U.S. patent application number 12/628055 was filed with the patent office on 2010-04-22 for sensitizing cells for apoptosis by selectively blocking cytokines.
Invention is credited to Giorgio Stassi, Matilde Todaro.
Application Number | 20100099742 12/628055 |
Document ID | / |
Family ID | 32605342 |
Filed Date | 2010-04-22 |
United States Patent
Application |
20100099742 |
Kind Code |
A1 |
Stassi; Giorgio ; et
al. |
April 22, 2010 |
Sensitizing Cells for Apoptosis by Selectively Blocking
Cytokines
Abstract
The invention refers to the use of a cytokine antagonist which
modulates the expression and/or the function of a cytokine,
particularly a Th2 helper cell cytokine, in a cell and causes the
down-regulation of anti-apoptotic proteins in said cell through the
cytokine modulation for sensitizing cells for apoptosis. In
particular, the cells that can be treated with the cytokine
antagonists are drug-resistant cancer cells which fail to undergo
apoptosis.
Inventors: |
Stassi; Giorgio; (Palermo,
IT) ; Todaro; Matilde; (Palermo, IT) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
32605342 |
Appl. No.: |
12/628055 |
Filed: |
November 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10544794 |
May 8, 2006 |
7645449 |
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PCT/EP2004/001177 |
Feb 9, 2004 |
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12628055 |
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Current U.S.
Class: |
514/44A ;
424/145.1; 424/649; 424/93.2; 514/1.1; 514/109; 514/171; 514/2.4;
514/251; 514/269; 514/27; 514/283; 514/34; 514/410; 514/449;
514/456; 514/49; 514/575; 514/641; 514/651 |
Current CPC
Class: |
A61K 33/24 20130101;
Y02A 50/30 20180101; A61P 35/02 20180101; A61P 15/00 20180101; Y02A
50/412 20180101; A61P 35/00 20180101; A61P 27/02 20180101; A61P
13/12 20180101; A61K 31/713 20130101; A61P 21/00 20180101; A61K
45/06 20130101; A61P 1/00 20180101; A61K 38/1793 20130101; A61P
7/00 20180101; A61K 31/704 20130101; A61P 1/18 20180101; A61P 37/06
20180101; A61K 31/337 20130101; A61P 19/00 20180101; A61P 25/00
20180101; A61P 13/08 20180101; A61K 31/7105 20130101; A61K 38/191
20130101; A61K 2039/505 20130101; A61P 1/16 20180101; A61K 38/19
20130101; A61P 11/00 20180101; A61P 17/00 20180101; A61K 38/19
20130101; A61K 2300/00 20130101; A61K 38/1793 20130101; A61K
2300/00 20130101; A61K 38/191 20130101; A61K 2300/00 20130101; A61K
31/704 20130101; A61K 2300/00 20130101; A61K 33/24 20130101; A61K
2300/00 20130101; A61K 31/337 20130101; A61K 2300/00 20130101; A61K
31/7105 20130101; A61K 2300/00 20130101; A61K 31/713 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/44.A ;
514/12; 424/145.1; 514/269; 514/251; 514/575; 514/109; 514/8;
514/34; 514/49; 424/649; 514/171; 514/283; 514/456; 514/27;
514/641; 514/651; 514/410; 514/449; 424/93.2 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 38/16 20060101 A61K038/16; A61K 31/7048 20060101
A61K031/7048; A61K 31/7052 20060101 A61K031/7052; A61K 31/353
20060101 A61K031/353 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 2003 |
EP |
03002603.3 |
Claims
1. Use of a cytokine antagonist which modulates the expression
and/or the function of a cytokine in a cell for the down-regulation
of a cell death preventing protein in a cell.
2. The use according to claim 1, wherein the cell is sensitized for
cell death.
3. The use according to claim 1 or 2, wherein the cell is a cancer
cell.
4. The use according to any of claims 1 to 3, wherein the cytokine
is selected from the group consisting of IL-4, IL-5, IL-6, IL-10
and IL-13, and combinations thereof, preferably IL-4, IL-10 and
IL-13, and combinations thereof.
5. The use according to any of claims 1 to 3, wherein the cell
death is caused by apoptosis and the cell death preventing protein
is an anti-apoptotic protein selected from the group consisting of
Bcl-2, cFLIP, Mcl-1, A1, BOO, NR-13, sentrin, TOSO, CPAN, PED,
DFF45, NAP, XIAP, cIAP-1, cIAP-2, ML-IAP, MAP, BIRC5, TIAP, Apollon
and fortilin, preferably Bcl-2, Bcl-x.sub.L, PED and cFLIP, and
combinations thereof, most preferably Bcl-2 and/or Bcl-x.sub.L.
6. The use according to any of claims 1 to 5, wherein the cytokine
antagonist is selected from the group consisting of a
transcriptional regulator of the cytokine/cytokine receptor gene,
an antisense nucleic acid molecule that is complementary to a
region of the cytokine/cytokine receptor gene, a dsRNA molecule
that is complementary to the cytokine/cytokine receptor mRNA, a
ribozyme that cleaves the cytokine/cytokine receptor mRNA, a
translational regulator of the cytokine/cytokine receptor mRNA, an
aptamer, which bind to the cytokine and/or cytokine receptor and
prevents or disrupts the interaction between the cytokine and its
receptor, an antibody that binds to the cytokine/cytokine receptor,
a receptor, a fragment or derivative thereof, of the cytokine,
preferably CD124, CD132, IL-13R.alpha.-2 and IL-10R.alpha., a
cytokine trap, and a cytokine mutein
7. The use according to claim 6, wherein the cytokine antagonist is
an antibody that binds to the cytokine/cytokine receptor.
8. The use according to claim 7, wherein the antibody is an
antibody that binds to IL-4, IL-10 or IL-13, and combinations
thereof.
9. The use according to any of claims 1 to 8, wherein the cytokine
antagonist is delivered to the proximity of or into the target
cell.
10. The use according to claim 9, wherein the cytokine antagonist
is delivered via a retroviral vector.
11. A method for the down-regulation of a cell death preventing
protein in a cell, the method comprising (a) providing a sample of
tissue or cells from a subject (b) contacting the cell or the
sample with a cytokine antagonist according to any of claims 6 to
8.
12. The method according to claim 11, wherein the cell is a cancer
cell.
13. Use of a cytokine antagonist, optionally in combination with
radiation therapy, for the manufacture of a medicament for the
treatment of cancer.
14. Use of a cytokine antagonist, optionally in combination with at
least one active compound, for the manufacture of a medicament for
the treatment of cancer.
15. The use according to claim 14, wherein the active compound is
selected from the group consisting of antimetabolites, preferably
cytarabine, fludarabine, 5-fluoro-2'-deoxyuiridine, gemcitabine,
hydroxyurea or methotrexate; DNA-fragmenting agents, preferably
bleomycin, DNA-crosslinking agents, preferably chlorambucil,
cisplatin, cyclophosphamide or nitrogen mustard; intercalating
agents preferably adriamycin (doxorubicin) or mitoxantrone; protein
synthesis inhibitors, preferably L-asparaginase, cycloheximide,
puromycin or diphteria toxin; topoisomerase I poisons, preferably
camptothecin or topotecan; topoisomerase II poisons, preferably
etoposide (VP-16) or teniposide; microtubule-directed agents,
preferably colcemid, colchicine, paclitaxel, vinblastine or
vincristine; kinase inhibitors preferably flavopiridol,
staurosporin, STI571 (CPG 57148B) or UCN-01
(7-hydroxystaurosporine); miscellaneous investigational agents,
preferably PS-341, phenylbutyrate, ET-18-OCH.sub.3, or farnesyl
transferase inhibitors (L-739749, L-744832); polyphenols preferably
quercetin, resveratrol, piceatannol, epigallocatechine gallate,
theaflavins, flavanols, procyanidins, betulinic acid; hormones
preferably glucocorticoids or fenretinide; hormone antagonists,
preferably tamoxifen, finasteride or LHRH antagonists;
plant-derived cytostatics (from Viscum and derivatives); alcaloids
preferably vindesine; podophyllotoxins preferably vinorelbin;
alkylants preferably nimustrine, carmustrine, lomustine,
estramustrine, melphalam, ifosfamide, trofosfamide, bendamustine,
dacarbazine, busulfane, procarbazine, treosulfane, tremozolamide,
thiotepa; cytotoxic antibiotics preferably aclarubicine,
daunorubicin, epirubicine, idarubicine, mitomycine, dactinomycine;
antimetabolites like folic acid analogs preferably methotrexate,
purine analogs preferably cladribin, mercaptopurin, tioguanine and
pyrimidine analogs preferably cytarabine, fluorouracil, docetaxel;
other antineoplastic, platinum compounds preferably thioplatin,
carboplatin, oxaliplatin; amsacrine, irinotecane,
interferon-.alpha., tretinoine, hydroxycarbamide, miltefosine,
pentostatine, aldesleukin; antineoplastic compounds derived from
organs, e.g. monoclonal antibodies preferably trastuzumab,
rituximab, or derived from enyzmes preferably pegaspargase;
endocrine effecting antineoplastic compounds belonging to hormones,
e.g. estrogens preferably polyestradiol, fosfestriol,
ethinylestradiol, gestagens preferably medroxyprogesterone,
gestonoroncaproat, megestrol, norethisterone, lynestrenol,
hypothalamus hormones preferably triptorelin, leuproreline,
busereline, gosereline, other hormones preferably testolactone,
testosterone; endocrine effecting antineoplastic compounds
belonging to hormone antagonists, e.g. antiestrogens preferably
toremifen; antiandrogens preferably flutamide, bicalutamide,
cyproterane; endocrine effecting antineoplastic compounds belonging
to enzyme inhibitors preferably anastrol, exemestane, letrozol,
formestane, aminoglutethimide, all of which can be occasionally
administered together with so-called protectives preferably
calciumfolinat, amifostin, lenograstin, molgromostin, filgrastin,
mesna or so-called additives preferably retinolpalmitate, thymus
D9, amilomer.
16. The use according to claim 15, wherein the active compound is
selected from the group consisting of paclitaxel, cisplatin, and
doxorubicin.
17. The use according to claim 14, wherein the active compound is a
death receptor agonist.
