2/neu has also emerged as an important target in breast
cancer therapeutics. HER2/neu overexpression occurs in
approximately half of DCIS cases, and 85-100% of high-grade,
comedo-type DCIS, the lesions associated with the highest risk of
progression (Wu Y, Tewari M, Cui S, Rubin R: Activation of the
insulin-like growth factor-I receptor inhibits tumor necrosis
factor-induced cell death. J Cell Physiol 168:499-509, 1996; Prisco
M, Hongo A, Rizzo M G, Sacchi A, Baserga R: The insulin-like growth
factor I receptor as a physiologically relevant target of p53 in
apoptosis caused by interleukin-3 withdrawal. Mol Cell Biol
17:1084-1092, 1997; and Tanno S, Tanno S, Mitsuuchi Y, Altomare D
A, Xiao G H, Testa J R: AKT activation up-regulates insulin-like
growth factor I receptor expression and promotes invasiveness of
human pancreatic cancer cells. Cancer Res 61:589-593, 2001). The
effectiveness of a monoclonal antibody that inhibits HER2/neu
indicates the potential of this RTK as an anti-cancer target,
although no small-molecule compounds specifically targeting
HER2/neu with efficacy in vivo have been reported.
[0076] NDGA has previously been shown to reduce growth and induce
apoptosis in a wide variety of cell lines including breast, lung,
pancreas cancers (Moody T W, Leyton J, Martinez A, Hong S,
Malkinson A, Mulshine J L: Lipoxygenase inhibitors prevent lung
carcinogenesis and inhibit non-small cell lung cancer growth. Exp
Lung Res 24:617-628, 1998; Chen X, Li N, Wang S, Hong J, Fang M,
Yousselfson J, Yang P, Newman R A, Lubet R A, Yang C S: Aberrant
arachidonic acid metabolism in esophageal adenocarcinogenesis, and
the effects of sulindac, nordihydroguaiaretic acid, and
alpha-difluoromethylomithine on tumorigenesis in a rat surgical
model. Carcinogenesis 23:2095-2102, 2002; Hausott B, Greger H,
Marian B: Naturally occurring lignans efficiently induce apoptosis
in colorectal tumor cells. J Cancer Res Clin Oncol 129:569-576,
2003; Wagenknecht B, Schulz J B, Gulbins E, Weller M: Crm-A, bcl-2
and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells
at the level of caspase 8 processing. Cell Death Differ 5:894-900,
1998; Schultze-Mosgau M H, Dale I L, Gant T W, Chipman J K, Kerr D
J, Gescher A: Regulation of c-fos transcription by chemopreventive
isoflavonoids and lignans in MDA-MB-468 breast cancer cells. Eur J
Cancer 34:1425-1431, 1998), but its anti-tumor action has not been
defined. The principal finding of the present study is therefore
the attribution of the described anti-cancer actions to the ability
of NDGA to inhibit cellular RTK activity. The ability of NDGA to
inhibit growth in normal culture conditions is consistent with
studies employing specific inhibition of IGF-1R signaling by
antibody or molecular techniques (Neuenschwander S, Roberts C T,
Jr., LeRoith D: Growth inhibition of MCF-7 breast cancer cells by
stable expression of an insulin-like growth factor I receptor
antisense ribonucleic acid. Endocrinology 136:4298-4303, 1995;
Arteaga C L, Osborne C K: Growth inhibition of human breast cancer
cells in vitro with an antibody against the type I somatomedin
receptor. Cancer Res 49:6237-6241, 1989). In addition, we have
demonstrated that NDGA was more effective at inhibiting growth
stimulated solely by IGF-1 than at inhibiting growth stimulated by
the complex milieu provided by fetal calf serum, or growth in the
absence of all exogenous growth factors. These results suggest that
inhibition of the IGF-1R comprises a major component of the
anti-mitogenic effects of NDGA in cell culture. The findings that
NDGA inhibition of the IGF-1R produces a subsequent reduction in
phosphorylation of the serine kinase Akt/PKB, and of the
pro-apoptotic protein BAD, provide evidence that the described
apoptotic effects of NDGA treatment result directly from an
inhibition of the cell survival pathway regulated by the
IGF-1R.
[0077] We also found that NDGA was able to reduce phosphorylation
of the IGF-1R and HER2/neu in tumors in vivo. This action against
these RTKs was associated with significant reductions in tumor cell
growth. While other studies have demonstrated the effectiveness of
NDGA against xenograft models of pancreatic and non-small cell lung
cancer (Tong W G, Ding X Z, Witt R C, Adrian T E: Lipoxygenase
inhibitors attenuate growth of human pancreatic cancer xenografts
and induce apoptosis through the mitochondrial pathway. Mol Cancer
Ther 1:929-935, 2002; Moody T W, Leyton J, Martinez A, Hong S,
Malkinson A, Mulshine J L: Lipoxygenase inhibitors prevent lung
carcinogenesis and inhibit non-small cell lung cancer growth. Exp
Lung Res 24:617-628, 1998), in the present study we were able to
demonstrate that chronic treatment of tumor-bearing mice resulted
in a reduction in the normal, physiological activation state of
both IGF-1 and c-erbB2/HER2/neu receptors. This effect was observed
16 hours after treatment, and is unlikely to be the result of an
acute effect of the previous injection. Thus, we believe that the
RTK-inhibitory actions of NDGA contribute greatly to its in vivo
anti-cancer properties as well.
[0078] The terminal half-life for a single injection of NDGA is
reported to be 135 minutes (Lambert J D, Meyers R O, Timmermann B
N, Dorr R T: Pharmacokinetic analysis by high-performance liquid
chromatography of intravenous nordihydroguaiaretic acid in the
mouse. J Chromatogr B Biomed Sci Appl 754:85-90, 2001), although
the half-life in tissues as well as the potency of potential
metabolites of NDGA are unknown. Although extracts of the creosote
bush have demonstrated toxic effects (Arteaga S, Andrade-Cetto A,
Cardenas R: Larrea tridentata (Creosote bush), an abundant plant of
Mexican and US-American deserts and its metabolite
nordihydroguaiaretic acid. J Ethnopharmacol 98:231-239, 2005), Pure
NDGA itself has minimal toxicity, has passed FDA approved
pre-clinical trials and is available for administration to humans
(G. Kelly, Insmed Inc., Glen Allen, Va., personal
communication).
[0079] The mechanism whereby NDGA inhibits RTK activity has not yet
been elucidated. Most small-molecule RTK inhibitors are competitive
inhibitors of ATP binding (Morin M J: From oncogene to drug:
development of small molecule tyrosine kinase inhibitors as
anti-tumor and anti-angiogenic agents. Oncogene 19:6574-6583,
2000), as is a recently reported IGF-1R-specific inhibitor
(Mitsiades C S, Mitsiades N S, McMullan C J, Poulaki V,
Shringarpure R, Akiyama M, Hideshima T, Chauhan D, Joseph M,
Libermann T A, Garcia-Echeverria C, Pearson M A, Hofmann F,
Anderson K C, Kung A L: Inhibition of the insulin-like growth
factor receptor-1 tyrosine kinase activity as a therapeutic
strategy for multiple myeloma, other hematologic malignancies, and
solid tumors. Cancer Cell 5:221-230, 2004; Garcia-Echeverria C,
Pearson M A, Marti A, Meyer T, Mestan J, Zimmermann J, Gao J,
Brueggen J, Capraro H G, Cozens R, Evans D B, Fabbro D, Furet P,
Porta D G, Liebetanz J, Martiny-Baron G, Ruetz S, Hofmann F: In
vivo antitumor activity of NVP-AEW541-A novel, potent, and
selective inhibitor of the IGF-IR kinase. Cancer Cell 5:231-239,
2004). However, NDGA does not share general structural homology
with ATP analogs. Interestingly, Blum et al. (Blum G, Gazit A,
Levitzki A: Substrate competitive inhibitors of IGF-1 receptor
kinase. Biochemistry 39:15705-15712, 2000: Blum G, Gazit A,
Levitzki A: Development of new insulin-like growth factor-1
receptor kinase inhibitors using catechol mimics. J Biol Chem
278:40442-40454, 2003) have investigated a compound that appears to
inhibit IGF-1R signaling by competing with the autophosphorylation
sites on the .beta.-subunit of the IGF-1R. This compound was
effective in inhibiting the growth of breast cancer cells in
culture. Interestingly, the di-catechol structure investigated by
this group shares considerable structural homology with NDGA,
differing only in length of the carbon chain linking the two
catechol rings. Modeling data generated by these investigators with
the closely related IR suggest that their lead compound competes
for binding with the tyrosine substrates at the kinase active site
(Blum G, Gazit A, Levitzki A: Substrate competitive inhibitors of
IGF-1 receptor kinase. Biochemistry 39:15705-15712, 2000). It is
unclear however whether NDGA interacts with a homologous site on
either IGF-1R or HER2/neu. The ability of NDGA to inhibit
autophosphorylation of intrinsically active HER2/neu suggests that
this compound acts directly on the enzymatic components of the
receptors and not by interfering with either ligand binding or
binding-dependent conformational changes. Thus, NDGA may represent
a potential new class of agents for the treatment of breast and
other cancers where the IGF-1R and/or HER2/neu play a role in the
oncogenic process.
Treating Neuroblastoma
[0080] Neuroblastoma is a common pediatric malignancy that
metastasizes to the liver, bone, and other organs, and is often
resistant to available treatments. Insulin-like growth factors
(IGFs) stimulate neuroblastoma growth, survival, and motility, and
are expressed by neuroblastoma cells and the tissues they invade.
Administration of formulations of the invention disrupt the effects
of IGFs on neuroblastoma tumorigenesis and thereby slow disease
progression. Nordihydroguaiaretic acid (NDGA), a phenolic compound
isolated from the creosote bush (Larrea divaricata), has anti-tumor
properties against a number of malignancies. NDGA inhibits the
phosphorylation and activation of the Her2/neu and IGF-I receptors
(IGF-IR). The present invention shows that NDGA inhibits
IGF-I-mediated activation of the IGF-IR in human neuroblastoma cell
lines. NDGA inhibits neuroblastoma growth and disrupts activation
of ERK and Akt signaling pathways induced by IGF-I. NDGA induces
apoptosis at higher doses, causing IGF-I-resistant activation of
caspase-3 and a large increase in the fraction of sub-G.sub.0
cells. NDGA inhibits the growth of xenografted human neuroblastoma
tumors in nude mice by 50%. The results provided show that small
molecules that prevent activation of the IGF-IR, such as NDGA, are
useful in the treatment of neuroblastoma.
[0081] Neuroblastoma affects an estimated 1 in 7000 children under
age 15 (Carlsen N L. Neuroblastoma: epidemiology and pattern of
regression. Problems in interpreting results of mass screening. Am
J Pediatr Hematol Oncol 1992;14:103-110), making it the second most
common solid tumor in children. Neuroblastoma tumors are believed
to arise from neural crest cells in the adrenal gland and spinal
ganglia. Neuroblastoma often regresses spontaneously in children
under 1 year of age, but neuroblastoma in older children is
difficult to treat with conventional radiation and chemical
therapies (Philip T. Overview of current treatment of
neuroblastoma. Am J Pediatr Hematol Oncol 1992;14:97-102).
Metastasis to bone, meninges, the liver, and other organs
contributes to the difficulty in eliminating the disease.
[0082] The development of effective treatments for neuroblastoma is
hampered by an incomplete understanding of the factors that lead to
neuroblastoma tumorigenesis, although several key abnormalities are
associated with a sinificant subset of aggressive tumors. Although
several chromosomal abnormalities have been described,
amplification of MYCN is still the best understood genetic
abnormality and is associated with advanced disease.
[0083] While a primary defect in growth factor signaling has not
been observed in neuroblastoma, growth factor responsiveness is
believed to support tumor growth, survival, and invasiveness. Thus,
therapeutic approaches that disrupt growth factor signaling may
have an impact on disease progression. Recently,
nordihydroguaiaretic acid (NDGA), a naturally occurring compound
isolated from creosote (Larrea divaricata), was found to inhibit
the activation of partially purified insulin-like growth factor I
(IGF-I) and her2/neu receptor tyrosine kinases.
[0084] NDGA has been extensively studied as an inhibitor of
arachidonic acid metablolism, where it blocks lipoxygenase
activity, but its ability to inhibit receptor tyrosine kinase
activation was previously unappreciated. In breast cancer cells,
NDGA inhibited ligand activation of the IGF-I and her2/neu
receptors and subsequent activation of signaling intermediates
downstream of these receptors. Both the in vitro and in vivo growth
of breast cancer cells is inhibited by NDGA, potentially via its
ability to suppress responsiveness to growth factors.
[0085] IGF-I and II are peptide growth factors that regulate cell
mitogenesis and survival. IGFs bind to the tyrosine kinase IGF-I
receptor (IGF-IR), causing receptor autophosphorylation that
initiates the mitogen activated protein kinase (MAPK) and
phosphatidylinositol 3-kinase (PI-3K) signaling pathways. MAPK
regulates mitogenesis (De Meyts P, Wallach B, Christoffersen C T,
et al. The insulin-like growth factor-I receptor. Structure,
ligand-binding mechanism and signal transduction. Horm Res
1994:42:152-169), while PI-3K activates targets that impact
apoptosis, such as Akt (Fresno Vara J A, Casado E, de Castro J,
Cejas P, Belda-Iniesta and Gonzalez-Baron M. PI3K/Akt signaling
pathway and cancer. Cancer Treat Rev 2004;30:193-204).
[0086] IGFs promote neuroblastoma tumorigencity by stimulating
proliferation, inhibiting apoptosis, and stimulating motility. IGFs
are expressed in all neuroblastoma tumor stages and in
neuroblastoma tumor lines (Martin D M, Yee D, Carlson R O and
Feldman E L. Gene expression of the insulin-like growth factors and
their receptors in human neuroblastoma cell lines. Brain Res Mol
Brain Res 1992;15:241-246), and can act as either autocrine or
paracrine mitogens (Martin D M and Feldman E L. Regulation of
insulin-like growth factor-II expression and its role in autocrine
growth of human neuroblastoma cells. J Cell Physiol
1993;155:290-300). IGF-I and IGF-IR expression prevent
neuroblastoma cells from undergoing apoptosis (Singleton J R, Dixit
V M and Feldman E L. Type I insulin-like growth factor receptor
activation regulates apoptotic proteins. J Biol Chem
1996;271:31791-31794) by regulating the activity of caspases and
Bcl proteins. IGFs also regulate the metastatic capabilities of
neuroblastoma cells by stimulating actin polymerization,
lamellipodium extension, and motility.
[0087] The present invention is based, in part, on an understanding
of the ability of NDGA to inhibit growth and IGF-IR-related
signaling events in breast cancer. The present invention shows the
anti-tumor effects of NDGA in three human neuroblastoma cell lines.
Results provided here quantify IGF-I- and serum-dependent growth of
neuroblastoma cells treated with NDGA, and characterize
IGF-I-dependent phosphorylation of IGF-IR, extracellular regulated
kinases (ERKs), and Akt in the presence of NDGA. Results provided
here show that IGF-IR blockade mediated by NDGA resulted in
decreased proliferation, increased apoptosis, decreased motility,
and decreased tumor growth in xenograft models. Further, the
results provided here show that NDGA is a potent inhibitor of
neuroblastoma growth and survival, and of IGF-I-stimulated
signaling, events associated with tumorigenesis in
neuroblastoma.
EXAMPLES
[0088] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
Breast Cancer
[0089] Materials and Methods
[0090] Materials.
[0091] NDGA and IGF-1 were gifts from Insmed Inc. (Glen Allen,
Va.). IGF-IR kinase domain peptide was obtained from Upstate, USA
(Charlottesville, Va.). Antibodies against the IGF-1R (C-20),
HER2/neu (C-18), and phosphospecific antibodies recognizing
phosphotyrosine (PY20), phosphoBAD (ser136), and pNeu (Tyr1248),
and HRP-conjugated anti-phosphotyrosine antibody (PY20HRP) were
obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).
.alpha.IR3, a monoclonal antibody against the IGF-1R, was obtained
from CalBiochem (San Diego, Calif.), and the phosphospecific
antibody pAkt(ser473) was obtained from Cell Signaling (Beverly,
Mass.). All other reagents were from Sigma (St. Louis, Mo.), except
as indicated below.
IGF-1R Peptide Autophosphorylation.
[0092] 1 .mu.g of IGF-1R kinase domain peptide was incubated +/-
varying concentrations of NDGA in 2% DMSO in 40 mM Tris, pH 7.4, 80
.mu.M EGTA, 0.25% 2-mercaptoethanol, 80 .mu.M Na.sub.3VO.sub.4, 10
mM MgCl.sub.2, and 2 mM MnCl.sub.2 for 20 min. ATP was then added
at a final concentration of 20 .mu.M. Autophosphorylation of
peptide was allowed to occur for 20 min at 22.degree. C. The
reaction was stopped by the addition of SDS-reducing buffer, and
the samples were run on SDS-PAGE. Following transfer to
nitrocellulose membrane, peptide autophosphorylation was determined
by western blotting employing an antibody against phosphotyrosine
diluted to 0.5 .mu.g/ml in phosphate buffered saline (PBS)
containing 3% milk. The membrane was incubated with the antibody
overnight at 4 C. Next, blots were washed 3 times with PBS, then
incubated with HRP conjugated sheep anti-rabbit IgG (Amersham,
Piscataway, N.J.) diluted 1:50,000 in PBS with 3% milk for 90
minutes at room temperature. After washing, blots were incubated
with SuperSignal (Pierce, Rockford, Ill.), and exposed to film.
Values were determined by scanning densitometry.
Preparation of Partially Purified RTKs by Wheat Germ Agglutin
(WGA)-Chromotography.
[0093] IGF-1 and HER2/neu receptors were partially purified by WGA
chromatography of cells overexpressing the protein of interest. To
isolate IGF-1R, CHO cells transfected with and overexpressing the
human IGF-1R (CHOIGF-1R) were plated in T-150 flasks and grown in
DMEM supplemented with 10% fetal calf serum (FCS) until 80%
confluent. Cells were harvested in the basal state and solubilized
in 50 mM HEPES, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 5 .mu.M
EGTA, 0.24 mg/ml aminoethyl-benzensulfonyl fluoride (AEBSF), 10
.mu.g/ml aprotinin, 25 mM benzamidine, 10 .mu.g soybean trypsin
inhibitor, and 5 .mu.g/ml leuptin for one hour at 4.degree. C.
Samples were centrifuged for 60 minutes at 100,000 g and
solubilized extract collected. Rodent HER2/neu receptors were
collected from MCNeuA cells grown under identical conditions and
processed similarly. Lysates from both cell lines were loaded onto
a 1 ml wheat germ agglutin (WGA) column (Pharmacia, Piscataway,
N.J.), washed with WGA buffer (50 mM HEPES pH 7.6, 150 mM NaCl,
0.1% Triton X-100, 1 mg/ml bacitracin, and 1 mM PMSF). Receptors
were then eluted with WGA buffer supplemented with 0.3 N
N-Acetyl-D-glucosamine. WGA fractions containing RTK of interest
were determined by SDS page and a western blot that employed
antibodies against either the IGF-1R or HER2/neu as
appropriate.
Determination of the Effects of NDGA on the Tyrosine Kinase
Activity of Partially Purified IGF-1 R.
[0094] The effects of NDGA on the ability of the IGF-1R to
phosphorylate exogenous substrates were determined by ELISA.
Tyrosine kinase substrate poly Glu4:Tyr1 (PGT) was coated on
Nunc-Immuno 96-well plate at 500 ng/well over night at 4 C. The
plate was washed and blocked with Superblock (Pierce, Rockford,
Ill.) for 30 min. WGA preparations enriched in IGF-1R from CHO-IGFR
cells were then incubated with or without NDGA in the presence of
10 uM ATP plus or minus 10 nM IGF-1 in Kinase Buffer (50 mM, pH
7.4, 150 mM NaCl, 0.1% Triton X-100, 0.1% gelatin, 5 mM MnCl2, 8 mM
MgCl2, and 1 mM PMSF). This reaction mixture was then added to the
substrate-coated wells to interact with PGT for 30 min at RT.
Plates were washed five times, then incubated with HRP-conjugated
anti-phosphotyrosine antibody (0.3 .mu.g/ml), diluted in Solution B
(50 mM HEPES, pH 7.6, 150 mM NaCl, 0.05% Tween-20, 1 mM PMSF, 2 mM
vanadate and 1 mg/ml bacitracin), for two hours at 22.degree. C.
Plates were washed and incubated with p-Tyr HRP conjugated
antibody. After washing, wells were incubated with
3,3',5,5'-tetramethly benzidine (TMB) peroxidase substrate. The
reaction was terminated with 1.0 M H.sub.3PO.sub.4. Values for
receptor autophosphorylation were deter mined by measuring
absorbance at 451 nm.
Determination of the Effects of NDGA on Autophosphorylation of
Isolated HER2/neu.
[0095] Autophosphorylation of HER2/neu in WGA preparations enriched
in this RTK were determined in the absence of ligand. Samples (1-5
.mu.l) were incubated in the presence of varying amounts of NDGA in
kinase buffer for 15 minutes at 4.degree. C. ATP (10 .mu.M final
concentration) was added to the reaction mixture for 45 minutes at
22.degree. C. The autophosphorylation reaction was stopped by the
addition of SDS-reducing buffer, and the samples were run on
SDS-PAGE. Following transfer to nitrocellulose membrane, receptor
autophosphorylation was determined by a western blot that employed
a phosphospecific antibody against the pNeu (Tyr1248) diluted
1:1000 in Superblock. Following an overnight incubation, membranes
were washed with 3 times with tris buffered saline with 0.5% Tween
20 (TBST), then incubated with HRP conjugated sheep anti-rabbit IgG
diluted 1:10,000 in Superblock for 90 minutes at room temperature.
After washing, blots were incubated with SuperSignal, and exposed
to film. Values were determined by scanning densitometry.
Determination of Ligand-Stimulated IGF-IR Signaling in Breast
Cancer Cells.
[0096] Assays were conducted with either MCF-7, MCNeuA, or SK-Br3
breast cancer cells. The MCNeuA cell line is a mammary carcinoma
cell line we have recently established from a spontaneously arising
tumor in a neu transgenic female mouse (Campbell M J, Wollish W S,
Lobo M, Esserman L J: Epithelial and fibroblast cell lines derived
from a spontaneous mammary carcinoma in a MMTV/neu transgenic
mouse. In Vitro Cell Dev Biol Anim 38:326-333, 2002) and is thus
driven by HER2/Neu overexpression. SK-Br3 human breast cancer cells
express both the IGF-1R and the HER2/Neu receptor. For the initial
screening, MCF-7 cells were grown in 96 well plates. For dose
effects of NDGA on cellular IGF-1R signaling, MCF-7, MCNeuA, or
SKBR3 cells were grown in 6-well plates. For all studies, when
cells reached 80% confluence they were serum-starved for 18 hr.
NDGA was dissolved in DMSO and diluted with culture medium before
being added to cells for 1 hour at 37.degree. C. The final
concentration of DMSO during the incubation was 0.3%. Cells were
then stimulated with 3 nM IGF-I for 10 minutes at 37.degree. C.
Reactions were terminated by rapidly aspirating medium and washing
cells three times with ice cold PBS. Cells were harvested and
solubilized in 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM
PMSF, and 2 mM vanadate for 1 hour at 4.degree. C. Protein was
deter mined by BCA assay (Pierce, Rockford, Ill.)
[0097] IGF-1R autophosphorylation was determined by ELISA as
described previously for the IR (Youngren J F, Goldfine I D,
Pratley R E: Decreased muscle insulin receptor kinase correlates
with insulin resistance in normoglycemic Pima Indians. Am J Physiol
273:E276-E283, 1997). Briefly, 20 .mu.g lysate protein was added to
duplicate wells in a 96-well plate coated with monoclonal antibody
to the IGF-IR (.alpha.IR3; 2 .mu.g/ml), and incubated 18 hours at
4.degree. C. ELISA color development was as described for the
substrate phosphorylation assay.
[0098] Inhibition of IGF-1-stimulated activation of serine kinase
Akt was determined in the lysates prepared for the IGF-1R
phosphotyrosine ELISA described above. 12 .mu.g of sample was
subjected to SDS-PAGE, transferred to a nitrocellulose membrane,
and phosphorylated HER2/neu quantified by blotting overnight at 4 C
with a phospho-specific antibody to Akt (ser473) diluted 1:1000 in
Superblock. Next, blots were washed with 3 times with TBST, then
incubated with HRP conjugated sheep anti-rabbit IgG diluted
1:10,000 in Superblock for 90 minutes at room temperature.
Phosphorylation of the apoptotic protein BAD was determined in the
same manner by blotting with a phospho-specific antibody to BAD
(ser136) (1:1,000).
Determination of NDGA Effects on Cellular HER2/neu
Autophosphorylation.
[0099] HER2/neu receptor autophosphorylation was determined in
serum-starved MCNeuA or SKBR3 cells, which were collected in the
basal state, due to the ligand-independent nature of HER2/neu
activation. Following a one-hour incubation in serum-free media,
soluble extracts of cells were prepared as above. HER2/neu
autophosphorylation was determined by a western blot that employed
a phosphospecific antibody, pNeu(Tyr1248) as described above.