18. The use according to claim 17, wherein the death receptor
agonist is a death receptor ligand selected from the group
consisting of TNF-.alpha., TNF-.beta., LT-.beta., TRAIL, CD95
ligand, TRAMP ligand, DR6 ligand, and fragments and derivatives
thereof.
19. The use according to claim 17, wherein the death receptor
agonist is an antibody against a death receptor, a derivative or
fragment thereof, selected from the group consisting of anti-CD95
antibody, anti-TRAIL-R1 antibody, anti-TRAIL-R2 antibody, anti-DR6
antibody, anti-TNF-R1 antibody and anti-TRAMP antibody.
20. The use according to claim 14, wherein the active compound is a
negative regulator of anti-apoptotic proteins, preferably IAPs.
21. The use according to any of claims 13 to 20, wherein the cancer
to be treated is selected from the group consisting of
neuroblastoma, intestine carcinoma preferably rectum carcinoma,
colon carcinoma, familiary adenomatous polyposis carcinoma and
hereditary non-polyposis colorectal cancer, esophageal carcinoma,
labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tong
carcinoma, salivary gland carcinoma, gastric carcinoma,
adenocarcinoma, medullary thyroid carcinoma, papillary thyroid
carcinoma, follicular thyroid carcinoma, anaplastic thyroid
carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian
carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium
carcinoma, chorion carcinoma, pancreatic carcinoma, prostate
carcinoma, testis carcinoma, breast carcinoma, urinary carcinoma,
melanoma, brain tumors preferably glioblastoma, astrocytoma,
meningioma, medulloblastoma and peripheral neuroectodermal tumors,
Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute
lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute
myeolid leukemia (AML), chronic myeloid leukemia (CML), adult
T-cell leukemia lymphoma, hepatocellular carcinoma, gall bladder
carcinoma, bronchial carcinoma, small cell lung carcinoma,
non-small cell lung carcinoma, multiple myeloma, basalioma,
teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyo
sarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma,
myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and
plasmocytoma.
22. The use according to any claim 21, wherein the cancer to be
treated is selected from the group consisting of thyroid carcinoma,
breast carcinoma, lung carcinoma, prostate carcinoma and colon
carcinoma.
23. The use according to any claim 21, wherein the cancer to be
treated is thyroid carcinoma.
24. A medicament for the treatment of cancer, comprising a cytokine
antagonist, optionally in combination with at least one active
compound, and a pharmaceutically acceptable carrier.
25. The use of a cytokine antagonist for diagnosing and monitoring
the cancer disease of a subject, comprising (a) providing a body
fluid sample or a sample of tissue or cells from a tumor of a
subject (b) contacting the sample with a labeled probe that binds
to a cytokine nucleic acid and/or with an antibody that binds to a
cytokine (c) determining the expression level of the cytokine in
the tissue or cells and comparing the expression level with healthy
control cells, and (d) correlating a better prognosis for the
subject with a low ratio of cytokine expression when compared to
the expression level in healthy control cells.
26. A diagnostic kit containing at least one cytokine antagonist,
optionally in combination with suitable buffers, enzymes and other
compounds.
27. The use according to any of claims 1 to 10, wherein the cell is
a non-lymphoid and a non-myeolid cancer cell.
28. The use according to claim 27, wherein the cytokine is IL-4
and/or IL-10 and/or IL-13.
29. The use according to claim 27 or 28, wherein the cytokine is
not produced by a lymphoid or a myeloid cell.
30. The use according to claim 29, wherein the cytokine is
autocrinely produced by the non-lymphoid and non-myeloid cancer
cell.
31. The use according to any of claims 27 to 30, wherein the
cell-death preventing protein is cFLIP and/or Bcl-2 and/or
Bcl-x.sub.L.
32. The use according to any of claims 27 to 31, wherein the
cytokine antagonist is an antibody that binds to IL-4 and/or IL 10
and/or IL-13.
33. The use according to any of claims 13 to 23, wherein the cancer
is a non-lymphoid and non-myeloid cancer.
34. The use according to claim 33, wherein the cytokine is IL-4
and/or IL-10 and/or IL-13.
35. The use according to claim 33 or 34, wherein the cytokine is
autocrinely produced by the non-lymphoid and non-myeloid
cancer.
36. The use according to any of claims 33 to 35, wherein the cancer
is selected from the group consisting of thyroid carcinoma, breast
carcinoma, lung carcinoma, prostate carcinoma, bladder carcinoma
and colon carcinoma.
37. The use according to claim 36, wherein the cancer is thyroid
carcinoma.
38. A medicament for the treatment of a non-lymphoid and/or
non-myeloid cancer, which autocrinely produce a cytokine,
comprising a cytokine antagonist as defined in any of claims 6 to
8, optionally in combination with at least one active compound, and
a pharmaceutically acceptable carrier.
39. A method for the down-regulation of an anti-apoptotic protein
in a non-lymphoid and/or non-myeolid cancer target cell which
autocrinely produces a cytokine, preferably an interleukin, the
method comprising contacting the target cell or the sample with a
cytokine antagonist as defined in any of claims 6 to 8.
40. The method according to claim 39, wherein the cytokine
antagonist is delivered to the proximity of or into the target
cell.
41. The method according to claim 40, wherein the cytokine
antagonist is delivered via a retroviral vector.
Description
[0001] The present invention relates to a method of sensitizing
cells for apoptosis by using compounds that selectively block
cytokines, in particular interleukins, and the use of said
compounds for the treatment of cancer and autoimmune diseases.
[0002] The molecular mechanisms controlling the balance between
cell survival and cell death play a key role in a number of
physiological and pathological processes. Crucial for the cellular
ability to induce death of supernumerary, misplaced or damaged
cells with high specificity and efficiency is the machinery of
so-called "apoptosis" or "programmed cell death".
[0003] Diseases and conditions in which apoptosis has been
implicated fall into two categories, those in which there is
increased cell survival (i.e. apoptosis is reduced) and those in
which there is excess cell death (i.e. apoptosis is increased).
Diseases in which there is an excessive accumulation of cells due
to increased cell survival include cancer, autoimmune disorders and
viral infections. For these and other conditions in which
insufficient apoptosis is believed to be involved, promotion of
apoptosis is desired. This can be achieved, for example, by
promoting cellular apoptosis or by increasing the sensitivity of
cells to endogenous or exogenous apoptotic stimuli, for example,
cell signaling molecules or other cytokines, cytotoxic drugs or
radiation. Promotion of or sensitization to apoptosis is believed
to have clinical relevance in sensitizing cancer cells to
chemotherapeutic drugs or radiation.
[0004] In the second category, AIDS and neurodegenerative disorders
like Alzheimer's or Parkinson's disease represent disorders for
which an excess of cell death due to promotion of apoptosis (or
unwanted apoptosis) is likely to be involved. Amyotrophic lateral
sclerosis, retinitis pigmentosa, and epilepsy are other neurologic
disorders in which apoptosis has been implicated. Apoptosis has
been reported to occur in conditions characterized by ischemia,
e.g. myocardial infarction and stroke. For these and other diseases
and conditions in which unwanted apoptosis is believed to be
involved, inhibitors of apoptosis are desired.
[0005] Currently, a major treatment for cancerous tumors is
surgical removal of the affected areas of the tissue, organ, or
gland. However, high recurrence rates are a major obstacle to the
complete eradication of cancerous cells. It is believed that
although the cancer cells in the malignant tumors can be removed
surgically, cancerous cells that have invaded the surrounding
tissue or lymph nodes frequently cause tumor recurrence. One reason
for frequent tumor recurrence may be that during the development of
the primary cancer, complete removal of all the cancer cells by
surgical procedures is extremely difficult. Although irradiation,
chemotherapy and appropriate hormone therapy all induce apoptosis
to some extent in tumor cells, higher doses of the drugs or
radiation may be required for suppressing the growth of cancer
cells, which, in turn, can cause severe side effects on
patients.
[0006] Thus, the problem underlying the present invention refers to
the identification of compounds that specifically modulate distinct
steps in the apoptosis pathway without causing the described
deleterious side effects.
[0007] The effective cure of patients suffering from cancer is
often difficult since many tumor cells have developed a resistance
to anti-cancer drugs used for chemotherapy. The described phenotype
involves a variety of strategies that tumor cells use to evade the
cytostatic effects of anti-cancer drugs. Mechanisms for drug
resistance include modifications in detoxification and DNA repair
pathways, changes in cellular sites of drug sequestration,
decreases in drug-target affinity, synthesis of specific drug
inhibitors within cells, and accelerated removal or secretion of
drugs. In addition, cancer cells commonly fail to undergo
apoptosis. Thus, apoptosis defects appear to be a major problem in
cancer therapy as they confer resistance to many tumors against
current treatment protocols, leading to tumor progression.
[0008] Apoptosis pathways involve diverse groups of molecules. One
set of mediators implicated in apoptosis are so-called caspases,
cysteine proteases that cleave their substrate specifically at
aspartate residues. Caspases convey the apoptic signal in a
proteolytic cascade, with caspases cleaving and activating other
caspases which subsequently degrade other cellular targets
eventually resulting in cellular breakdown. Caspase activation
itself can be triggered by external stimuli affecting certain
self-surface receptors, known to the person skilled in the art as
so-called death receptors, or by intracellular stress response via
the mitochondria leading to the release of mitochondrial proteins.
Known death receptors mediating apoptosis include members of the
tumor necrosis factor (TNF) receptor super family such as, e.g.
CD95 (APO-1/Fas) or TRAIL (TNF-related apoptosis inducing ligand)
receptors 1 and 2. Stimulation of death receptors with
apoptosis-inducing substances leads, among others, to the
activation of caspase 8, which in turn activates other
downstream-acting caspases.
[0009] The induction or inhibition of apoptosis is controlled in
part by the Bcl-2 family members. A number of such genes, including
Bcl-2 and Bcl-x.sub.L, counteract apoptosis by preserving
mitochondrial membrane integrity and preventing cytochrome c
release in the cytoplasm. In contrast, the pro-apoptotic members
such as Bax and Bad antagonize the function of Bcl-2 and
Bcl-x.sub.L inducing heterodimer formation and mitochondrial
membrane permeabilization with cytochrome c release.