Effects of NDGA on Proliferation of Breast Cancer Cells.
[0100] The inhibitory effects of NDGA on breast cancer cell growth
were determined using a CyQUANT cell proliferation assay kit
(Molecular Probes, Eugene, Oreg.). MCF-7 or MCNeuA cells were
plated in 96 well plates (5.times.10.sup.3 cells/well) in DMEM
supplemented with 10% FCS. One plate was prepared for each harvest
day. Cells were allowed to adhere overnight and were then treated
with various concentrations of NDGA or DMSO as a vehicle control.
Microplate cultures were harvested on days 0, 1, 2, and 3 by
inverting the microplate onto paper towels with gentle blotting, to
remove growth medium without disrupting adherent cells. Each plate
was kept at -80.degree. C. until the end of the experiment (day 3)
when all of the plates were thawed and assayed together. After
thawing, 200 .mu.l of CyQUANT GR solution was added to each well
and the plates were incubated in the dark for two to five minutes.
Fluorescence was measured with a SpectraMax Gemini XS fluorescence
microplate reader (Molecular Devices, Sunnyvale, Calif.) with
480-nm excitation and 520-nm emission. Proliferation index was
calculated as the percent of nucleotide content versus control
cells at day 0.
Effects of NDGA on IGF-1 Stimulated Proliferation of Breast Cancer
Cells.
[0101] MCF-7 cells were harvested at an early passage number by
washing three times with PBS and trypsinizing with 1 mL 0.05%
trypsin. Cells were resuspended in 5 mL defined medium (1:1 Ham's
F12: DMEM 4.5 g/L glucose; 1 mg/mL BSA; 10 ug/mL Transferrin; 15 mM
HEPES pH 7.2; 2 mM L-glutamine; 100 units/mL Penicillin G; 100
mcg/mL Streptomycin SO4; 2.5 ug/mL Fungizone) containing 200 ug
soybean trypsin inhibitor. Cells were plated in 96 well collagen
coated plates (Sigma, St. Louis, Mo.) at a density of 5000
cells/well in 100 ul medium. Twenty hours later defined medium with
or without IGF-I at varying concentrations was added. Four hours
later NDGA diluted in defined medium was added. Plates were
harvested on day 3, as described above, for determination of cell
number by CyQUANT assay.
In Vivo Studies
[0102] All animal studies complied with protocols approved by of
the Institutional Animal Care and Use Committee of the University
of California, San Francisco. Our syngeneic model studies utilized
the FVB/N-TgN(MMTVneu)202 mouse strain developed by Muller and
colleagues (Guy C T, Webster M A, Schaller M, Parsons T J, Cardiff
R D, Muller W J: Expression of the neu protooncogene in the mammary
epithelium of transgenic mice induces metastatic disease. Proc Natl
Acad Sci USA 89:10578-10582, 1992). This strain, denoted hereafter
as neuTg, expresses the wild-type rat neu proto-oncogene (a
homologue of human HER2) under the control of the mouse mammary
tumor virus (MMTV) long terminal repeat (LTR) on an FVB mouse
background. The MCNeuA mammary carcinoma cell line employed in
these studies was derived from a spontaneously arising tumor in a
neuTg female mouse (Campbell M J, Wollish W S, Lobo M, Esserman L
J: Epithelial and fibroblast cell lines derived from a spontaneous
mammary carcinoma in a MMTV/neu transgenic mouse. In Vitro Cell Dev
Biol Anim 38:326-333, 2002). These cells are tumorigenic when
transplanted back into neuTg mice. Female mice were injected
subcutaneously with 10.sup.5 MCNeuA tumor cells on day 0. Treatment
with NDGA began on day 9. One group of mice received 37.5 mg/kg
NDGA prepared in 20% warm (37.degree. C.) ethanol, administered
intraperitonealy (i.p.) three times per week. The general
preparation of NDGA into vehicle involved heating 100% ethanol and
deionized distilled water to 37.degree. C. The desired amount of
NDGA was then dissolved in 100% ethanol and diluted with water drip
by drip for a final solution of 20% ethanol/80% water. This
solution was kept warm at 37.degree. C. until given to mice by i.p.
injection. A second group of mice received an oral dose of 100
mg/kg by gavage. NDGA was prepared for oral delivery by dissolving
it in carboxymethylcellulose. Control mice received i.p. injections
of ethanol/water vehicle only. Tumor growth was measured at the
time of drug delivery on alternate treatment days with calipers and
volume calculated using the equation:
(length.times.width.sup.2)*(.pi./6).
RTK Activation in Tumors
[0103] At the end of the study, approximately 16 hrs following a
final i.p. dose of NDGA, tumors were excised from the mice in order
to assess the autophosphorylation state of IGF-1 and HER2/neu
receptors. Tumors were homogenized in 50 mM HEPES, pH 7.6, 150 mM
NaCl, 1 mM PMSF, 2 .mu.M leupeptin, 2 .mu.M Pepstatin A, and 2 mM
Na.sub.3VO.sub.4, and solubilized by the addition of 1% Triton
x-100. Soluble lysates were prepared as described for cell culture
studies. Tyrosine phosphorylation of IGF-1R was determined by ELISA
as described for MCF-7 cells. Phosphorylation of HER2/neu was
determined by western blot employing the phospho-specific antibody
as in the cell culture studies. The degree of phosphorylation for
each receptor was normalized to total receptor protein content in
the tumor samples as determined by western blotting with antibodies
against the human IGF-1R and the rodent HER2/neu.
Statistics
[0104] Statistics were calculated using MedCalc statistical
software (MedCalc Software, Mariakerke, Belgium). Growth of breast
cancer cells in culture and in vivo was analyzed by two-way
analysis of variance for treatment, time, and interaction effects
with post-hoc analysis by Student's t-test. Dose effects of NDGA on
receptor phosphorylation were analyzed by one-way analysis of
variance with post-hoc analysis by Student's t-test. Significance
was set at P<0.05.
Results
Effects of NDGA on Isolated RTKs.
[0105] NDGA (FIG. 1) has previously been shown to directly inhibit
ligand-stimulated PDGFR autophosphorylation (Domin J, Higgins T,
Rozengurt E: Preferential inhibition of platelet-derived growth
factor-stimulated DNA synthesis and protein tyrosine
phosphorylation by nordihydroguaiaretic acid. J Biol Chem
269:8260-8267, 1994) and a homologous compound is an inhibitor of
the IGF-1R (Blum G, Gazit A, Levitzki A: Substrate competitive
inhibitors of IGF-1 receptor kinase. Biochemistry 39:15705-15712,
2000; Blum G, Gazit A, Levitzki A: Development of new insulin-like
growth factor-1 receptor kinase inhibitors using catechol mimics. J
Biol Chem 278:40442-40454, 2003).
[0106] The present invention shows that NDGA has a direct effect on
both IGF-1R and HER2/neu receptors. More specifically, the present
invention shows the ability of NDGA to inhibit autophosphorylation
and/or substrate tyrosine kinase activity of both these receptors
in vitro. The ability of NDGA to directly inhibit the kinase domain
of the receptor was shown using a synthetic peptide consisting of
the 379 terminal amino acids of the IGF-1R beta subunit. This
peptide, which displays intrinsic autophosphorylation and substrate
tyrosine kinase activity, was incubated with increasing
concentrations of NDGA prior to the addition of 20 .mu.M ATP. Under
these conditions, NDGA inhibited activation of the IGF-1R kinase
domain at a concentration at 1 .mu.M and the results are shown in
the Western blot image of FIG. 8.
[0107] The effects of NDGA against the intact IGF-1 and HER2/neu
receptors were shown using wheat germ affinity chromatography to
obtain fractions enriched in either the human IGF-1R or the rodent
HER2/neu receptor extracted from cells overexpressing these
proteins. In IGF-1R preparations, we determined the ability of NDGA
to inhibit IGF-1 stimulated phosphorylation of the synthetic
tyrosine kinase substrate, poly Glu4:Tyr1. Incubation of IGF-1R
preparations with NDGA for 20 minutes prior to the addition of 10
nM IGF-1 produced dramatic reductions in tyrosine phosphorylation
of the substrate (FIG. 3). This effect was observed at
concentrations of NDGA as low as 0.3 .mu.M.
[0108] Because HER2/neu is intrinsically active in cells in the
absence of ligand binding, basal autophosphorylation is observed in
isolated receptor preparations (FIG. 4). Incubation of HER2/neu
preparations with 10 .mu.M ATP produced a further increase in
ligand-independent HER2/neu autophosphorylation. However,
preincubation with NDGA for 20 minutes prior to the addition of ATP
abolished this increase in HER2/neu autophosphorylation (FIG.
4).
Effects of NDGA on IGF-1R and HER2/neu Signaling in Cells.
[0109] Treatment of MCF-7 cells with increasing concentrations of
NDGA for one hour prior to the addition of 3 nM IGF-1 produced a
dose-dependent decrease in IGF-1R autophosphorylation with an
IC.sub.50 of 31.+-.12 .mu.M (FIG. 5A). Similar effects of NDGA on
IGF-1R autophosphorylation were observed in SK-Br3 breast cancer
cells (data not shown). In order to determine the impact of NDGA on
downstream signaling of the IGF-1R, we studied the Akt/BAD pathway
that regulates cellular apoptosis. Incubation of MCF-7 cells with 1
nM IGF-1 dramatically increased phosphorylation of Akt, and, to a
lesser extent, BAD. These effects were reduced in a dose-dependent
manner by concentrations of NDGA similar to those that inhibited
IGF-1R autophosphorylation (FIG. 5B). These data demonstrate that
NDGA treatment results in transition into a pro-apoptotic state for
MCF-7 cells.
[0110] Because MCF-7 cells do not express HER2/neu, we employed
MCNeuA cells, a breast cancer cell line derived from transgenic
mice overexpressing the rodent form of this receptor. Exposure of
these cells to increasing concentrations of NDGA produced a
dose-dependent inhibition of HER2/neu autophosphorylation.
Ligand-independent tyrosine phosphorylation of HER2/neu was
inhibited by NDGA with an IC.sub.50 of 15.+-.4 (FIG. 6). Similar
results were observed in SK-Br3 cells, which express relatively
large amounts of the human HER2/neu receptor (data not shown).
Effects of NDGA on Growth of Breast Cancer Cells in Culture:
[0111] Given the ability of NDGA to inhibit the function of the
IGF-1R and the HER2/neu receptor, we tested its effect on the
proliferation of MCF-7 and MCNeuA breast cancer cells under normal
culture conditions. When MCF-7 cells were grown in 10% fetal calf
serum and then incubated with varying concentrations of NDGA for up
to 3 days, the rate of proliferation was significantly reduced by
NDGA concentrations as low as 15 .mu.M (FIG. 7A). At 60 .mu.M, cell
number was dramatically reduced within 24 hours. As numerous growth
factors are present in serum, we examined the ability of NDGA to
specifically inhibit growth of these cells mediated by IGF-1 alone.
In MCF-7 cells grown in media supplemented only with 10 nM IGF-1,
NDGA inhibited proliferation with an IC.sub.50 of approximately 10
.mu.M (FIG. 7B). In the presence of 10% fetal calf serum, NDGA also
inhibited growth of MCNeuA cells, for which growth is largely due
to the ligand-independent activity of the overexpressed HER2/neu
receptor. and SK-Br3 cells, which express relatively high levels of
both IGF-1 and HER2/neu receptors with potencies similar to that
observed for MCF-7 cells (data not shown).
Effects of NDGA on IGF-1R and HER2/neu Receptor Activation of
Breast Tumors in Mice:
[0112] In order to assess the ability of NDGA to inhibit activation
of the IGF-1R and HER2/neu receptor in vivo, we employed a
syngeneic mouse model of breast cancer featuring a cell line that
expresses both RTKs. In these studies, MCNeuA breast cancer cells
were injected subcutaneously into female neuTg mice, the strain
from which this cell line was originally obtained. NDGA was
administered three times a week either as an i.p. dose of 37.5
mg/kg or as an oral dose of 100 mg/kg, prepared in
carboxymethylcellulose. Administration of NDGA began nine days
after implantation of tumor cells. Twenty nine days
post-implantation, and 16 hours following the final administration
of NDGA tumors were excised from intraperitonealy-treated and
control mice at the end of the study and analyzed. We observed that
autophosphorylation of both receptors was reduced in tumors from
NDGA-treated mice compared to vehicle-treated controls (FIG. 8).
NDGA treatment had no effect on the total cellular content of
either the IGF-1 or HER2/neu receptor.
[0113] FIG. 8 shows that NDGA Directly Inhibits Autophosphorylation
of IGF-1R Kinase Domain. A peptide corresponding to the kinase
domain of IGF-1R was incubated with varying concentrations of NDGA
prior to the addition of ATP. Inhibition of peptide tyrosine
phosphorylation by NDGA was determined by western blots that
employed an anti-phosphotyrosine antibody. A representative blot is
shown.
[0114] Inhibition of signaling by both RTKs through treatment with
NDGA in vivo was associated with a reduced tumor growth rate. There
were no differences in tumor growth rates between animals receiving
oral or i.p. administration of NDGA. Data combined from both
treatment groups demonstrated that NDGA significantly reduced tumor
growth from 21 days post-implantation through the remainder of the
study (FIG. 9).
[0115] FIG. 9 shows that NDGA Directly Inhibits Tyrosine Kinase
Activity of Isolated IGF-1R. IGF-1R were partially purified by
affinity chromatography from cells overexpressing human IGF-1R. The
IGF-1R was incubated with varying concentrations of NDGA prior to
the addition of IGF-1. ATP-induced tyrosine phosphorylation of the
immobilized substrate, poly Glu4:Tyr1 was determined by ELISA
employing an anti-phosphotyrosine antibody to readout. IGF-1
stimulation of substrate phosphorylation was calculated as the O.D.
value in the absence of added IGF-1 subtracted from the O.D. value
in each experimental condition. Values represent mean.+-.SEM of 3
experiments.
[0116] FIG. 10 shows that NDGA Directly Inhibits
Autophosphorylation of Isolated HER2/neu. HER2/neu receptors were
partially purified by affinity chromatography from cells
overexpressing mouse HER2/neu. Incubation of HER2/neu receptors
with NDGA prior to the addition of ATP reduced autophosphorylation
as determined by western blot employing a phosphospecific antibody,
pNeu(Tyr1248). A representative blot of 3 experiments is shown.
[0117] FIG. 11 shows that NDGA Inhibits the IGF-1R Signaling
Pathway in MCF-7 Cells. MCF-7 breast cancer cells were incubated
with varying concentrations of NDGA for 1 hr. Soluble extracts were
then collected in the basal state or following a 10 minute
incubation with 3 nM IGF-1. (A) Tyrosine phosphorylation of the
IGF-1R was then determined by specific ELISA (.box-solid. basal
state; 3 nM IGF-1). Values represent mean.+-.SEM of 3 experiments,
normalized to cells treated with vehicle alone. *Values
significantly reduced vs. vehicle treated controls. (B) Serine
phosphorylation of PKB and BAD determined by western blot.
Representative blots of 3 experiments are shown.
[0118] FIG. 12 shows that NDGA Inhibits HER2/neu
Autophosphorylation in MCNeuA Cells. MCNeuA breast cancer cells
were incubated with varying concentrations of NDGA for 1 hr.
Tyrosine phosphorylation of HER2/neu receptors in soluble extracts
was determined by western blot employing a phosphospecific
antibody. A representative blot of four experiments is shown.
[0119] FIG. 13 shows that NDGA Inhibits Serum and IGF-1 Stimulated
Proliferation of MCF-7 Breast Cancer Cell Lines. (A) MCF-7 cells
were grown in media supplemented with 10% fetal calf serum.
Beginning on day 0, cells were incubated with varying
concentrations of NDGA. Cell number was estimated by determination
of nucleotide content (CyQUANT assay) on days 0, 1, 2, and 3.
Proliferation index was calculated as the percent difference in
cell number vs. day 0. (B) MCF-7 cells were initially plated in
basal media. Growth continued in basal media or media supplemented
with 10 nM IGF-1. 24 hours after plating (day 0), cells were
incubated with varying concentrations of NDGA or DMSO alone. Plates
were harvested on day 3 for CyQuant assay. Values shown are O.D.
values reflecting total nucleic acid content per well. All values
represent mean.+-.SEM of 3 experiments. *Proliferation
significantly reduced vs. vehicle treated controls. *Cell number
significantly reduced from day 0.
[0120] FIG. 14 shows that Chronic NDGA Administration Inhibits RTK
Activation in Tumors In Vivo. Following 21 days of treatment, and
16 hrs after the final intraperitoneal administration of NDGA,
MCNeuA tumors were excised and soluble protein extracts prepared.
Tyrosine phosphorylation of IGF-1R (black bars) was deter mined by
ELISA. Phosphorylation of HER2/neu (gray bars) was determined by
western blot. Tumor content of both RTKs was not different across
treatment groups as determined by western blot. Values represent
mean phosphorylation level normalized to control values.+-.SEM for
4-5 animals per group. *Receptor tyrosine phosphorylation
significantly reduced vs. vehicle treated controls.
[0121] FIG. 15 shows that Growth Inhibition of MCNeuA Cells in vivo
By NDGA Administered Orally and by Intraperitoneal Injection.
MCNeuA cells were injected into NeuTG mice on day 0. Treatment with
NDGA began on Day 9, with NDGA administered 3.times. week, either
orally in carboxymethylcellulose (100 mg/kg) (.tangle-solidup.) or
injected intraperitoneally (37.5 mg/kg) (.box-solid.). Values
represent mean tumor volume.+-.SEM for 4-5 animals per group.
*Tumor volume significantly reduced for combined treatment groups
vs. vehicle treated controls.
Example 2
Neuroblastoma
Materials and Methods
[0122] Cell culture and reagents. Human SH-SY5Y, SHEP, and Kelly
neuroblastoma cells were cultured in Dulbecco's modified Eagle
medium (DMEM) with 10% calf serum and maintained in a humidified
incubator with 10% CO.sub.2 at 37.degree. C. NDGA from Insmed
Corporation (Richmond, Va.) was dissolved immediately before each
experiment in DMSO to make a 1000.times. solution, which was then
added to the cell culture medium. IGF-I was purchased from GroPep
(Adelaide, S A, Australia). Anti-IGF-IR antibody (.alpha.IR-3) was
purchased from Calbiochem (San Diego, Calif.). Anti-phosphotyrosine
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz,
Calif.). Anti-Akt, anti-phospho-Akt, anti-Erk1/2,
anti-phospho-Erk1/2, and anti-cleaved caspase-3 antibodies were
purchased from Cell Signaling Technologies (Beverly, Mass.).
Horseradish peroxidase conjugated goat anti-rabbit IgG was
purchased from Zymed Laboraties (South San Francisco, Calif.).
CyQuant was purchased from Molecular Probes (Eugene, Oreg.).
Propidium iodide was purchased from Sigma (St. Louis, Mo.).
[0123] IGF-IR phosphorylation ELISA. SH-SY5Y and SHEP cells were
grown to 80% confluence in DMEM/10% calf serum, then serum-starved
for 4 h. Cultures were then treated with DMSO or 60 uM NDGA and
incubated for 1 h. Some cultures were then treated with 1 nM IGF-I
for 10 min. The medium was removed, cultures rinsed 3.times. in
cold PBS, and lysis buffer (120 mM HEPES, 300 mM NaCl 2 mM sodium
orthovanadate, and 1 mM phenylmethylsufonylfluoride (PMSF)) was
added. Cultures were rocked in lysis buffer at 4.degree. C. for 1
h. 96-well plates were coated with .alpha.IR-3 antibody in 50 mM
NaHCO.sub.3, pH 9.0, for 2 h at RT. Plates were rinsed 3x in
tris-buffered saline+0.1% Tween (TBST), then blocked with
SuperBlock (Pierce, Rockford, Ill.) for 30 min at RT. Each well of
the ELISA plate received 30 .mu.g of lysate protein from the cell
cultures, followed by 24 h incubation at 4.degree. C. Plates were
rinsed 5.times. with TBST, and HRP-conjugated anti-phosphotyrosine
antibody was added (1:2000, diluted in 120 mM HEPES, 300 mM NaCl, 2
mM sodium orthovanadate, and 1 mM PMSF, 1% bovine serum albumin, 1
mg/ml bacitracin, and 0.5% Tween-20) for 2 h at RT. Plates were
again rinsed 5.times. in TBST, and TMB (Pierce) was added until
blue color was sufficiently developed. Absorbance at 451 nm was
quantified. Each lysate was run in triplicate, and the experiment
was repeated 3 times.
[0124] CyQUANT assay for cell growth. Cells were plated on four
96-well tissue culture plates, in DMEM/10% calf serum at a density
of 8000 cells/well and incubated for 24 h. In one set of
experiments, the medium was switched to serum free DMEM
supplemented with 1% bovine serum albumin (to provide osmotic
support). IGF-I (10 nM) was added to some samples on a daily basis
for up to 3 days. In a second set of experiments, the cells
continued to be cultured in DMEM/10% calf serum for the duration of
the experiment, with no additional IGF-I. For all experiments, DMSO
or different concentrations of NDGA were added to the cultures at 0
h (the day after plating). The media from one plate was immediately
removed, and the plate was frozen at -80.degree. C. This plate
served as the baseline for the experiment. Single plates were
frozen at 24, 48, and 72 h following addition of drugs. DNA content
of each well was quantified by staining with CyQUANT according to
the manufacturer's instructions and measuring CyQUANT absorbance
with a fluorimeter. Each condition was run in triplicate, and the
experiment was repeated three times.
[0125] Detection of phospho-Akt and phospho-Erk. Cells were grown
to 80% confluence, serum starved for 4 h, and treated with DMSO or
different concentrations of NDGA for 1 h. Then, some cultures were
treated with 10 nM IGF-I for 15 min. Cultures were immediately
placed on ice, the medium was removed, and the cells were lysed in
modified RIPA buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 1%
deoxycholate). Fifty .mu.g of protein from each sample were
separated via SDS-PAGE and transferred to nitrocellulose. Phospho-
and total-Akt and -Erk were detected by immunoblotting.
[0126] Propidium iodide staining of apoptotic cells. SH-SY5Y cells
were cultured in DMEM with 10% calf serum. Cultures were treated
with DMSO or NDGA for 24 h. The supernatant was collected to save
detached cells. Attached cells were removed from the plate via
trypsinization, and pelleted by centrifugation in combination with
the cells in the supernatant. The cell pellet was fixed by
drop-wise addition of cold 70% ethanol while vortexing gently, and
stored at 4.degree. C. The pellet was washed twice and resuspended
in PBS and stained with 1 .mu.g/ml propidium iodide. Propidium
iodide fluorescence was measured in 30,000 cells per sample using a
Becton-Dickinson (Franklin Lakes, N.J.) Facscalibur flow cytometer.
The percentage of cells in each stage of the cell cycle, as well as
the percentage of cells that were apoptotic (sub-G0) was determined
by analyzing the data with ModFit software. The experiment was
repeated three times.
[0127] Detection of Caspase-3 cleavage. Neuroblastoma cells were
grown to 80% confluence and treated with DMSO or NDGA for 6 h.
Alternatively, cells were serum starved for 4 h and cultured with
or without 60 .mu.M NDGA and with or without 10 nM IGF-I for 3 h.
Lysates were collected as described above, and the 14/17 kD
cleavage fragments of caspase-3 were detected via SDS-PAGE followed
by immunoblotting with anti-cleaved caspase-3 antibody.
[0128] Measurement of cell motility. Neuroblastoma cells were
plated on gold particle-coated coverslips (prepared as described in
(30)) in serum-free media at a density of 25,000 cells per
coverslip. The cells were incubated for 2 h to allow adhesion to
the coverslip. Then, wells were treated with DMSO or 30 .mu.M NDGA
for 1 h. 1 nM IGF-I was then added to some wells. Incubation
continued for 6 h, followed by fixation with 3.5% glutaraldehyde.
Coverslips were mounted on glass slides, then viewed on a Lietz
Orthoplan inverted microscope attached to a Sony videoprocessor.
Digital images of the tracks etched into the gold by the cells from
3 separately treated coverslips per condition were collected at
200.times. magnification using Adobe Photoshop software. For each
condition, the areas of 120 tracks made by individual cells were
measured with NIH Image 1.61 software.
[0129] Treatment of xenografted nude mice with NDGA. Nude mice
xenografted with human Kelly neuroblastoma cells were treated with
NDGA to determine if NDGA can affect tumor growth in vivo. Briefly,
7.times.10.sup.6 Kelly human neuroblastoma cells were resuspended
in a 1:1 mixture of PBS and Matrigel (BD Clontech Inc.) and 100
.mu.l of the mixture was injected subcutaneously into the flanks of
6-12 week-old BALB/c nude mice. After 1 cm tumors were established
(.about.10-14 days post implantation), animals were injected
subcutaneously with either vehicle (DMSO) or NDGA, (50 mg/kg,
suspended in DMSO) daily for 10 days. Tumors were then harvested,
weighed and measured. The formula width.sup.2.times.length/2 was
used to calculate tumor volumes.