[0010] In human cancers, a high expression of the anti-apoptotic
members of the Bcl-2 family is commonly found and contributes to
both neoplastic cell expansion and resistance to the therapeutic
action of chemotherapeutic drugs. Overexpression of Bcl-2 can
render cells resistant to apoptosis, thereby favoring malignant
growth. Moreover, since many chemotherapeutic agents kill tumor
cells by inducing apoptosis, overexpression of Bcl-2 or Bcl-x.sub.L
can lead to a multi-drug resistant phenotype.
[0011] The expression of a variety of genes involved in the
survival or death of different target cells, including members of
the Bcl-2 family, is regulated by so-called cytokines. Cytokines
belong to a diverse group of soluble, non-antibody proteins
secreted by a variety of cell types of the immune system, which
modulate the functional activities of individual cells by
interaction with specific cell surface receptors, e.g. interferon,
interleukin. The person skilled in the art knows two functionally
distinct subsets of so-called T-helper cells that have been
characterized on the basis of cytokine production. One subset, Th1
cells, secrete IFN-.gamma. and other cytokines associated with
inflammation and cell-mediated immune responses, whereas Th2 cells
promote humoral response releasing IL-4, IL-5 and IL-10.
[0012] It is known in the art that cancer types of lymphoid origin
(and probably also of myeloid origin) autocrinely produce cytokines
such as IL-1 and IL-6, and that e.g. IL-4 enhances the survival of
B cells in chronic lymphocytic leukaemia (CLL) in vitro by
inhibiting apoptosis (see e.g. Lu Zhao Yan et al., Blood, 1995,
Vol. 86, pages 3123-3131; Mainou-Fowler et al., Leukemia and
Lymphoma, 1996, Vol. 21, pages 369-377). On the other hand,
cytokines produced by T or B lymphocytes in the surrounding of a
tumor, e.g. thyroid cancer, promote the survival and/or growth of
said tumor (Stassi et al., Nature Reviews Immunology, 2002, Vol. 2,
pages 195-204).
[0013] With respect to solve the problem underlying the present
invention, namely the identification of compounds that specifically
modulate distinct apoptosis steps, the inventors have surprisingly
found that thyroid cancer cells autocrinely produce high levels of
IL-4 and IL-10, as compared with normal tissues, while IFN-.gamma.
was barely detectable in those cancer cells. Thyroid cancer is the
most common endocrine malignancy, responsible for about 60% of the
death secondary to endocrine cancer. Three major types of malignant
tumors originate from the thyroid epithelium. The more
differentiated papillary (PTC) and follicular (FTC) thyroid
carcinomas account for the vast majority of malignant tumors, while
the undifferentiated anaplastic carcinomas (UTC) are extremely
rare. The high levels of IL-4 and IL-10 in thyroid cancer cells
correlated with an overexpression of Bcl-x.sub.L and Bcl-2 which in
turn protects thyroid cancer cells against the cytotoxic effect of
chemotherapeutic drugs suggesting a potential role of these
anti-apoptotic proteins in thyroid cancer resistance from
drug-induced cytotoxicity.
[0014] The person skilled in the art is aware of the fact that
thyroid cancer cells belong to so-called epithelial cancers which
are clearly distinguished from either lymphoid or myeloid cancer
types.
[0015] Thus, a first object of the present invention refers to the
use of a cytokine antagonist which modulates the expression and/or
the function of a cytokine in a cell for the down-regulation of a
cell death preventing protein in a cell.
[0016] As a result of the down-regulation of a cell death
preventing protein the cell is sensitized for cell death. In the
context of the present invention, the term "cell death" refers to
any mechanism and process which can cause a cell to die. The
skilled artisan distinguishes two processes named apoptosis and
necrosis both of which are addressed within the scope of the
present invention. However, the use of a cytokine antagonist
according to the present invention is particularly effective if the
death process the cell should be sensitized for is apoptosis. Thus,
in a preferred embodiment of the present invention the "cell death
preventing" proteins refer to "anti-apoptotic" proteins.
[0017] In a particular embodiment of the present invention the term
"cell" refers to cells, that fail to undergo apoptosis as described
in the introduction. In this respect, the cells encompass, for
example, cancer cells and self-reacting cells of the immune system.
Most preferably, the cell of the present invention is a cancer
cell.
[0018] In a particularly preferred embodiment, the cancer cell is a
non-lymphoid and/or a non-myeloid cancer cell, most preferably an
epithelial cancer.
[0019] If the cell is a cancer cell, the defect in undergoing
apoptosis may have rendered the cell resistant to various treatment
strategies exploiting anti-neoplastic compounds and/or radiation
therapies. The cancer cell to which the cytokine is preferably
applied to can also be resistant to compounds which do not
necessarily lead to cell death directly, but which sensitize these
cells for apoptosis. The skilled artisan knows that such compounds
include naturally occurring agonists for death receptors, i.e.
receptor ligands or agonistic antibodies to said death receptors,
as well as chemotherapeutic drugs.
[0020] The "cytokine" of the present invention belongs to the group
of cytokines that are predominantly secreted by Th2 helper cells.
More preferably, the cytokine is selected from the group consisting
of IL-4, IL-5, IL-6, IL-10, and IL-13, as well as combinations
thereof. For the efficient use of the cytokine antagonist of the
present invention it is most preferred, if the cytokine is IL-4,
IL-10 and/or IL-13, as well as combinations thereof.
[0021] Within the scope of the present invention, "anti-apoptotic
proteins" include members of the Bcl family such as Bcl-2,
Bcl-x.sub.L, cFLIP, Mcl-1, Bcl-w, A1/BFL1, BOO/DIVA, NR-13,
sentrin, TOSO, CPAN, PED, DFF45, and the like. The anti-apoptotic
proteins of the present invention also include so-called
"Inhibitors of Apoptosis Proteins" (IAPs). IAPs bind to early
active caspases, thereby preventing the ongoing of the apoptosis
process. They are expressed at high levels in many tumors and, by
inhibition of caspases, contribute to the resistance of cancers
against apoptosis induction. Examples of IAPs include NAIP, XIAP
(hILP), cIAP-1, cIAP-2, ML-IAP (livin), KIAP, BIRC5 (survivin),
TIAP, and Apollon. Finally, anti-apoptotic proteins can be others
such as fortilin, and the like.
[0022] In a preferred embodiment of the present invention, the
anti-apoptotic proteins include PED, cFLIP, Bcl-2 and Bcl-x.sub.L,
and combinations thereof. Most preferably, the anti-apoptotic
proteins which are down-regulated by the cytokine antagonist are
Bcl-2 and/or Bcl-x.sub.L.
[0023] The term "cytokine antagonist" refers to any compound that
is capable of directly modulating the expression and/or the
function of the cytokine, thus leading to the down-regulation of
anti-apoptotic proteins. It is further contemplated within the
scope of the present invention that the cytokine antagonist refers
to any compound that modulates the expression and/or the function
of a cytokine indirectly, namely by affecting the expression and/or
the function of the respective cytokine receptor. It is obvious to
the person skilled in the art that a down-regulation of the
cytokine receptor directly interferes with the function of the
cytokine itself. Therefore, the hereinafter described mechanisms
and molecules, respectively, that modulate the expression and/or
the function of a cytokine may also be extrapolated to cytokine
receptors. In this respect, the term "cytokine" encompasses also
cytokine receptors, unless otherwise indicated.
[0024] In the context of the present invention, the modulation of
the expression and/or the function of the cytokine/cytokine
receptor, hereinafter referred to as the "modulation", by the use
of the cytokine antagonist according to the present invention can
occur on the protein and/or on the nucleic acid level.
[0025] If the modulation occurs on the nucleic acid level, the
cytokine antagonist according to the present invention can be a
peptide or a nucleic acid that regulates the transcription of the
cytokine gene by binding to up-stream and/or down-stream regulatory
sequences of the coding region of the cytokine. Such regulatory
sequences are known to the person skilled in the art and include
so-called promoter, operator, enhancer or silencer regions. For
example, the cytokine antagonist may interfere with the binding of
the RNA polymerase to the promoter region of the cytokine gene,
either by binding directly to the RNA polymerase binding region, by
binding to the polymerase itself or by binding to other factors,
e.g. transcription factors, which are required for efficient RNA
polymerase binding and function. Furthermore, the cytokine
antagonist may bind to the operator region and act as a so-called
repressor of cytokine gene expression.
[0026] In a further embodiment of the present invention, the
modulation on the nucleic acid level can occur by the use of
nucleic acid molecules that hybridize to, and are therefore
complementary to the coding sequence of the cytokine. These nucleic
acid molecules may encode or act as cytokine gene antisense
molecules useful, for example, in cytokine gene regulation. With
respect to cytokine gene regulation, such techniques can be used to
modulate, for example, the phenotype and metastatic potential of
cancer cells. The use of antisense molecules as inhibitors is a
specific, genetically based therapeutic approach. The present
invention provides the therapeutic and prophylactic use of nucleic
acids of at least six nucleotides that are antisense to a gene or
cDNA encoding one of the aforementioned cytokines.
[0027] Similarly, a cytokine antagonist of the present invention
that modulates the expression and/or the function of a cytokine on
the nucleic acid level can be a dsRNA molecule which is
complementary to the cytokine mRNA. Such molecules are also known
in the art as small interfering RNA (siRNA). This technology to
inhibit the expression of certain mRNAs is known to the person
Skilled in the art as RNA interference (RNAi). Preferably, the
dsRNA molecules which are complementary to the mRNA of the
cytokines of the present invention have a length between 10 and 30
base pairs, more preferably, they have a length between 19 and 25
base pairs. The cytokine antagonist being siRNA may be delivered to
the target cell by any method known to the one of skilled art.
Applicable is, for instance, the delivery by using cationic
liposome reagents. It is also conceivable that the siRNA directed
against the cytokine mRNA is obtained by using the DNA encoding it.
In this case, a DNA construct comprising both a stretch of 19 to 25
nucleotides of the desired cytokine coding region, and the
antisense stretch being separated from the sense stretch by a
suitable linker which is able to form a hairpin loop, is inserted
into a vector. The vector can be introduced into the target cell by
methods well known the skilled artisan. The design of such a
construct is further described e.g. in Brummelkamp et al. (Science
2002 Vol. 296, pages 550-553).
[0028] Furthermore, the present invention encompasses so-called
ribozymes as cytokine antagonists. Ribozymes are naturally
occurring RNA fragments that can be designed as human therapeutics
to recognize, bind and digest any disease-causing mRNA sequence, in
this case the cytokine mRNA. Ribozymes are designed to target the
cytokine mRNA through complementary base pair hybridization. After
binding to the target, the enzymatic activity of the ribozyme
cleaves the cytokine mRNA thus preventing its translation into
protein. The cytokine mRNA ribozymes can be chemically synthesized
to selectively inhibit the cytokine production. In addition, the
ribozymes may be chemically modified allowing the ribozymes to be
more stable and active. Included are also ribozymes that do not
only cleave cytokine-specific RNA molecules but also faun
carbon-carbon bonds in a covalent fashion, which raises the
possibility of ribozymes that can catalyze other types of chemical
reactions.
[0029] In a further embodiment of the present invention the
translation of the cytokine gene can be reduced or eliminated by
binding of an RNA-binding protein to one or more operator sequences
in the 5'-UTR of the cytokine mRNA transcript. The bound
RNA-binding protein interferes with translation, likely by
preventing ribosome assembly or blocking the movement of the
ribosome along the transcript from 5' to 3'. Such RNA-binding
proteins may be multimeric, e.g. dimers of a particular RNA-binding
protein. It is also possible within the scope of the present
invention that the cytokine antagonist inhibits the cytokine
expression by promoting or at least being involved in the
degradation of cytokine mRNA.
[0030] If the modulation occurs on the protein level, the present
invention encompasses antibodies or fragments thereof capable of
specifically recognizing one or more epitopes of the cytokine gene
products, epitopes of conserved variants of the cytokine gene
products, epitopes of mutant cytokine gene products, or peptide
fragments of cytokine gene products. Such antibodies may include,
but are not limited to, polyclonal antibodies, monoclonal
antibodies (mAbs), human, humanized or chimeric antibodies,
single-chain antibodies, Fab fragments, F(ab')2 fragments, Fv
fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments
of any of the above. The cytokine antagonist being an antibody as
described above can be used to capture and neutralize excessive
amounts of cytokines that are overexpressed in drug-resistant
cancer cells. It may be desirable for the present invention if the
antibody recognizes more than one of the above mentioned cytokines.
In order to capture and neutralize more than one overexpressed
cytokine, the antibody used as a cytokine antagonist of the present
invention can possess more than one specificities, i.e. being, for
example, bispecific, trispecific or multispecific.
[0031] Epitopes and antigenic regions useful for generating
antibodies can be found within the cytokine amino acid sequences
(e.g. SWISS-PROT numbers P05112 for IL-4, P22301 for IL-10 or
P35225 for IL-13) by procedures available to one of skill in the
art. For example, short, unique peptide sequences can be identified
in the amino acid sequences that have little or no homology to
known amino acid sequences. Preferably the region of a protein
selected to act as a peptide epitope or antigen is not entirely
hydrophobic; hydrophilic regions are preferred because those
regions likely constitute surface epitopes rather than internal
regions of the present proteins and polypeptides. These surface
epitopes are more readily detected in samples tested for the
presence of the present proteins and polypeptides.
[0032] Peptides can be made by any procedure known to one of skill
in the art, for example, by using in vitro translation or chemical
synthesis procedures. Short peptides which provide an antigenic
epitope but which by themselves are too small to induce an immune
response may be conjugated to a suitable carrier. Suitable carriers
and methods of linkage are well known in the art. Suitable carriers
are typically large macromolecules such as proteins,
polysaccharides and polymeric amino acids. Examples include serum
albumins, keyhole limpet hemocyanin, ovalbumin, polylysine and the
like. One of skill in the art can use available procedures and
coupling reagents to link the desired peptide epitope to such a
carrier. For example, coupling reagents can be used to form
disulfide linkages or thioether linkages from the carrier to the
peptide of interest. If the peptide lacks a disulfide group, one
may be provided by the addition of a cysteine residue.
Alternatively, coupling may be accomplished by activation of
carboxyl groups.
[0033] The minimum size of peptides useful for obtaining antigen
specific antibodies can vary widely. The minimum size must be
sufficient to provide an antigenic epitope which is specific to the
protein or polypeptide. The maximum size is not critical unless it
is desired to obtain antibodies to one particular epitope. For
example, a large polypeptide may comprise multiple epitopes, one
epitope being particularly useful and a second epitope being
immunodominant.
[0034] In a preferred embodiment of the present invention, the
cytokine antagonist refers to an antibody against IL-4, IL-5, IL-6,
IL-10, and/or IL-13, as well as combinations thereof. Even more
preferred, the cytokine antagonist refers to an antibody against
IL-4, IL-10, and/or IL-13, as well as combinations thereof. Most
preferably, the cytokine antagonist refers to an antibody against
IL-4 and/or IL-10, and combinations thereof. It is understood that
the antibody being used as a cytokine antagonist can possess more
than one specificities, as described supra, i.e. being directed to
more than one of the mentioned IL, e.g. a bispecific antibody to
IL4 and IL10.
[0035] In a further embodiment of the present invention, the
cytokine antagonist that modulates the expression and/or the
function of the cytokine can be a so-called aptamer, either a
peptide-based aptamer or an oligonucleotide-based aptamer. Peptide
aptamers are defined as protein-based recognition agents that
consist of constrained combinatorial peptide libraries displayed on
the surface of a scaffold protein. Peptide aptamers function in
trans, interacting with and inactivating gene products without
mutating the DNA that encodes them. In principle, combinatorial
libraries of peptide aptamers should contain aptamers that interact
with any given gene product, thus allowing peptide aptamers to be
generated against an organism's entire proteome.
Oligonucleotide-based aptamers being used as cytokine antagonist
according to the present invention comprise DNA as well as RNA
aptamers. In this respect, the present invention encompasses also
mirror-image L-DNA or L-RNA aptamers, so-called spiegelmers.
[0036] The aptamers that are useful as cytokine antagonists for the
present invention include those which interact with specific
proteins and thus prevent or disrupt the specific protein
interaction between the cytokine and its receptor. They can
interact with the cytokine itself, preferably with that region of
the cytokine that is involved in the receptor binding. The aptamers
can also prevent/disrupt the interaction between the cytokine and
its receptor by binding to the receptor, preferably with that
region of the receptor that is involved in the cytokine binding. It
is also possible that the aptamers bind to other factors/proteins
that are required for successful cytokine/receptor interaction.
[0037] In the context of the described aptamers, it is also
feasable the cytokine antagonist comprises so-called small molecule
inhibitors that may exhibit similar properties as aptamers, namely
binding to either the cytokine or to the cytokine receptor, thereby
inhibiting their proper interaction and, thus, function. The small
molecule inhibitor can be a peptide or a small chemical compound,
which has been identified by methods known to the skilled artisan,
e.g. by computational combinatorial chemistry in combination with
screening of compound libraries.
[0038] In a further embodiment of the present invention the
cytokine antagonist that modulates the expression and/or the
function of the cytokine, comprises at least one receptor, a
derivative or fragment thereof, of any of the cytokines included in
the present invention. Similarly to the proposed and described
effect for using antibodies as cytokine antagonists, the cytokine
receptor, a fragment or derivative thereof, can be used to capture
and neutralize excessive amounts of cytokines which are
overexpressed in drug-resistant cancer cells. Examples for suitable
receptors and receptor subunits, respectively, include CD124 which
binds both IL-4 and IL-13 (data base accession number P24394),
CD132 which represents the common gamma subunit shared by IL-2,
IL-4, IL-7, IL-9, and IL-15 receptors (data base accession number
P31785), IL-13 receptor alpha-2 chain (data base accession number
Q14627) and IL-10 receptor alpha chain (data base accession number
Q13651).
[0039] In the context of the present invention the term "derivative
or fragment" of a cytokine receptor refers to peptides the length
of which and/or the amino acid composition of which can differ from
the originally disclosed amino acid sequence, provided that the
function of the receptor, namely the binding of the cytokine, is
neither reduced nor eliminated. Therefore, the term "derivative or
fragment" includes peptides which are extended or shortened on
either the amino- or the carboxyterminal end or which possess
deletions or insertions internally. In addition, the term
"derivative or fragment" includes peptides with one or more amino
acids being different from the originally disclosed sequence.
Particularly advantageous for the present invention, especially if
the receptor is used therapeutically, are soluble receptors lacking
the transmembrane region. In this case, the receptor comprises the
proposed extracellular binding domain, a fragment or derivative
thereof, optionally being directly or via a spacer linked to the
proposed intracellular domain or to the Fc part of an antibody.
[0040] With respect to receptors, derivatives or fragments thereof,
the present invention also comprises so-called cytokine traps,
which make use of the fact that the signalling cascade triggered by
cytokines is initiated with the cytokine binding to a first
subunit, said binding leading to the recruitment of the second
subunit, whereby only the complex of the cytokine bound to both
receptor subunit chains initiate the subsequent cascade. As
described in Nature Medicine 2003, Vol. 9, pages 20-22 and pages
47-52, cytokine traps consist of the two relevant receptor subunits
which are linked together by fusion with the Fc portion (complement
binding domain) of the immunoglobulin IgG1. Therefore, the cytokine
antagonist of the present invention can be a so-called
"heterodimeric trap" consisting of two receptor subunits each of
which is fused to the Fc portion of an antibody comprising the
heavy chain constant regions CH2 and CH3 and the hinge region of
IgG1, whereby the constructs are paired via disulfide bridges
between the hinge regions. The cytokine antagonist of the present
invention can also be a so-called "inline trap", where the two
receptor extracellular domains are fused in-line followed by the
human IgG1 Fc. For example, a cytokine antagonist for the cytokine
IL-4 would consist of the extracellular domains of CD124, as
specified above, and CD132, as specified above, linked to IgG1 in
the described manner.
[0041] Furthermore, the modulation of the cytokine can be achieved
by using so-called muteins of the cytokines. Muteins are
derivatives of biologically active proteins the amino acid
composition of which has been artificially altered. The muteins of
the present invention are still able to bind to their respective
cell surface receptor, but are incapable of triggering an internal
signal cascade which would lead to the up-regulation of
anti-apoptotic proteins. In this respect, the muteins compete with
the endogenously expressed cytokines for the binding sites on the
respective receptor. The muteins can be made via bacterial
expression of mutant genes that encode the muteins that have been
synthesized from the genes for the parent proteins by
oligonucleotide-directed mutagenesis.