Results
[0130] IGF-I stimulated IGF-IR phosphorylation in neuroblastoma
cells is inhibited by NDGA. NDGA was previously shown to inhibit
the autophosphorylation of the IGF-IR in partially purified
preparations of the receptor, and in breast cancer cell lines. To
test the effects of NDGA on IGF-IR activation in neuroblastoma
cells, serum-starved SHEP and SH-SY5Y neuroblastoma cells were
treated with either DMSO (vehicle control) or 60 M NDGA for 1 h
(FIGS. 16A and 16B). The cultures were then treated for 10 min with
or without 1 nM IGF-I. Protein lysates were collected and the
degree of IGF-IR phosphorylation in the lysate samples was
quantified using ELISA. IGF-I induced an increase in IGF-IR
tyrosine phosphorylation in both cell lines. This IGF-I stimulated
receptor phosphorylation was inhibited by NDGA. SH-SY5Y (A) cells
showed a higher response to IGF-I than SHEP cells (B), consistent
with the increased basal levels of IGF-IR found in SH-SY5Y cells
(Kim B. van Golen C M and Feldman E L. Insulin-like growth factor-I
signaling in human neuroblastoma cells. Oncogene
2004;23:130-141).
[0131] FIGS. 16A and 16B show that NDGA inhibits activation of the
IGF-IR by IGF-I. SH-SY5Y (A.) and SHEP (B.) neuroblastoma cells
were serum starved and treated with DMSO or 60 .mu.M NDGA for 1 h.
Then, 1 nM IGF-I was added to half the cultures for 10 min. IGF-IR
phosphorylation was detected by an ELISA (Materials and Methods).
Results are means +/- SEM for measurements collected in all
experiments expressed as a percentage of IGF-IR phosphorylation in
unstimulated DMSO-treated cells. The experiment was repeated three
times, with each condition run in triplicate within each
experiment. *p<0.01 vs. DMSO+IGF-I.
[0132] NDGA inhibits neuroblastoma proliferation The amount of
cellular DNA present in neuroblastoma cultures was measured to
quantify the cell proliferation or cell death that occurred during
a three day treatment with NDGA. SH-SY5Y and Kelly neuroblastoma
cells were cultured on four 96-well plates in serum-free media
supplemented with 10 nM IGF-I for up to 72 h. At 0 h, the cultures
were treated with 15-120 .mu.M NDGA, or DMSO as control. The cell
content of each plate was measured by staining for total DNA, using
CyQUANT dye. In both SH-SY5Y and Kelly cells, treatment with NDGA
inhibited proliferation, and caused cell death at higher doses
(FIGS. 17A and 17B).
[0133] FIGS. 17A and 17B show that NDGA inhibits neuroblastoma
growth in serum-free medium supplemented with 10 nM IGF-I. SH-SY5Y
(A.) and Kelly (B.) neuroblastoma cells were cultured in serum-free
medium with 10 nM IGF-I and treated with DMSO or NDGA (15-120
.mu.M). DNA content was measured at 0, 24, 48, and 72 h using
CyQUANT staining (Materials and Methods). Means +/- SEM from three
separate experiments are expressed as a percentage of absorbance at
0 h. *p<0.05 vs. DMSO-treated control at the same time
point.
[0134] To determine if NDGA would still have an inhibitory effect
on neuroblastoma growth in serum, where other factors could
contribute to neuroblastoma mitogenesis and survival in addition to
IGFs, the experiment was repeated using SHEP and SH-SY5Y cells
cultured in medium containing 10% serum (FIGS. 18A and 18B). NDGA
inhibited the proliferation of SH-SY5Y and SHEP cells cultured in
serum up to 72 h. Higher doses of NDGA caused cell death, as the
amount of DNA in these cultures is less than the amount at 0 h.
These results demonstrate that NDGA inhibits the growth and
survival of neuroblastoma cells supported by either serum or
IGF-I.
[0135] FIGS. 18A and 18B show that NDGA inhibits neuroblastoma
growth in serum. SH-SY5Y (A.) and SHEP (B.) neuroblastoma cells
were cultured in serum and treated with DMSO or NDGA (30-120
.mu.M). Samples were collected and processed for CyQUANT absorbance
as in FIG. 2. Each bar represents the mean +/- SEM of three
separate experiments, and each condition was run in triplicate.
*p<0.05 vs. DMSO-treated control at the same time point.
[0136] NDGA prevents IGF-I activation of the MAPK pathway.
Neuroblastoma mitogenesis is regulated by IGFs via the activation
of the MAPK signaling pathway, leading to the phosphorylation and
activation of ERK 1 and 2. The effects of NDGA treatment on
IGF-stimulated ERK phosphorylation were investigated in SI-WP and
SH-SY5Y neuroblastoma cells. Serum starved cells were treated for 1
h with DMSO or increasing concentrations of NDGA, and then
stimulated with 10 nM IGF-I for 15 min. Lysates were collected and
proteins separated by SDS-PAGE as described in Materials and
Methods. ERK phosphorylation was assessed by immunoblotting with
anti-phospho ERK1/2 antibody. ERK phosphorylation was increased by
IGF-I in SH-SY5Y cells (FIG. 19A). NDGA causes a dose-dependent
inhibitory effect on IGF-stimulated phosphorylation of ERK. The
total ERK content of each lane is shown for comparison. Similar
results were obtained in SHEP cells.
[0137] Akt phosphorylation is inhibited by NDGA. IGFs also signal
through the PI-3K pathway in neuroblastoma cells, leading to the
activation of Akt. The effect of NDGA on IGF-stimulated Akt
activation was assessed in serum-starved SH-SY5Y and SHEP cells,
via SDS-PAGE and Western immunoblotting. Similar to the effects on
ERK phosphorylation, NDGA caused a dose-dependent inhibition of
IGF-stimulated Akt phosphorylation in SH-SY5Y and SHEP cells (FIG.
19B). Total Akt content of each lane is shown for control.
[0138] FIGS. 19A and 19B show that IGF-I-stimulated ERK and Akt
activation are blocked by NDGA. SH-SY5Y cells were serum starved
and treated with DMSO or 3, 30, or 60 .mu.M NDGA for 1 h, then
given 10 nM IGF-I for 15 min. Lysates were collected and ERK (A.)
or Akt (B.) phosphorylation was detected via Western blot analysis.
A. Upper panel shows ERK phosphorylation. Lower panel is total ERK
to show equal loading of lanes. B. Upper panel shows phospho-Akt,
while lower panel shows total Akt as a loading control.
Representatives of three separate experiments are shown.
[0139] Caspase-3 is activated by NDGA. Akt activation supports
neuroblastoma survival by suppressing apoptosis, in part by
preventing the catalytic activation of caspase-3. Disruption of Akt
signaling increases activation of caspase-3 driving neuroblastoma
cells into apoptosis.
[0140] To determine if NDGA causes caspase-3 activation, SH-SY5Y
neuroblastoma cells were cultured in serum and treated with NDGA
for 6 h. Caspase-3 activation was assessed by SDS-PAGE and
immunoblotting with anti-cleaved caspase-3 antibody, which detects
the small cleavage fragments of caspase-3 that are released upon
its activation. GAPDH expression was detected for loading control.
NDGA causes dose-dependent caspase-3 activation (FIG. 20A). To
determine if exogenous IGF-I was able to prevent this activation,
SH-SY5Y cells were cultured in serum-free media containing 10 nM
IGF-I and simultaneously treated with NDGA or DMSO as a control.
Caspase-3 activation was still detectable when NDGA-treated cells
were given IGF-I (10 nM) (FIG. 20B). Caspase-3 activation was not
detected in serum-starved cells cultured in the absence of IGF-I,
while NDGA treated SH-SY5Y cells cultured in the absence of IGF-I,
which secrete their own IGF-II, showed strong caspase activation.
This shows that NDGA is capable of both pushing the cells into an
apoptotic state and suppressing IGF-mediated rescue.
[0141] NDGA causes neuroblastoma cells to undergo apoptosis.
SH-SY5Y cells cultured in serum were treated with DMSO or NDGA
(30-120 .mu.M). After 24 h, the cells were harvested and subjected
to flow cytometric cell cycle analysis as described in Materials
and Methods. NDGA causes a dramatic, dose-dependent increase in the
percentage of sub-G.sub.0 cells, the fraction of cells undergoing
apoptosis (FIG. 20C).
[0142] FIGS. 20A, 20B and 20C show that NDGA causes caspase
activation and apoptosis in neuroblastoma cells. A. SH-SY5Y cells
grown in serum containing-medium were treated with DMSO or 0.3-60
.mu.M NDGA for 12 h. Activated caspase-3 fragments were detected
using western blot analysis. Upper panel shows the 14/17 kD
cleavage fragments of caspase-3, while the lower panel shows GAPDH
expression as a loading control. A representative of three separate
experiments is shown. B. Serum-starved SH-SY5Y cells were treated
with DMSO or 60 .mu.M NDGA for 12 h. Some cultures included 10 nM
IGF-I for the entire treatment period. Lysates were collected and
caspase-3 cleavage fragments were detected as above. C. SH-SY5Y
cells were treated with DMSO or NDGA (30-120 .mu.M) for 24 h,
fixed, stained with propidium idodide, and subjected to flow
cytometric analysis of cell cycle. Bars represent the mean +/- SEM
percentage of cells in the sub-G.sub.0 apoptotic phase from five
separate experiments. *p<0.05 vs. DMSO.
[0143] IGF-stimulated cell motility is inhibited by NDGA. IGFs
increase the motility of neuroblastoma cells, in part through PI-3K
signaling. The ability of NDGA to impact neuroblastoma motility was
assessed by measuring the motility of serum starved SHEP and
SH-SY5Y cells treated with or without 1 nM IGF-I, over a 6 h
period. Motility was quantified by plating the cells on coverslips
coated with fine particles, and then measuring the areas cleared of
particles by the cells after they moved during the 6 h incubation.
IGF-I increased the motility of SH-SY5Y and SHEP cells, and 30
.mu.M NDGA strongly suppressed this increase in motility (FIG.
21A).
[0144] NDGA inhibits tumor growth in a xenograft model of
neuroblastoma. To determine if the anti-tumorigenic effects of NDGA
would be observed in vivo, nude mice with established Kelly cell
xenografts were treated with NDGA (50 mg/kg i.p. daily) or vehicle
(n=4 per treatment group). After 10 d of treatment, all mice were
sacrificed because the tumors in the vehicle treated mice had grown
so large that our institutional animal care rules required the
animals be sacrificed. NDGA, however, had inhibited tumor growth by
50% (FIG. 21B).
[0145] FIGS. 21A, 21B and 21C show that NDGA inhibits IGF-I
stimulated motility and in vivo neuroblastoma tumor growth. A.
SH-SY5Y and SHEP cells were plated on gold particle-coated
coverslips in serum-free conditions. After adhering, the cells were
treated with DMSO or 30 .mu.M NDGA for 1 h. Half the cultures were
then treated with 1 nM IGF-I, and incubation continued for 6 h. The
track areas of cells that were etched into the gold particle
coating were measured using NIH Image software. Each bar represents
the mean +/- SEM of 120 individual track areas collected from three
separate experiments. *p<0.001 vs. DMSO+1 nM IGF-I. B. Kelly
neuroblastoma cells were implanted subcutaneously in nude mice as
described in Materials and Methods. When palpable tumors formed
(day 12), mice were treated with daily i.p. injections of DMSO
(vehicle, solid line) or 50 mg/kg NDGA (dashed line). On day 22,
the animals were sacrificed and tumor volume was measured with
calipers. N=4 animals in each treatment group.
Discussion
[0146] The IGF signaling system has become a target of increasing
interest in cancer therapy research. A variety of approaches to
disrupting the system have been proposed and investigated,
including use of anti-receptor antibodies, anti-sense nucleotides,
and ligand mimicking compounds (Foulstone E, Prince S, Zaccheo O,
et al. Insulin-like growth factor ligands, receptors, and binding
proteins in cancer. J Pathololgy 2005;205:145-153). Decreasing IGF
availability to tumors is also being considered. A fusion protein
of the IGF binding proteins 3 and 6 is reported to sequester
autocrine IGF-II and decrease rhabdomyosarcoma thymidine uptake
(Dake B L, Boes M, Bach L A and Bar R S. Effect of an insulin-like
growth factor binding protein fusion protein on thymidine
incorporation in neuroblastoma and rhabdomyosarcoma cell lines.