[0042] In line with the above disclosures, the present invention
furthermore refers to a method for the down-regulation of a cell
death preventing protein in a cell, the method comprising [0043]
(a) providing a sample of tissue or cells from a subject [0044] (b)
contacting the cell or the sample with a cytokine antagonist
[0045] In a particularly preferred embodiment of the present
invention the cell, to which the disclosed method should be applied
to, is a cancer cell.
[0046] In a particular embodiment, the present invention refers to
a method for the down-regulation of an anti-apoptotic protein in a
non-lymphoid and/or non-myeloid cancer target cell which
autocrinely produces high levels of a cytokine, preferably an
interleukin, the method comprising contacting the target cell or
the sample with a cytokine antagonist as defined supra.
[0047] In order to act properly as a cytokine antagonist and in
order to perform the described method it is desirable that the
cytokine antagonist is delivered to the site of action namely to
the proximity of a cell and/or into a cell. The person skilled in
the art is aware of a variety of methods how to deliver the
disclosed cytokine antagonists into or in the proximity of the
target cell. In general, the appropriate method depends on whether
the cytokine antagonist is a nucleic acid or a peptide.
Furthermore, if the cytokine antagonist is a peptide it can be
delivered into or in the proximity of the target cell by
introducing the nucleic acid encoding it either to the target cell
itself or to other cells being suitable to produce the peptide. For
peptide production, both eukaryotic and prokaryotic host cells are
contemplated.
[0048] There are several well-known methods of introducing nucleic
acids into animal cells, any of which may be used in the present
invention and which depend on the host. Typical hosts include
mammalian species, such as humans, non-human primates, dogs, cats,
cattle, horses, sheep, and the like. At the simplest, the nucleic
acid can be directly injected into the target cell/target tissue,
or by so-called microinjection into the nucleus. Other methods
include fusion of the recipient cell with bacterial protoplasts
containing the nucleic acid, the use of compositions like calcium
chloride, rubidium chloride, lithium chloride, calcium phosphate,
DEAE dextran, cationic lipids or liposomes or methods like
receptor-mediated endocytosis, biolistic particle bombardment
("gene gun" method), infection with viral vectors, electroporation,
and the like.
[0049] For the introduction of the cytokine antagonist,
respectively the nucleic acid encoding it, into the cell and its
expression it can be advantageous if the nucleic acid is integrated
in an expression vector. The expression vector is preferably a
eukaryotic expression vector, or a retroviral vector, a plasmid,
bacteriophage, or any other vector typically used in the
biotechnology field. If necessary or desired, the nucleic acid
encoding the cytokine antagonist can be operatively linked to
regulatory elements which direct the transcription and the
synthesis of a translatable mRNA in pro- or eukaryotic cells. Such
regulatory elements are promoters, enhancers or transcription
termination signals, but can also comprise introns or similar
elements, for example those, which promote or contribute to the
stability and the amplification of the vector, the selection for
successful delivery and/or the integration into the host's genome,
like regions that promote homologous recombination at a desired
site in the genome. For therapeutic purposes, the use of retroviral
vectors has been proven to be most appropriate to deliver a desired
nucleic acid into a target cell.
[0050] If the cytokine antagonist is a peptide that shall be
directly introduced into the target cell it can be fused to a
carrier peptide that mediates the cellular uptake of the peptide.
Appropriate carriers are known to the person skilled in the art and
include TAT, fibroblast growth factor, galparan (transportan),
poly-arginine, and Pep-1, and functional fragments and derivatives
of any of said carriers. Furthermore, the cytokine may be fused to
a ligand for a cell surface receptor, or a functional portion
thereof, and thus internalized by receptor-mediated
endocytosis.
[0051] The cytokine antagonist as disclosed in the present
invention can be used as a pharmaceutical, optionally in
combination with at least one active compound, for the treatment of
cancer. This is a further embodiment of the present invention. The
term "active compound" refers to a compound other than the cytokine
antagonist which is able to induce or sensitize for cell death,
preferably apoptosis, or which inhibits cell proliferation. Active
compounds which are able to induce or sensitize for cell death,
preferably apoptosis are known to the person skilled in the
art.
[0052] First, the phrase "active compound" refers to the use of
electromagnetic or particulate radiation in the treatment of
neoplasia. Radiation therapy is based on the principle that
high-dose radiation delivered to a target area will result in the
death of reproductive cells in both tumor and normal tissues. The
radiation dosage regimen is generally defined in terms of radiation
absorbed dose (rad), time and fractionation, and must be carefully
defined by the oncologist. The amount of radiation a patient
receives will depend on various consideration but the two most
important considerations are the location of the tumor in relation
to other critical structures or organs of the body, and the extent
to which the tumor has spread. Examples of radiotherapeutic agents
are provided in, but not limited to, radiation therapy and is known
in the art (Hellman, Principles of Radiation Therapy, Cancer, in
Principles and Practice of Oncology, 24875 (Devita et al., ed., 4th
ed., vl, 1993). Recent advances in radiation therapy include
three-dimensional conformal external beam radiation, intensity
modulated radiation therapy (IMRT), stereotactic radiosurgery and
brachytherapy (interstitial radiation therapy), the latter placing
the source of radiation directly into the tumor as implanted
"seeds." These newer treatment modalities deliver greater doses of
radiation to the tumor, which accounts for their increased
effectiveness when compared to standard external beam radiation
therapy. Beta-emitting radionuclides are considered the most useful
for radiotherapeutic applications because of the moderate linear
energy transfer (LET) of the ionizing particle (electron) and its
intermediate range (typically several millimeters in tissue). Gamma
rays deliver dosage at lower levels over much greater distances.
Alpha particles represent the other extreme; they deliver very high
LET dosage, but have an extremely limited range and must,
therefore, be in intimate contact with the cells of the tissue to
be treated. In addition, alpha emitters are generally heavy metals,
which limits the possible chemistry and presents undue hazards from
leakage of radionuclide from the area to be treated. Depending on
the tumor to be treated all kinds of emitters are conceivable
within the scope of the present invention.
[0053] Generally, radiation therapy can be combined temporally with
other active compounds listed below to improve the outcome of
treatment. There are various terms to describe the temporal
relationship of administering radiation therapy together with other
active compounds, and the following examples are the preferred
treatment regimens and are generally known by those skilled in the
art and are provided for illustration only and are not intended to
limit the use of other combinations. Administration of radiation
therapy with other active compounds can be "sequential", i.e.
separately in time in order to allow the separate administration,
"concomitant" which refers to the administration on the same day,
and, finally, "alternating" which refers to the administration of
radiation therapy on the days in which other active compounds would
not have been administered.
[0054] Another class of active compounds are chemical compounds
having a cytostatic or anti-neoplastic effect ("cytostatic
compound"). Cytostatic compounds included in the present invention
comprise, but are not restricted to (i) antimetabolites, such as
cytarabine, fludarabine, 5-fluoro-2'-deoxyuiridine, gemcitabine,
hydroxyurea or methotrexate; (ii) DNA-fragmenting agents, such as
bleomycin, (iii) DNA-crosslinking agents, such as chlorambucil,
cisplatin, cyclophosphamide or nitrogen mustard; (iv) intercalating
agents such as adriamycin (doxorubicin) or mitoxantrone; (v)
protein synthesis inhibitors, such as L-asparaginase,
cycloheximide, puromycin or diphteria toxin; (vi) topoisomerase I
poisons, such as camptothecin or topotecan; (vii) topoisomerase II
poisons, such as etoposide (VP-16) or teniposide; (viii)
microtubule-directed agents, such as colcemid, colchicine,
paclitaxel, vinblastine or vincristine; (ix) kinase inhibitors such
as flavopiridol, staurosporin, STI571 (CPG 57148B) or UCN-01
(7-hydroxystaurosporine); (x) miscellaneous investigational agents
such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH.sub.3, or
farnesyl transferase inhibitors (L-739749, L-744832); polyphenols
such as quercetin, resveratrol, piceatannol, epigallocatechine
gallate, theaflavins, flavanols, procyanidins, betulinic acid and
derivatives thereof; (xi) hormones such as glucocorticoids or
fenretinide; (xii) hormone antagonists, such as tamoxifen,
finasteride or LHRH antagonists.
[0055] Other cytostatic compounds include plant-derived cytostatics
(from Viscum and derivatives); alcaloids such as vindesine;
podophyllotoxins such as vinorelbin; alkylants such as nimustrine,
carmustrine, lomustine, estramustrine, melphalam, ifosfamide,
trofosfamide, bendamustine, dacarbazine, busulfane, procarbazine,
treosulfane, tremozolamide, thiotepa; cytotoxic antibiotics such as
aclarubicine, daunorubicine, epirubicine, idarubicine, mitomycine,
dactinomycine; antimetabolites like folic acid analogs such as
methotrexate, purine analogs such as cladribin, mercaptopurin,
tioguanine and pyrimidine analogs such as cytarabine, fluorouracil,
docetaxel; platinum compounds such as carboplatin, oxaliplatin;
amsacrine, irinotecane, interferon-.alpha., tretinoine,
hydroxycarbamide, miltefosine, pentostatine, aldesleukin;
antineoplastic compounds derived from organs, e.g. monoclonal
antibodies such as trastuzumab, rituximab, or derived from enyzmes
such as pegaspargase; endocrine effecting antineoplastic compounds
belonging to hormones, e.g. estrogens such as polyestradiol,
fosfestriol, ethinylestradiol, gestagens such as
medroxyprogesterone, gestonoroncaproat, megestrol, norethisterone,
lynestrenol, hypothalamus hormones such as triptoreline,
leuproreline, busereline, gosereline, other hormones such as
testolactone, testosterone; endocrine effecting antineoplastic
compounds belonging to hormone antagonists, e.g. antiestrogens such
as toremifen; antiandrogens such as flutamide, bicalutamide,
cyproterane; endocrine effecting antineoplastic compounds belonging
to enzyme inhibitors such as anastrol, exemestane, letrozol,
formestane, aminoglutethimide, all of which can be occasionally
administered together with so-called protectives such as
calciumfolinat, amifostin, lenograstin, molgromostin, filgrastin,
mesna or so-called additives such as retinolpalmitate, thymus D9,
amilomer.