Endocrinology 2004;145:3369-3374). Small molecular inhibitors of
the IGF-I receptor are another approach that is attracting much
attention. A related pair of highly specific and potent inhibitors
of the IGF-IR, NVP-ADW742 and NVP-AEW541, inhibit the growth of a
wide variety of tumors in vitro, as well as fibrosarcoma growth in
vivo (Mitsiades C S, Mitsiades N S, McMullan C J, et al. Inhibition
of the insulin-like growth factor receptor-1 tryosine kinase
activity as a therapeutic strategy for multiple myeloma, other
hematologic malignancies, and solid tumors. Cancer Cell
2004;5:221-230; Garcia Echeverria C, Pearson M A, Marti A, et al.
In vivo antitumor activity of NVP-AEW541-A novel, potent, and
selective inhibitor of the IGF-IR kinase. Cancer Cell
2004;5:231-239). Still, relatively few agents have been identified
that have affects against the IGF-IR. Considering the promising
pre-clinical results of anti-IGF treatment in numerous
malignancies.
[0147] NDGA is a naturally occurring compound that has been
extensively studied for its anti-lipoxygenase activity. It will
inhibit the tyrosine phosphorylation of partially purified IGF-I
and her2/neu receptors, as well as these same receptors
endogenously expressed in breast cancer cells. While it is quite
potent at inhibiting these receptors, NDGA does not show high
selectivity for a single receptor, in contrast to NVP-ADW742 and
NVP-AEW541. NDGA inhibits the activation of the PDGF receptor and
PDGF-stimulated DNA synthesis (Domin J, Higgins T, and Rozengurt E.
Preferential inhibition of platelet-derived growth
factor-stimulated DNA synthesis and protein tyrosine
phosphorylation by nordihydroguaiaretic acid. J Biol Chem
1994:269:8260-8267). Seufferlein, et al., found no affect of NDGA
on EGF receptor phosphorylation. suggesting it has some selectivity
(Seufferlein T, Seckl M J, Schwarz E, et al. Mechanisms of
nordihydroguaiaretic acid-induced growth inhibition and apoptosis
in human cancer cells. Brit J Cancer 2002:86:1188-1196).
[0148] Due to NDGA's effects on the structurally similar insulin
receptor, a diabetic phenotype is one logical toxicity to expect.
Paradoxically, NDGA has an anti-diabetic effect on rats, decreasing
serum glucose and triglycerides without affecting insulin levels.
NDGA was previously considered for treatment of diabetes because of
its inhibition of prostaglandin synthesis. Thus. NDGA's inhibition
of insulin receptors may not result in a diabetes-like toxicity
because of its concomitant effects on prostaglandin synthesis. NDGA
analogs are being developed in an attempt to achieve better
specificity, and some have been tested for efficacy against lung
cancer (Moody T W, Leyton J, Martinez A, Hong S, Malkinson A and
Mulshine J L. Lipoxygenase inhibitors prevent lung carcinogenesis
and inhibit non-small cell lung cancer growth. Exp Lung Res
1998:24:617-628). Further characterization of these analogs may
lead to the discovery of agents more specific for individual
receptor tyrosine kinases.
[0149] NDGA has already been tested as a potential anti-cancer
agent in several in vitro and in vivo studies, often with an
underlying hypothesis that suppressing prostaglandin synthesis will
suppress tumor growth without directly evaluating this mechanism of
action. NDGA is effective in vitro against numerous tumor cell
types, where it induces apoptosis and suppresses mitogenesis
(Seufferlein T, Seckl M J, Schwarz E, et al. Mechanisms of
nordihydroguaiaretic acid-induced growth inhibition and apoptosis
in human cancer cells. Brit J Cancer 2002;86:1188-1196; Tong W -G,
Ding X -Z, Witt R C and Adrian T E. Lipoxygenase inhibitors
attenuate growth of human pancreatic cancer xenografts and induce
apoptosis through the mitochondrial pathway. Mol Cancer Therap
2002;1:929-935; Hoferova Z, Fedorocko P, Hofer M, Hofmanova J,
Kozubik A and Eliasova V. Lipoxygenase inhibitors suppress
proliferation of G5:113 fibrosarcoma cells in vitro but they have
no anticancer activity in vivo. Neoplasm 2003;50:102-109; Vondracek
J, Stika J V, Soucek K, et al. Inhibitors of arachidonic acid
metabolism potentiate tumor necrosis factor-alpha-induced apoptosis
in HL-60 cells. Eur J Pharmacol 2001;424:1-11). Cancers that are
highly responsive to IGF, including lung (Moody T W, Leyton J,
Martinez A, Hong S, Malkinson A and Mulshine J L. Lipoxygenase
inhibitors prevent lung carcinogenesis and inhibit non-small cell
lung, cancer growth. Exp Lung Res 1998;24:617-628) and breast,
respond to NDGA treatment in vivo.
[0150] Considering its potency in inhibiting IGF-IR
phosphorylation, NDGA could be effective in suppressing the growth
of neuroblastoma tumors, which are highly responsive to IGFs. Both
paracrine and autocrine IGFs stimulate neuroblastoma mitogenesis.
Neuroblastoma cell lines that secrete IGF-II are capable of
serum-independent growth. Additionally, cell lines that express
high levels of the IGF-IR are more aggressively tumorigenic. IGFs
strongly activate the MAPK signaling pathway, which culminates in
phosphorylation of ERK 1 and 2. Thus, interrupting IGF signaling at
the level of the receptor can be used to prevent the growth of
neuroblastoma tumors.
[0151] The present invention shows that NDGA at low doses (15-30
.mu.M) completely blocks neuroblastoma growth over a period of
several days in vitro, both in serum and serum-free conditions
where added and autocrine IGFs support neuroblastoma growth. The
growth of Kelly neuroblastoma tumor xenografts in nude mice is also
suppressed by NDGA. A single daily dose of an NDGA formulation of
the invention, was very well tolerated by the mice, and inhibited
tumor growth by 50%. NDGA prevents IGF-I-mediated activation of
both the IGF-IR and ERK 1 and 2 in neuroblastoma cells at the same
doses that inhibit growth in vitro. NDGA was similarly effective at
inhibiting IGF-IR signaling and the growth of breast cancer cells
in vitro and in xenografts. The effects of NDGA on other molecular
targets notwithstanding, the ability of NDGA to inhibit mitogenesis
in these experiments is likely attributable at least in part to
blocking IGF-IR activation.
[0152] IGFs are also potent stimulators of neuroblastoma survival,
causing strong activation of Akt and Bcl-2 while suppressing
caspase-3 activation. Results provided here show that NDGA causes
neuroblastoma death as the dose is increased and is strongly
apoptotic, causing caspase-3 activation and a large increase in
sub-G.sub.0 cells. IGF-I normally can prevent caspase activation in
neuroblastoma cells, but IGF-I activation of Akt was inhibited in
cells treated with NDGA. Additionally, IGF-I was only partially
able to mitigate NDGA-induced caspase-3 activation. NDGA appears to
prevent the survival-promoting effects of IGFs by interrupting
signaling pathways. Similar results are seen in breast cancer cell
lines treated with NDGA, where Akt activation is suppressed and BAD
activation is increased. NDGA and other lipoxygenase inhibitors
cause caspase-3 activation in pancreatic cancer cells; thus, a
lipoxygenase-related mechanism is also possible in
neuroblastoma.
[0153] IGF-I stimulates neuroblastoma cells to undergo organized
actin polymerization and lamellipodium extension, resulting in
increased cell motility. Increased cell motility, along with the
ability to digest extracellular matrix, affords cancer cells
greater ability to invade tissues and blood vessels, leading to
metastasis and diffuse tissue dissemination. This is of particular
concern with neuroblastoma, where tumor invasion of bone, a site of
high IGF production, is associated with poor response to therapy.
NDGA effectively inhibits IGF-I stimulated motility of
neuroblastoma cells at a low dose.
[0154] The present invention shows that NDGA effectively suppresses
neuroblastoma growth in vitro and in vivo, and inhibits the
motility and promotes the apoptosis of neuroblastoma cells in
culture. These effects are attributable, at least in part, to the
prevention of IGF-IR activation by IGFs, an important event in the
regulation of neuroblastoma growth, survival, and motility. The
present invention also shows that NDGA administration to animals is
well-tolerated and effective. The NDGA formulation of the invention
can be co-administered with anti-myc agents and/or radiation, so as
to be even more effective in treatments that affect other aspects
of neuroblastoma tumorigenesis. NDGA, in combination with other
agents that affect IGF action, such as IGF binding proteins or
anti-PI-3K agents, can provide increased tumor kill with decreased
general toxicities.
Example 3
[0155] Materials and methods
Materials
[0156] NDGA and IGF-1 were a gift from Insmed Inc. (Glen Allen,
Va.). Antibodies against the IGF-1R (C-20), HER2 (C-18), and
phosphospecific antibodies recognizing phosphotyrosine (PY20), and
pNeu (Tyr1248), and HRP-conjugated anti-phosphotyrosine antibody
(PY20HRP) were all obtained from Santa Cruz Biotechnology (Santa
Cruz, Calif.). Alpha IR3, a monoclonal antibody against the IGF-1R,
was obtained from CalBiochem (San Diego, Calif.). Phosphospecific
antibodies pIGF-IR (Y1131) and pAkt(ser473) were obtained from Cell
Signaling (Beverly, Mass.). All other reagents were from Sigma (St.
Louis, Mo.), except as indicated below. Gefitinib (Iressa) was a
gift of Mark Moasser, University of California San Francisco.
Herceptin was purchased from a commercial pharmacy.
Cell Culture
[0157] MCF-7 cells stably transfected with the full length HER2
cDNA (MCF-7/HER2-18) or control vector (MCF-7/neo) were generously
provided by Dr. Christopher Benz (Buck Institute for Age Research,
Novato, Calif.) and were maintained at 37 oC, 5% CO2 in DMEM+10%
FCS (DMEM-10) supplemented with 200 .mu.g/ml Geneticine.
Preparation of Cell Lysates
[0158] For dose effects of RTK inhibitors (NDGA or gefitinib) on
cellular IGF-1R and HER2 signaling, cells were grown in 6-well
plates to .about.80% confluency, then serum-starved for 18 hr. RTK
inhibitors were dissolved in DMSO and diluted with culture medium
before being added to cells for 1.5 hour at 37.degree. C. The final
concentration of DMSO during the incubation was 0.3%. For some
studies, cells were also stimulated with 3 nM IGF-I for 10 minutes
at 37.degree. C. Reactions were terminated by rapidly aspirating
medium and washing cells three times with ice cold PBS. Cells were
harvested and solubilized in 50 mM HEPES, 150 mM NaCl, 1% Triton
X-100, 1 mM PMSF, and 2 mM vanadate for 1 hour at 4.degree. C.
Protein concentrations were determined by BCA assay (Pierce,
Rockford, Ill.). Enzyme linked immunosorbent assays (ELISA) for
phosphorylated IGF-1R and HER2 IGF-1R phosphorylation was
determined by ELISA as described previously for the insulin
receptor [Youngren J F, Goldfine I D and Pratley R E: Decreased
muscle insulin receptor kinase correlates with insulin resistance
in normoglycemic Pima Indians. Am J Physiol 273: E276-83, 1997].
Briefly, 10 .mu.g lysate protein were added to triplicate wells in
a 96-well plate coated with monoclonal antibody to the IGF-IR (IR3;
2 .mu.g/ml), and incubated for 18 hours at 4.degree. C. Plates were
washed five times and then HRP-conjugated anti-phosphotyrosine
antibody (0.3 .mu.g/ml), diluted in Solution B (50 mM HEPES, pH
7.6, 150 mM NaCl, 0.05% Tween-20, 1 mM PMSF, 2 mM vanadate and 1
mg/ml bacitracin), was added for two hours at 22.degree. C. Plates
were washed five times prior to color development with TMB
substrate, which was terminated with 1.0 M H3PO4. Values for
receptor phosphorylation were determined by measuring absorbance at
450 nm.
[0159] HER2 phosphorylation was also determined by ELISA as above,
using 2 .mu.g of lysate protein per well and 2 .mu.g/ml Herceptin
as the capturing antibody.
Western Blot Analysis
[0160] Total protein extracts (10 .mu.g), prepared as described
above from cells cultured in the presence or absence of IGF-1
and/or RTK inhibitors, were subjected to SDS-PAGE and subsequently
transferred to nitrocellulose membranes. Membranes were incubated
overnight at 4 oC with primary antibody diluted in Superblock
(Pierce) containing 0.1% Tween 20 (Bio-Rad). The membranes were
washed 3 times with TBS-T, then incubated with HRP-conjugated
secondary antibody diluted in Superblock/Tween 20 for 90 min at
room temperature. Membranes were washed again and bound antibodies
detected by enhanced chemiluminescence (Pierce). Primary antibodies
against the following proteins were used at the indicated
dilutions: IGF-1R used at 0.2 .mu.g/ml, HER2 used at 0.2 .mu.g/ml,
p-IGF-1R (Y1131) used at 1:1000, pNeu (Tyr1248) used at 0.2
.mu.g/ml, and pAkt(ser473) used at 1:1000. Secondary HRP-conjugated
antibodies were directed against the appropriate species of origin
of the primary antibody.