[0056] In a preferred embodiment of the present invention, the
active compound having a cytostatic effect is selected from the
group consisting of cisplatin, doxorubicin and paclitaxel
(taxol).
[0057] Another class of active compounds which can be used in the
present invention are those which are able to sensitize for or
induce apoptosis by binding to death receptors ("death receptor
agonists"). Agonists of death receptors include death receptor
ligands such as tumor necrosis factor .alpha. (TNF-.alpha.), tumor
necrosis factor .beta. (TNF-.beta., lymphotoxin-.alpha.), LT-.beta.
(lymphotoxin-.beta.), TRAIL (Apo2L, DR4 ligand), CD95 (Fas, APO-1)
ligand, TRAMP (DR3, Apo-3) ligand, DR6 ligand as well as fragments
and derivatives of any of said ligands. Furthermore, death
receptors agonists comprise agonistic antibodies to death receptors
such as anti-CD95 antibody, anti-TRAIL-R1 (DR4) antibody,
anti-TRAIL-R2 (DR5) antibody, anti-DR6 antibody, anti TNF-R1 (p55
TNF-R) antibody and anti-TRAMP (DR3) antibody as well as fragments
and derivatives of any of said antibodies. Preferably, the
agonistic antibodies are selected from the group consisting of
anti-TRAIL-R1 antibody, anti-TRAIL-R2 antibody, anti TNF-R1
antibody and fragments and derivatives of any of said
antibodies.
[0058] Another class of active compounds which can be used in
combination with the cytokine antagonist are peptides, proteins or
small molecule inhibitors which negatively regulate or inhibit the
above described anti-apoptotic proteins. Examples of negatively
regulating peptides include Smac/DIABLO, NRAGE and TAK1, fragments
and derivatives thereof, which particularly inhibit the above
described IAPs. These peptides may be modified in a way that they
can be rapidly internalized into tumor cells by cellular uptake.
The modification can occur by attaching a carrier peptide that
mediates cellular uptake as disclosed above to the active
compound.
[0059] The cytokine antagonist can be administered alone or in
combination with one or more active compounds. The latter can be
administered before, after or simultaneously with the
administration of the cytokine antagonist. The dose of either the
cytokine antagonist or the active compound as well as the duration
and the temperature of incubation can be variable and depends on
the target that is to be treated.
[0060] A further object of the present invention are pharmaceutical
preparations which comprise an effective dose of at least one
cytokine antagonist, optionally in combination with at least one
active compound and a pharmaceutically acceptable carrier, i.e. one
or more pharmaceutically acceptable carrier substances and/or
additives.
[0061] The pharmaceutical according to the invention can be
administered orally, for example in the form of pills, tablets,
lacquered tablets, sugar-coated tablets, granules, hard and soft
gelatin capsules, aqueous, alcoholic or oily solutions, syrups,
emulsions or suspensions, or rectally, for example in the fruit of
suppositories. Administration can also be carried out parenterally,
for example subcutaneously, intramuscularly or intravenously in the
form of solutions for injection or infusion. Other suitable
administration forms are, for example, percutaneous or topical
administration, for example in the form of ointments, tinctures,
sprays or transdermal therapeutic systems, or the inhalative
administration in the form of nasal sprays or aerosol mixtures, or,
for example, microcapsules, implants or rods. The preferred
administration form depends, for example, on the disease to be
treated and on its severity.
[0062] The preparation of the pharmaceutical compositions can be
carried out in a manner known per se. To this end, the cytokine
antagonist and/or the active compound, together with one or more
solid or liquid pharmaceutical carrier substances and/or additives
(or auxiliary substances) and, if desired, in combination with
other pharmaceutically active compounds having therapeutic or
prophylactic action, are brought into a suitable administration
form or dosage form which can then be used as a pharmaceutical in
human or veterinary medicine.
[0063] For the production of pills, tablets, sugar-coated tablets
and hard gelatin capsules it is possible to use, for example,
lactose, starch, for example maize starch, or starch derivatives,
talc, stearic acid or its salts, etc. Carriers for soft gelatin
capsules and suppositories are, for example, fats, waxes, semisolid
and liquid polyols, natural or hardened oils, etc. Suitable
carriers for the preparation of solutions, for example of solutions
for injection, or of emulsions or syrups are, for example, water,
physiological sodium chloride solution, alcohols such as ethanol,
glycerol, polyols, sucrose, invert sugar, glucose, mannitol,
vegetable oils, etc. It is also possible to lyophilize the cytokine
antagonist and/or the active compound and to use the resulting
lyophilisates, for example, for preparing preparations for
injection or infusion. Suitable carriers for microcapsules,
implants or rods are, for example, copolymers of glycolic acid and
lactic acid.
[0064] The pharmaceutical preparations can also contain additives,
for example fillers, disintegrants, binders, lubricants, wetting
agents, stabilizers, emulsifiers, dispersants, preservatives,
sweeteners, colorants, flavorings, aromatizers, thickeners,
diluents, buffer substances, solvents, solubilizers, agents for
achieving a depot effect, salts for altering the osmotic pressure,
coating agents or antioxidants.
[0065] The dosage of the cytokine antagonist, in combination with
one or more active compounds to be administered, depends on the
individual case and is, as is customary, to be adapted to the
individual circumstances to achieve an optimum effect. Thus, it
depends on the nature and the severity of the disorder to be
treated, and also on the sex, age, weight and individual
responsiveness of the human or animal to be treated, on the
efficacy and duration of action of the compounds used, on whether
the therapy is acute or chronic or prophylactic, or on whether
other active compounds are administered in addition to the cytokine
antagonist.
[0066] The cytokine antagonists according to the present invention,
respectively the medicaments containing the latter, can be used for
the treatment of all cancer types which are resistant to apoptosis
due to the expression of anti-apoptotic proteins. Examples of such
cancer types comprise neuroblastoma, intestine carcinoma such as
rectum carcinoma, colon carcinoma, familiary adenomatous polyposis
carcinoma and hereditary non-polyposis colorectal cancer,
esophageal carcinoma, labial carcinoma, larynx carcinoma,
hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma,
gastric carcinoma, adenocarcinoma, medullary thyroid carcinoma,
papillary thyroid carcinoma, follicular thyroid carcinoma,
anaplastic thyroid carcinoma, renal carcinoma, kidney parenchym
carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus
carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic
carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma,
urinary carcinoma, melanoma, brain tumors such as glioblastoma,
astrocytoma, meningioma, medulloblastoma and peripheral
neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma,
Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic
leukemia (CLL), acute myeolid leukemia (AML), chronic myeloid
leukemia (CML), adult T-cell leukemia lymphoma, hepatocellular
carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell
lung carcinoma, non-small cell lung carcinoma, multiple myeloma,
basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma,
rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma,
myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and
plasmocytoma.
[0067] In a particularly preferred embodiment, the cytokine
antagonists according to the present invention, respectively the
medicaments containing the latter, can be used for the treatment of
non-lymphoid and non-myeloid, preferably epithelial cancers.
[0068] Examples of cancer types where the use of the cytokine
antagonists according to the present invention, respectively the
medicaments containing the latter, is particularly advantageous
include all forms of thyroid carcinomas (medullary thyroid
carcinoma, papillary thyroid carcinoma, follicular thyroid
carcinoma, anaplastic thyroid carcinoma), breast carcinoma, lung
carcinoma, prostate carcinoma and colon carcinoma. Most preferably,
the cytokine antagonists are useful for the treatment of thyroid
carcinomas.
[0069] The cytokine antagonists according to the present invention,
respectively the medicaments containing the latter, can also be
used for the treatment of all autoimmune diseases which are
resistant to apoptosis due to the expression of anti-apoptotic
proteins. Examples of such autoimmune diseases are collagen
diseases such as rheumatoid arthritis, Lupus erythematodes
disseminatus, Sharp syndrome, CREST syndrome (calcinosis, Raynaud
syndrome, esophageal dysmotility, teleangiectasia),
dermatomyositis, vasculitis (Morbus Wegener) and Sjogren syndrome,
renal diseases such as Goodpasture syndrome, rapidly-progressing
glomerulonephritis and membrane-proliferative glomerulonephritis
type II, endocrine diseases such as type-I diabetes, autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
autoimmune parathyreoidism, pernicious anemia, gonad insufficiency,
idiopathic Morbus Addison, hyperthyreosis, Hashimoto thyroiditis
and primary myxedemia, skin diseases such as Pemphigus vulgaris,
bullous pemphigoid, Herpes gestationis, Epidermolysis bullosa and
Erythema multiforme major, liver diseases such as primary biliary
cirrhosis, autoimmune cholangitis, autoimmune hepatitis type-1,
autoimmune hepatitis type-2, primary sclerosing cholangitis,
neuronal diseases such as multiple sclerosis, Myastenia gravis,
myasthenic Lambert-Eaton syndrome, acquired neuromyotony,
Guillain-Barre syndrome (Muller-Fischer syndrome), Stiff-man
syndrome, cerebellar degeneration, ataxia, opsoklonus, sensoric
neuropathy and achalasia, blood diseases such as autoimmune
hemolytic anemia, idiopathic thrombocytopenic purpura (Morbus
Werlhof), infectious diseases with associated autoimmune reactions
such as AIDS, Malaria and Chagas disease.
[0070] A further object of the present invention is the use of the
cytokine antagonist hybridizing with or binding to the cytokine, or
the nucleic acid encoding it, as a diagnostic tool to detect and
quantify the expression level of a cytokine present in the
drug-resistant tumor cell. It is also possible to detect and
quantify the expression level of a cytokine and thus, the
susceptibility for cancer, by analyzing any of a potential
patient's body fluid, such as serous effusions (blood), semen,
vaginal secretions, saliva, cerebrospinal fluid, pleural and
pericardial fluid, peritoneal fluid, synovial fluid and amniotic
fluid.
[0071] Thus, the present invention refers to the use of a cytokine
antagonist for diagnosing and monitoring the cancer disease of a
subject, comprising [0072] (a) providing a body fluid sample or a
sample of tissue or cells from a non-lymphoid and/or non-myeloid
tumor of a subject [0073] (b) contacting the sample with a labeled
probe that binds to a cytokine nucleic acid and/or with an antibody
that binds to a cytokine [0074] (c) determining the expression
level of the cytokine in the tissue or cells and comparing the
expression level with healthy control cells, and [0075] (d)
correlating a better prognosis for the subject with a low ratio of
cytokine expression when compared to the expression level in
healthy control cells.
[0076] The cytokine antagonist may therefore be useful to predict
whether a patient suffering from a certain cancer type would be
susceptible to a certain therapy and whether it would be required
to change the treatment strategies. Binding and hybridization
assays can be used to detect, prognose, diagnose, or monitor
disease (including conditions and disorders) associated with the
overexpression of the cytokines in tumor cells or body fluids. This
requires the detection of nucleic acids that encode the cytokines,
and the detection of the cytokine proteins.
[0077] Cytokine nucleic acids are detected and quantified herein by
any of a number of means well known to those of skill in the art.
Appropriate detection methods include biochemical methods such as
spectrophotometry, radiography, gel electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography, and
the like, and various immunological methods such as fluid or gel
precipitation reactions, immunodiffusion, immunoelectrophoresis,
radioimmunoassays (RIAs), enzyme-linked immunosorbent assays
(ELISA), immunofluorescence assays, tissue array, and the like.
[0078] Hybridization techniques are frequently used for detecting
nucleic acids and the present invention contemplates all available
hybridization techniques, including Southern, Northern and in situ
hybridization techniques, dot blot analysis, cDNA arrays.
Expression of cytokine mRNAs may be detected, for example, by
Northern analysis, or by reverse transcription and amplification by
PCR. Also contemplated are nucleic acid detection and
quantification methods which employ signal moieties that are
conjugated to nucleic acid probes, e.g. by incorporation of
radioactively labeled nucleotides. Nucleic acids in a sample can be
immobilized on a solid support and hybridized to such probes. The
signal moiety can be detected directly, for example by
fluorescence. Alternatively, the signal moiety may be detected
indirectly by its enzymatic activity, for example in an ELISA or
other colorimetric assay.
[0079] Hybridization techniques are usually performed by providing
a sample of tissue or cells, contacting the sample with a labeled
probe, that binds to said nucleic acid molecule, and determining
the presence or amount of the probe bound to said nucleic acid
molecule, thereby determining the presence or amount of the nucleic
acid molecule in said sample.
[0080] Methods to quantify the presence and amount of a cytokine
protein in a given sample are well known to the person skilled in
the art. Briefly, a sample is provided, said sample is contacted
with an antibody that immunospecifically binds to a given cytokine
and the presence or amount of antibody bound to said cytokine is
determined, whereby the presence or amount of cytokines in said
sample is determined. Methods to determine the amount and presence
of polypeptides comprise, among others, FACS, Western blotting,
immunoprecipitation, ELISA, and RIA. It is advantageous if the
antibody used for detection is conjugated to a molecule that
enables and contributes to the detection. Suitable molecules
comprise biotin, horseradish peroxidase, alkaline phosphatase,
fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), diamidinophenylindol (DAPI) and
phycoerythrin.
[0081] Thus, the present invention finally embodies a diagnostic
kit containing at least one cytokine antagonist being a nucleic
acid or a peptide/protein, optionally in combination with suitable
buffers, enzymes and other compounds facilitating the detection and
quantification of the cytokine in a drug-resistant tumor cell or in
a body fluid such as serous effusions (blood), semen, vaginal
secretions, saliva, cerebrospinal fluid, pleural and pericardial
fluid, peritoneal fluid, synovial fluid and amniotic fluid.
[0082] The invention is further illustrated in the following
examples:
EXAMPLES
Example 1
Thyroid Cancer Cells are Resistant to Chemotherapy-Induced Cell
Death
[0083] Although clinical trials with single agents or with
combinations of chemotherapeutic drugs have produced rare and
limited positive response, without increase in median and mean
survival time in comparison with the natural history of the
disease, some compounds have shown a few beneficial effects in
terms of partial response rates and reduction of metastatic tumor
expansion.
[0084] To investigate the sensitivity of the different histological
variants of thyroid epithelial carcinomas to the conventional
chemotherapeutic drugs, the viability of freshly purified normal
and neoplastic thyrocytes exposed to cisplatin (300 ng/ml),
doxorubicin (5 .mu.M) and taxol (5 .mu.M) was measured, using
dosages compatible with the in vivo levels observed during cancer
treatment. In line with the modest clinical efficacy reported in
clinical trials, primary neoplastic cells derived from all the
histological variants of thyroid epithelial carcinomas showed a
considerable resistance to chemotherapeutic drugs as compared with
normal thyrocytes (FIG. 1). Such resistance persisted for some days
and was generally lost after eight to ten days of in vitro culture
(results not shown).
Example 2
Thyroid Cancer Cells Express Bcl-2 and Bcl-x.sub.L
[0085] Refractoriness to chemotherapy of thyroid carcinoma cells
may result from the inhibitory action of anti-apoptotic genes.
Therefore, the expression of relevant anti-apoptotic proteins,
potentially able to protect thyroid cancer cells from the cytotoxic
activity of chemotherapeutic drugs was evaluated.
Immunohistochemical analysis of PTC, FTC and UTC paraffin embedded
sections showed that Bcl-2 and Bcl-x.sub.L were considerably
upregulated in thyroid carcinoma cells (FIGS. 2a and b). To
determine more accurately the difference between normal and
malignant thyrocytes, freshly purified control and neoplastic
thyroid cells were lysed and analyzed by immunoblot. As shown in
FIG. 2c and d, Bcl-x.sub.L was weakly expressed in normal cells and
four to five fold upregulated in all the histological cancer
variants, while Bcl-2 was found about threefold higher in FTC cells
and twofold higher in PTC and UTC cells, as compared with normal
thyrocytes. Hearts from Bcl-x.sub.L, and Bcl-2 transgenic mice were
used as positive control. The ability of Bcl-x.sub.L and Bcl-2
overexpression to protect some cell types against the cytotoxic
effect of chemotherapeutic drugs suggests a potential role of these
anti-apoptotic proteins in thyroid cancer resistance from
drug-induced cytotoxicity.
Example 3
Exogenous Bcl-2 and Bcl-x.sub.L Protect Thyrocytes from Cell Death
Induced by Chemotherapeutic Agents
[0086] To prove that Bcl-x.sub.L and Bcl-2 up-regulation protect
thyrocytes from apoptosis induced by chemotherapeutic drugs and may
be responsible for thyroid cancer cell survival, normal thyrocytes
were transduced with a retroviral vector (PINCO) that carried the
green fluorescent protein (GFP) as a reporter gene. After
infection, thyrocytes transduced with empty vector, Bcl-x.sub.L and
Bcl-2 were sorted by flow cytometry and exposed to cisplatinum,
doxorubicin and taxol to evaluate the extent of
chemotherapy-induced apoptosis. The infections were monitored by
immunoblot analysis to confirm the efficiency of gene delivery
(FIG. 3a). Thyrocytes transduced with either Bcl-x.sub.L or Bcl-2
were almost completely protected from the cytotoxic effects of
chemotherapeutic agents (FIG. 3b and c), indicating that
overexpression of any of the two genes was sufficient to prevent
thyroid cancer cell destruction. Thus, Bcl-x.sub.L and Bcl-2
represent likely candidates for mediating refractoriness of thyroid
cancer cells to chemotherapy.
Example 4
Autocrine Production of IL-4 and IL-10 in Thyroid Cancer Cells
[0087] To investigate whether the tumor microenvironment can
influence thyroid cancer cell phenotype and function, the presence
of those cytokines previously found to modulate thyrocyte
susceptibility to apoptosis was next evaluated. The presence of Th1
and Th2 cytokines in the neoplastic thyroid gland was investigated
by immunohistochemistry on paraffin embedded sections of thyroid
carcinomas and by immunocytochemistry and immunoblot analysis on
freshly isolated thyroid carcinoma cells. All the histological
variants analyzed by immunohistochemistry, exhibited a high
reactivity for IL-4 and IL-10, as compared with normal tissues,
while IFN-.gamma. was barely detectable (FIG. 4a). Interestingly,
the reactivity against Th2 cytokines localized in thyroid
follicles, suggesting that neoplastic thyroid cells were the source
of production for both IL-4 and IL-10 (FIG. 4a). To rule out the
possibility that these cytokines were released by infiltrating T
cells, freshly purified thyroid cancer cells were analyzed by
immunocytochemistry and immunoblot for expression of Th1 and Th2
cytokines. As observed in the immunohistochemistry experiments,
purified thyroid cancer cells showed intense reactivity for both
IL-4 and IL-10, while no expression of IFN-.gamma. was detectable
(FIG. 4b and c). Twenty nanograms of recombinant human IL-4, IL-10
and IFN-.gamma. were used as positive controls for the immunoblot
analysis. The comparison between positive controls and cancer
samples indicated that malignant thyroid cells produce considerable
amounts of those Th2 cytokines that have shown anti-apoptotic
activity on thyroid follicular cells.
Example 5
IL-4 and IL-10 Protect Thyrocytes from Cell Death Induced by
Chemotherapeutic Agents
[0088] It was next investigated whether IL-4 and IL-10 can modulate
the sensitivity to chemotherapy-induced apoptosis and the
expression of anti-apoptotic proteins in thyroid cells.
Interestingly, both IL-4 and IL-10 drastically prevented death of
normal thyrocytes exposed to cisplatinum, doxorubicin and taxol
(FIG. 5a), suggesting that autocrine production of these cytokines
in thyroid cancer cells is responsible for refractoriness to
chemotherapy. Furthermore, both IL-4 and IL-10 upregulated
Bcl-x.sub.L, and Bcl-2 after 48 hours of culture (FIG. 5b), while
IFN-.gamma. was not effective. Thus, it is likely that increased
expression of anti-apoptotic proteins and subsequent protection of
tumor cells from chemotherapy are mediated by the autocrine release
of IL-4 and IL-10.