Cell Growth Assays
[0161] The inhibitory effects of RTK inhibitors (NDGA and
gefitinib) on breast cancer cell growth were determined using a
CyQuant cell proliferation assay kit (Molecular Probes, Eugene,
Oreg.). MCF-7/neo or MCF-7/HER2-18 cells were plated in 96 well
plates (4.times.103 cells/well) in 100 .mu.l/well of DMEM-10
medium. Cells were allowed to adhere overnight and were then
treated with various concentrations of NDGA, gefitinib, or DMSO as
a vehicle control in 100 .mu.l/well of serum free DMEM (SF-DMEM),
making the final serum concentration 5%. Media with inhibitors was
refreshed on day 3 and the cultures were harvested on day 6. The
plates were inverted onto paper towels with gentle blotting to
remove growth medium without disrupting adherent cells. Each plate
was kept at -80.degree. C. until assayed for cell growth. After
thawing the plate at room temperature, 200 .mu.l of CyQuant GR
solution was added to each well and the plates were incubated in
the dark for five minutes. Fluorescence was measured with a
SpectraMax Gemini XS fluorescence microplate reader (Molecular
Devices) with 480-nm excitation and 520-nm emission.
[0162] The growth inhibitory effects of tamoxifen, in the presence
or absence of NDGA, were assessed in MCF-7/HER2-18 cells using a
CyQuant assay as described above with a few modifications to the
protocol. Cells were estrogen-starved for three days in DMEM
containing 10% charcoal-dextrin-stripped FCS (CDSS) prior to their
plating in 96 well plates. Cells were plated in 100 .mu.l of the
same media, allowed to adhere overnight, and then were switched to
DMEM+10% CDSS supplemented with 100 pM estrogen. Tamoxifen (100 nM
final concentration) and/or NDGA (10-20 .mu.M final concentration)
was added in 100 .mu.l of SF-DMEM to yield a final concentration of
5% CDSS in DMEM. The media and inhibitors were refreshed on day 3,
the cultures were harvested on day 6, and a CyQuant assay was
performed as described above.
Results
HER2 Receptor, But Not the IGF-1R, is Overexpressed in Tamoxifen
Resistant MCF-7/HER2-18 Cells.
[0163] Various studies have demonstrated reduced IGF-1R expression
in antiestrogen-resistant cell lines [Brockdorff B L, Heiberg I and
Lykkesfeldt A E: Resistance to different antiestrogens is caused by
different multi-factorial changes and is associated with reduced
expression of IGF receptor Ialpha. Endocr Relat Cancer 10: 579-90,
2003; Frogne T, Jepsen J S, Larsen S S, Fog C K, Brockdorff B L and
Lykkesfeldt A E: Antiestrogen-resistant human breast cancer cells
require activated protein kinase B/Akt for growth. Endocr Relat
Cancer 12: 599-614, 2005; McCotter D, van den Berg H W, Boylan M
and McKibben B: Changes in insulin-like growth factor-I receptor
expression and binding protein secretion associated with tamoxifen
resistance and estrogen independence in human breast cancer cells
in vitro. Cancer Lett 99: 239-45, 1996; van den Berg H W, Claffie
D, Boylan M, McKillen J, Lynch M and McKibben B: Expression of
receptors for epidermal growth factor and insulin-like growth
factor I by ZR-75-1 human breast cancer cell variants is inversely
related: the effect of steroid hormones on insulin like growth
factor I receptor expression. Br J Cancer 73: 477-81, 1996]. We
examined the levels of the IGF-1R and HER2 proteins in parental
MCF-7/neo and MCF-7/HER2-18 cells using Western blot analyses.
Compared to the MCF-7/neo cells, the level of IGF-1R was decreased
in MCF-7/HER2-18 cells (FIG. 22), consistent with the reports
described above.
[0164] In contrast, the HER2 protein was abundant in the
MCF-7/HER2-18 cells but undetectable in MCF-7/neo cells (FIG. 22).
This observation is in agreement with the original report that
MCF-7/HER2-18 cells overexpress the HER2 protein compared to
parental MCF-7 cells [Benz C C, Scott G K, Sarup J C, Johnson R M,
Tripathy D, Coronado E, Shepard H M and Osborne C K:
Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7
cells transfected with HER2/neu. Breast Cancer Res Treat 24: 85-95,
1993].
NDGA, But Not Gefitinib, Equally Inhibits the Growth of Both
Parental and Tamoxifen Resistant MCF-7/HER2-18 Cells
[0165] Gefitinib, an EGFR inhibitor, has been shown to inhibit the
growth of HER2-overexpressin breast cancer cell lines, including
MCF-7/HER2-18 [Anderson N G, Ahmad T, Chan K, Dobson R and Bundred
N J: ZD1839 (Iressa), a novel epidermal growth factor receptor
(EGFR) tyrosine kinase inhibitor, potently inhibits the growth of
EGFR-positive cancer cell lines with or without erbB2
overexpression. Int J Cancer 94: 774-82, 2001; Moasser M M, Basso
A, Averbuch S D and Rosen N: The tyrosine kinase inhibitor ZD1839
("Iressa") inhibits HER2-driven signaling and suppresses the growth
of HER2-overexpressina tumor cells. Cancer Res 61: 7184-8, 2001;
Moulder S L, Yakes F M, Muthuswamy S K, Bianco R, Simpson J F and
Arteaga C L: Epidermal growth factor receptor (HER1) tyrosine
kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu
(erbB2)-overexpressing breast cancer cells in vitro and in vivo.
Cancer Res 61: 8887-95, 2001]. NDGA inhibits both HER2 and IGF-1R
activities and has been shown to inhibit the growth of
HER2-negative (MCF-7) as well as HER2-positive (SKBr3) breast
cancer cells [Youngren J F, Gable K, Penaranda C, Maddux B A,
Zavodovskaya M, Lobo M, Campbell M, Kerner J and Goldfine I D:
Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and
cerbB2/HER2/neu receptors and suppresses growth in breast cancer
cells. Breast Cancer Res Treat 94: 37-46, 2005]. We compared the
effects of both tyrosine kinase inhibitors on the growth of
parental MCF-7/neo and MCF-7/HER2-18 cells. Gefitinib (FIG. 23A)
was more effective at inhibiting the growth of HER2-overexpressing
MCF-7/HER2-18 cells when compared to MCF-7/neo cells. In contrast,
NDGA was equally effective in both cells lines, with a one half
maximal effect occurring at 12.5 .mu.M (FIG. 23B).
NDGA Inhibits Both the IGF-1R and HER2 Receptor and Downstream
Signaling in Tamoxifen Resistant MCF-7/HER2-18 Cells
[0166] The tamoxifen resistant MCF-7/HER2-18 cell line expresses
both IGF-1R and HER2, two receptor tyrosine kinases (RTK) that play
a role in breast cancer. We have previously shown that NDGA
inhibits the kinase activities of IGF-1R in MCF-7 cells and HER2 in
SKBR-3 cells [Youngren J F, Gable K, Penaranda C, Maddux B A,
Zavodovskaya M, Lobo M, Campbell M, Kerner J and Goldfine I D:
Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and
cerbB2/HER2/neu receptors and suppresses growth in breast cancer
cells. Breast Cancer Res Treat 94: 37-46, 2005]. We next compared
the effects of NDGA with gefitinib on the activities of these
receptor kinases in MCF-7/HER2-18 cells.
[0167] Employing a sensitive ELISA [Youngren J F, Goldfine I D and
Pratley R E: Decreased muscle insulin receptor kinase correlates
with insulin resistance in normoglycemic Pima Indians. Am J Physiol
273: E276-83, 1997] we observed that gefitinib had only a weak
inhibitory effect on IGF-1 stimulated phosphorylation of the IGF-1R
(FIG. 24A). In contrast, gefitinib significantly inhibited HER2
phosphorylation with a half maximal effect occurring at less than 5
.mu.M (FIG. 24A). Whereas gefitinib suppressed HER2 but not IGF-1R
activity, NDGA strongly inhibited both kinases as measured by
phosphotyrosine specific ELISAs (FIG. 24B) and by Western blot
(FIG. 24B, inset).
[0168] The serine kinase AKT/PKB is activated by receptor tyrosine
kinases, including IGF-1R and HER2, and mediates cell growth [Ahmad
S, Singh N and Glazer R I: Role of AKT1 in 17 beta-estradiol- and
insulin-Eke growth factor I (IGF-I)-dependent proliferation and
prevention of apoptosis in MCF-7 breast carcinoma cells. Biochem
Pharmacol 58: 425-30, 1999; Martin M B, Franke T F, Stoica G E,
Chambon P, Katzenellenbogen B S, Stoica B A, McLemore M S, Olivo S
E and Stoica A: A role for Akt in mediating the estrogenic
functions of epidermal growth factor and insulin-like growth factor
I. Endocrinology 141: 4503-11, 2000; Mitsiades C S, Mitsiades N and
Koutsilieris M: The Akt pathway: molecular targets for anticancer
drug development. Curr Cancer Drug Targets 4: 235-56, 2004: Stoica
G E, Franke T F, Wellstein A, Czubayko F, List H J, Reiter R,
Morgan E, Martin M B and Stoica A: Estradiol rapidly activates Akt
via the ErbB2 signaling pathway. Mol Endocrinol 17: 818-30, 2003].
We measured the effects of NDGA on the phosphorylated state of this
protein in MCF-7/HER2-18 cells. In the absence of IGF-1, AKT/PKB
was phosphorylated and the level of phospho-AKT/PKB was decreased
by treatment with NDGA (FIG. 25). Addition of 3 nM IGF-1 increased
the amount of phospho-AKT/PKB and NDGA also inhibited this IGF-1
stimulated phosphorylation of AKT/PKB. NDGA did not alter the
content of the AKT/PKB protein in MCF-7/HER2-18 cells (data not
shown).
NDGA Attenuates Tamoxifen Resistance in HER2 Overexpressing MCF-7
Cells
[0169] Several reports have demonstrated cross-talk between IGF-1R
and HER2 signaling pathways [Gee J M, Robertson J F, Gutteridge E,
Ellis I O, Pinder S E, Rubini M and Nicholson R I: Epidermal growth
factor receptor/HER2/insulin-like growth factor receptor signalling
and oestrogen receptor activity in clinical breast cancer. Endocr
Relat Cancer 12 Suppl 1: S99-S111, 2005; Lu Y, Zi X, Zhao Y and
Pollak M: Overexpression of ErbB2 receptor inhibits IGF-I induced
Shc-MAPK signaling pathway in breast cancer cells. Biochem Biophys
Res Commun 313: 709-15, 2004; Nahta R, Yuan L X, Zhang B, Kobayashi
R and Esteva F J: Insulin-like growth factor-I receptor/human
epidermal growth factor receptor 2 heterodimerization contributes
to trastuzumab resistance of breast cancer cells. Cancer Res 65:
11118-28, 2005] as well as between ER signaling and these RTKs [Lee
A V, Weng C N, Jackson J G and Yee D: Activation of estrogen
receptor-mediated gene transcription by IGF-I in human breast
cancer cells. J Endocrinol 152: 39-47, 1997: Martin M B and Stoica
A: Insulin-like growth factor-I and estrogen interactions in breast
cancer. J Nutr 132: 3799S-3801S, 2002; Yee D and Lee A V: Crosstalk
between the insulin-like growth factors and estrogens in breast
cancer. J Mammary Gland Biol Neoplasia 5: 107-15, 2000]. Given this
cross-talk, we next examined whether NDGA (which inhibits both HER2
and IGF-1R activities) could overcome tamoxifen resistance in
MCF-7/HER2-18 cells.
[0170] Tamoxifen at 100 nM inhibited the growth of MCF-7/neo cells
by over 50% (FIG. 26A). In contrast, tamoxifen at 100 nM had less
of an effect on MCF-7/HER2-18 cells (24% growth inhibition) (FIG.
26B), consistent with other reports demonstrating tamoxifen
resistance of MCF-7/HER2-18 cells [Benz C C, Scott G K, Sarup J C,
Johnson R M, Tripathy D, Coronado E, Shepard H M and Osborne C K:
Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7
cells transfected with HER2/neu. Breast Cancer Res Treat 24: 85-95,
1993].