Example 6
Blocking Autocrine IL-4 and IL-10 Activity Primes Thyroid Cancer
Cell for Chemotherapy-Mediated Destruction
[0089] To test whether autocrine IL-4 and/or IL-10 release by
thyroid tumors is responsible for upregulation of anti-apoptotic
proteins, tumor cells were treated for two days with neutralizing
Abs specific for IL-4 and/or IL-10 and measured Bcl-x.sub.L and
Bcl-2 expression. As shown in FIG. 6a, the levels of both proteins
dramatically decreased in thyroid tumor cells exposed to
neutralizing Abs against IL-4 and IL-10, while the blockade of a
single cytokine had a very limited effect. To test whether
cytokine-mediated increase in Bcl-x.sub.L and Bcl-2 levels was
responsible for thyroid tumor cell resistance to chemotherapy, PTC,
FTC and UTC cells were treated for two days with neutralizing
anti-IL-4 and anti-IL10 Abs and analyzed for viability and
sensitivity to Chemotherapeutic drugs. A significant percentage of
thyroid tumor cells from all the histological variants underwent
spontaneous apoptosis after 48-hour exposure to anti-IL-4 and
anti-IL-10 Abs (FIG. 6a), indicating that these cytokines indeed
act as survival factors for thyroid cancer cells. Moreover, these
cells acquired sensitivity to chemotherapy-induced cytotoxicity and
showed massive death after 24-hour treatment with cisplatinum,
doxorubicin or taxol (FIG. 6b). Thus, neutralization of IL-4 and
IL-10 released by thyroid cancer cells allows their destruction
through the use of chemotherapeutic drugs.
Example 7
Down-Regulation of Anti-Apoptotic Proteins Sensitizes Cells to
TRAIL-Induced Cell Death
[0090] To determine the potential of TRAIL-mediated apoptosis in
vivo TRAIL-Receptor (TR) expression in normal and thyroid carcinoma
cells was documented. To determine the presence of TRAIL-R1,
TRAIL-R2, TRAIL-R3 and TRAIL-R4 immunohistochemical stainings of
paraffin embedded thyroid tissue sections from patients affected by
PTC, FTC and UTC were performed and compared with sections from
normal thyroid lobes contralateral to the cancerous lobe in
patients with thyroid cancer. It was found that TRAIL-R1-TR4 were
strongly expressed in all the papillary tumors analysed and
completely absent in follicular and anaplastic tumors (data not
shown). To test whether autocrine IL-4 and IL-10 release is
responsible for TRAIL-induced apoptosis resistance in all the
histological thyroid cancer variants examined carcinoma cells were
treated for two days with IL-4- and IL-10-neutralizing antibodies
and then tumor cell resistance to TRAIL-induced apoptosis was
measured. A significant percentage of tumor cells was apoptotic
after 48 hours' exposure to anti-IL-4 and anti-IL-10 Abs,
indicating that these cytokines act as survival factors for these
cells (data not shown). Thus, downregulation of anti-apoptotic
proteins such as FLIP, Bcl-x.sub.L and Bcl-2, through the
inhibition of Th2 cytokines, sensitizes these cells to
TRAIL-induced cell death.
Example 8
IL-4 Inhibits Chemotherapy- and CD95-Induced Apoptosis in Bladder
Cancer Cells
[0091] A decreased Th1/Th2 ratio has been observed in a wide
variety of human cancers, where it has been proposed to correlate
with the stage and grade of malignancy. IL-4 released from
tumor-associated Th2 lymphocytes has been shown to promote the
growth of pulmonary metastatic cancer in mice, suggesting a
specific role of this cytokine in influencing tumor cell survival.
Therefore, the ability of IL-4 to modulate apoptosis sensitivity of
cells derived from different human solid tumors was
investigated.
[0092] Anticancer treatment using cytotoxic drugs mediates cell
death by activating the intracellular apoptotic program. To
determine whether IL-4 was able to inhibit apoptosis induced by
chemotherapeutic drugs, bladder tumor cells (RT112) were pretreated
for two days with different concentrations of IL-4 and subsequently
exposed to camptothecin or etoposide. While a considerable number
(.about.60%) of tumor cells underwent apoptosis in response to
chemotherapeutic agents, cells preincubated with IL-4 were
significantly protected from drug-induced death (FIG. 7A),
suggesting that IL-4 interferes with the apoptotic program
activated by anticancer agents in tumor cells. The protective
effect of IL-4 was already consistent after 24 hours of
pretreatment and remained stable from the second day of treatment
unless IL-4 was removed from the culture medium (FIG. 7B and data
not shown).
[0093] The observation that tumor cells treated with IL-4 display a
reduced sensitivity to chemotherapeutic drugs led us to investigate
whether this cytokine would influence the expression of apoptosis
regulatory genes. Western blot analysis of apoptosis-related
proteins was performed on cancer cell lines treated for 48 hours
with IL-4 as compared to untreated or IL-2-treated controls. Among
the proteins examined, it was found that levels of Bcl-X.sub.L and
cFlip/FLAME-1 were increased in tumor cells treated with IL-4,
while levels of caspases and other pro- and anti-apoptotic Bcl-2
family members remained unchanged (FIG. 7C-D and data not shown).
The increased expression of cFlip/FLAME-1 prompted us to
investigate if IL-4 could protect tumor cells from death receptor
stimulation. CD95 ligand (CD95L) is one of the major effector
molecules of cytotoxic T lymphocytes and NK cells. The CD95 pathway
has been demonstrated to be involved in tumor clearance in vivo.
CD95 mutation or downmodulation has been found in several tumors,
and the development of resistance to CD95-induced cell death has
been suggested to contribute to immune evasion of malignant cells.
As shown in FIG. 7E, IL-4 treatment was able to significantly
inhibit CD95-induced apoptosis in bladder cancer cells, suggesting
that IL-4 can negatively affect the immune response against
tumors.
Example 9
IL-4 Inhibits Chemotherapy- and CD95-Induced Apoptosis in Prostate
and Breast Cancer Cells
[0094] It was hypothesized that the ability of IL-4 to protect
bladder cancer cells from chemotherapy and anti-CD95-induced
apoptosis could be also displayed by other tumors characterized by
the presence of IL-4, such as prostate and breast cancer.
Therefore, tumor cell lines derived from prostate (LNCaP) and
breast (MDA-MB-231) carcinomas were pretreated for two days with
IL-4 and subsequently exposed to those antineoplastic agents that
showed the highest cytotoxic activity towards each cell line.
Whilst a considerable percentage of prostate and breast tumor cells
cultured in medium alone or pretreated with IL-2 underwent
apoptosis in response to chemotherapeutic agents, cells
preincubated with IL-4 were significantly protected from
drug-induced death (FIG. 8A-B). Similarly, IL-4 greatly reduced
anti-CD95-induced apoptosis (FIG. 8C), suggesting that the presence
of IL-4 in the tumor infiltrate may protect cancer cells from
cytotoxic therapy and immune response. Conversely, pretreatment of
cells with IL-2 did not exert any protective effect, demonstrating
the specificity of IL-4-mediated signals in the inhibition of
apoptosis initiated by CD95 in cancer cells. Notably, cells
pretreated with IL-4 not only displayed an increased survival to
cytotoxic drugs and CD95 stimulation, but regained full
proliferative activity when the cytotoxic stimulus was removed
(data not shown). Therefore, it appears that IL-4 is able to exert
a significant effect on survival and growth of tumor cells,
including a faster expansion of cells that survive chemotherapy
treatment.
Example 10
IL-4 Upregulates the Expression of Anti-Apoptotic Proteins in
Prostate and Breast Cancer Cells
[0095] The observation that prostate and breast tumor cells treated
with IL-4 display a reduced sensitivity to death induced by CD95
and chemotherapeutic drugs led us to investigate whether this
cytokine would influence the expression of apoptosis regulatory
genes. As for bladder tumor cells, it was found that levels of
Bcl-X.sub.L and cFlip/FLAME-1 were considerably upregulated
following IL-4 treatment in LNCap prostate cancer cells (FIG. 9A),
thus providing a possible explanation for IL-4-mediated protection
from apoptotic events initiated by chemotherapy and CD95 receptor.
Differently, only a modest increase in cFlip/FLAME-1 was not
observed in MDA-MB-231 cells, where Bcl-X.sub.L likely represents
the primary effector of IL-4-mediated protection from apoptosis
(FIG. 9B). Therefore, IL-4-mediated increased survival in bladder,
prostate and breast cancer cells is associated with upregulation of
anti-apoptotic proteins.
Example 11
Exogenous Expression of Bcl-x.sub.L or cFlip/FLAME-1 Protects Tumor
Cells from Drug-Induced Apoptosis
[0096] Anti-apoptotic Bcl-2 family members regulate the release of
cytochrome c from mitochondria and have been implicated in
apoptosis modulation of tumor cells exposed to chemotherapeutic
drugs. Increased expression of cFlip/FLAME-1 has been reported to
inhibit CD95-dependent apoptosis of malignant cells, thus resulting
in tumor escape from T cell immunity in vivo. To determine whether
increased expression of these two anti-apoptotic effectors was able
to protect tumor cells from apoptosis induced by chemotherapeutic
drugs, RT112, MDA-MB-231 and LNCaP cells were transduced with a
retroviral vector containing the cDNA for Bcl-X.sub.L or
cFlip/FLAME-1 and the GFP as a reporter gene. Retroviral infection
yielded 100% GFP-positive cell populations, which were then
analyzed for expression of the transduced genes. By titrating viral
supernatant and modulating the number of infection cycles, it was
possible to obtain levels of Bcl-X.sub.L and cFlip/FLAME-1 in
transduced cells comparable to those of IL-4-treated cells, as
shown by immunoblot analysis and densitometry quantification (FIG.
9C-E). Cells were then exposed to chemotherapeutic drugs, and
protection from apoptosis exerted by exogenous Bcl-X.sub.L or
cFlip/FLAME-1 was evaluated in relation to non-transduced cells
pretreated with IL-4 or with the control cytokine IL-2. Whereas
control cells were efficiently killed by cytotoxic drugs, exogenous
expression of Bcl-X.sub.L protected tumor cells from drug-induced
apoptosis at levels similar to those produced by IL-4 (FIG. 10). A
partial inhibition of drug-induced death was also observed in cells
overexpressing cFlip/FLAME-1 (FIG. 10), possibly reflecting the
ability of cFlip/FLAME-1 to activate anti-apoptotic signals through
NF-kB or alternatively indicating the involvement of death
receptor-mediated events in chemotherapy-induced apoptosis.
Example 