[0171] Both NDGA and tamoxifen had antiproliferative effects on the
tamoxifen sensitive MCF-7/neo cells (FIG. 26A). NDGA at 10 and 15
.mu.M inhibited growth by 23% and 55%, respectively. However, when
combined, NDGA treatment did not enhance the growth inhibitory
effects of tamoxifen in these cells (FIG. 26C). In contrast to
MCF-7/neo cells, NDGA treatment significantly enhanced the
antiproliferative effects of tamoxifen in these anti-estrogen
resistant cells (FIG. 26D). NDGA alone, at 10 and 15 .mu.M,
inhibited the growth of MCF-7/HER2-18 cells by 9 and 38%,
respectively. While tamoxifen alone induced a 24% reduction in
growth, the combination of tamoxifen with 10 or 15 .mu.M NDGA
resulted in 40 and 60% growth inhibition, respectively, indicating
additive effects of NDGA and tamoxifen.
Discussion
[0172] Interference with growth factor signals that drive cell
proliferation and survival is an attractive strategy for cancer
treatment. Initial explorations have concentrated on types of
cancers in which growth factor signaling is elevated and plays a
dominant role in driving cell proliferation. A well known example
is breast cancer with overexpression of the HER2 receptor which
responds to interventions that block HER2 action, such as gefitinib
[Benz C C, Scott G K, Sarup J C, Johnson R M, Tripathy D, Coronado
E, Shepard H M and Osborne C K: Estrogen-dependent,
tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected
with HER2/neu. Breast Cancer Res Treat 24: 85-95, 1993; Agrawal A,
Gutteridge E, Gee J M, Nicholson R I and Robertson J F: Overview of
tyrosine kinase inhibitors in clinical breast cancer. Endocr Relat
Cancer 12 Suppl 1: S135-44, 2005; Arteaga C L, Moulder S L and
Yakes F M: HER (erbB) tyrosine kinase inhibitors in the treatment
of breast cancer. Semin Oncol 29: 4-10, 2002; Arteaga C L and
Truica C I: Challenges in the development of anti-epidermal growth
factor receptor therapies in breast cancer. Semin Oncol 31: 3-8,
2004; Herbst R S and Kies M S: Gefitinib: current and future status
in cancer therapy. Clin Adv Hematol Oncol 1: 466-72, 2003; Johnston
S R: Clinical trials of intracellular signal transductions
inhibitors for breast cancer--a strategy to overcome endocrine
resistance. Endocr Relat Cancer 12 Suppl 1: S145-57, 2005:
Kaklamani V and O'Regan R M: New targeted therapies in breast
cancer. Semin Oncol 31: 20-5, 2004; Konecny G E, Wilson C A and
Slamon D J: Is there a role for epidermal growth factor receptor
inhibitors in breast cancer prevention? J Natl Cancer Inst 95:
1813-5, 2003; Penne K, Bohlin C, Schneider S and Allen D: Gefitinib
(Iressa, ZD1839) and tyrosine kinase inhibitors: the wave of the
future in cancer therapy. Cancer Nurs 28: 481-6, 2005; Von Pawel J:
Gefitinib (Iressa, ZD1839): a novel targeted approach for the
treatment of solid tumors. Bull Cancer 91: E70-6, 2004; Wakeling A
E: Inhibitors of growth factor signalling. Endocr Relat Cancer 12
Suppl 1: S183-7, 2005] or Herceptin [Baselga J, Carbonell X,
Castaneda-Soto N J, Clemens M, Green M, Harvey V, Morales S, Barton
C and Ghahramani P: Phase II study of efficacy, safety, and
pharmacokinetics of trastuzumab monotherapy administered on a
3-weekly schedule. J Clin Oncol 23: 2162-71, 2005; Marty M,
Cognetti F, Maraninchi D, Snyder R, Mauriac L, Tubiana-Hulin M,
Chan S, Grimes D, Anton A, Lluch A, Kennedy J, O'Byrne K, Conte P,
Green M, Ward C, Mayne K and Extra J M: Randomized phase II trial
of the efficacy and safety of trastuzumab combined with docetaxel
in patients with human epidermal growth factor receptor 2-positive
metastatic breast cancer administered as first-line treatment: the
M77001 study group. J Clin Oncol 23: 4265-74, 2005; Slamon D J,
Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T,
Eiermann W, Wolter J, Pegram M, Baselga J and Norton L: Use of
chemotherapy plus a monoclonal antibody against HER2 for metastatic
breast cancer that overexpresses HER2. N Engl J Med 344: 783-92,
2001; Vogel C L, Cobleigh M A, Tripathy D, Gutheil J C, Harris L N,
Fehrenbacher L, Slamon D J, Murphy M, Novotny W F, Burchmore M,
Shak S, Stewart S J and Press M: Efficacy and safety of trastuzumab
as a single agent in first-line treatment of HER2-overexpressing
metastatic breast cancer. J Clin Oncol 20: 719-26, 2002]. Herein we
have examined the action of NDGA, an agent that blocks signaling
through HER2 receptors, as well as IGF-1 receptors [Youngren J F,
Gable K, Penaranda C, Maddux B A, Zavodovskaya M, Lobo M, Campbell
M, Kerner J and Goldfine I D: Nordihydroguaiaretic acid (NDGA)
inhibits the IGF-1 and cerbB2/HER2/neu receptors and suppresses
growth in breast cancer cells. Breast Cancer Res Treat 94: 37-46,
2005], and compared it with that of gefitinib which is directed
exclusively at EGFR/HER2 receptors. Both of these drugs work at the
level of preventing receptor auto-phosphorylation, a factor that
was monitored in our studies.
[0173] We studied the actions of these drugs on MCF-7/neo cells, an
estrogen receptor positive human breast cancer cell line that is
sensitive to the antiestrogen tamoxifen, and MCF-7/HER2-18 cells
that overexpress the HER2 receptor and demonstrate a reduced
sensitivity to tamoxifen. MCF-7/neo cells express IGF-1 receptors
but not HER2, whereas the MCF-7/HER2-18 cell line expresses both
IGF-1R and HER2.
[0174] Our studies show that gefitinib, as expected from its mode
of action, inhibits the growth of MCF-7/HER2-18 cells, but has less
potency on MCF-7/neo cells. These observations are consistent with
the notion that inhibition of HER2 receptors is likely to be most
effective on cells whose proliferation is driven by enhanced
activity of this receptor pathway. In contrast to the selective
efficacy of gefitinib, we found that NDGA is equally effective in
inhibiting cell proliferation of both MCF-7/neo cells and
MCF-7/HER2-18 cells. Inhibition of MCF-7/HER2-18 cells by NDGA is
accompanied by an increase in apoptosis (data not shown) as well as
a decrease in downstream signaling (Akt phosphorylation). The
kinetics of inhibition of cell proliferation of MCF-7/HER2-18 cells
match those of NDGA inhibition of IGF-1 receptor phosphorylation
with half-maximal effects in the range of 10-20 .mu.M. Inhibition
of HER2 receptor phosphorylation was also evident, with
half-maximal effects in the range of 30 .mu.M. Breast cancers are
either estrogen-dependent or -independent. A subset of breast
cancers, despite the presence of estrogen receptors, do not respond
to endocrine therapy and it has been reported that HER2 expression
is associated with a reduced response rate to hormone therapy of
metastatic breast cancer [Wright C, Angus B, Nicholson S, Sainsbury
J R, Cairns J, Gullick W J, Kelly P, Harris A L and Home C H:
Expression of c-erbB-2 oncoprotein: a prognostic indicator in human
breast cancer. Cancer Res 49: 2087-90, 1989]. Transfection of
ER-positive cells with a HER2 cDNA, resulting in overexpression of
this RTK, also results in resistance to tamoxifen treatment [Benz C
C, Scott G K, Sarup J C, Johnson R M, Tripathy D, Coronado E,
Shepard H M and Osborne C K: Estrogen-dependent,
tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected
with HER2/neu. Breast Cancer Res Treat 24: 85-95, 1993]. The
mechanism of this resistance includes signaling from the HER2
receptor to ER alpha which results in phosphorylation and enhanced
action of the hormone independent transcriptional activation
function one (AF1) of the receptor. Additionally HER2 signaling
leads to phosphorylation and enhanced action of the major
coactivator for ERalpha driven gene expression, the amplified in
breast cancer 1 (AIB 1) coactivator [Shou J, Massarweh S, Osborne C
K, Wakeling A E, Ali S, Weiss H and Schiff R: Mechanisms of
tamoxifen resistance: increased estrogen receptor-HER2/neu
cross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst
96: 926-35, 2004]. In addition to these effects downstream of HER2,
a feed forward loop in which the liganded ERalpha stimulates the
HER2 pathway is activated. The result is that tamoxifen mimics
estrogen and drives proliferation.
[0175] In view of the disruption of tamoxifen action in
HER2-overexpressing cells, studies have examined the effects of
blocking HER2 signaling in tamoxifen resistant breast cancer cells.
It has been shown that gefitinib can overcome tamoxifen resistance,
or prevent its development, both in vitro and in a mouse xenograft
model [Shou J, Massarweh S, Osborne C K, Wakeling A E, All S, Weiss
H and Schiff R: Mechanisms of tamoxifen resistance: increased
estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast
cancer. J Natl Cancer Inst 96: 926-35, 2004; Gee J M, Harper M E,
Hutcheson I R, Madden T A, Barrow D, Knowlden J M, McClelland R A,
Jordan N, Wakeling A E and Nicholson R I: The antiepidermal growth
factor receptor agent gefitinib (ZD1839/Iressa) improves
antihormone response and prevents development of resistance in
breast cancer in vitro. Endocrinology 144: 5105-17, 2003; Kurokawa
H and Arteaga C L: Inhibition of erbB receptor (HER) tyrosine
kinases as a strategy to abrogate antiestrogen resistance in human
breast cancer. Clin Cancer Res 7: 4436s-4442s; discussion
4411s-4412s, 2001]. However, continuous treatment eventually leads
to acquired resistance to gefitinib which is associated with
increased signaling via the IGF-1R [Jones H E, Goddard L, Gee J M,
Hiscox S, Rubini M, Barrow D, Knowlden J M, Williams S, Wakeling A
E and Nicholson R I: Insulin-like growth factor-I receptor
signalling and acquired resistance to gefitinib (ZD1839; Iressa) in
human breast and prostate cancer cells. Endocr Relat Cancer 11:
793-814, 2004].
[0176] In addition to interactions between ER and HER2 signaling
pathways, cross-talk between IGF-1R and HER2 in breast cancer cells
has been reported [Gee J M, Robertson J F, Gutteridge E, Ellis I O,
Pinder S E, Rubini M and Nicholson R I: Epidermal growth factor
receptor/HER2/insulin-like growth factor receptor signalling and
oestrogen receptor activity in clinical breast cancer. Endocr Relat
Cancer 12 Suppl 1: S99-S111, 2005; Lu Y, Zi X, Zhao Y and Pollak M:
Overexpression of ErbB2 receptor inhibits IGF-I induced Shc-MAPK
signaling pathway in breast cancer cells. Biochem Biophys Res
Commun 313: 709-15, 2004; Nahta R, Yuan L X, Zhang B, Kobayashi R
and Esteva F J: Insulin-like growth factor-I receptor/human
epidermal growth factor receptor 2 heterodimerization contributes
to trastuzumab resistance of breast cancer cells. Cancer Res 65:
11118-28, 2005]. We also have observed of cross-talk between IGF-1R
and HER2 in MCF-7/HER2-18 cells (unpublished data). Given that
IGF-1R signaling plays a role in the development of gefitnib
resistance in tamoxifen resistant cells, and that there is
cross-talk between ER, IGF-1R, and HER2 signaling pathways, we
examined the interaction of NDGA (which inhibits both IGF-1R and
HER2) with tamoxifen on the growth of tamoxifen resistant
MCF-7/HER2-18 cells. NDGA alone inhibited the proliferation of both
tamoxifen sensitive MCF-7/neo and tamoxifen resistant MCF-7/HER2-18
cells. Notably, NDGA combined with tamoxifen demonstrated additive
growth inhibitory effects on MCF-7/HER2-18 cells. The development
of acquired resistance to anti-hormonal therapies such as tamoxifen
is a major therapeutic problem in breast cancer. These results
suggest that NDGA might be clinically useful, in conjunction with
anti-hormonal agents, in the treatment of hormone-resistant breast
cancer, or possibly in preventing the development of acquired
resistance to these agents.
[0177] In summary, we demonstrated that NDGA inhibits the kinase
activities of the IGF-1 receptor and the HER2 receptor and blocks
cellular proliferation of both MCF-7/neo and MCF-7/HER2-18 cells.
NDGA also attenuated tamoxifen resistance in the
HER2-overexpressing MCF-7/HER2-18 cell line. These data raise the
possibility that NDGA, and similar agents that target multiple
growth factor pathways, will have a broad spectrum of action on
breast cancers with a variety of perturbations in their signaling
pathways.
[0178] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *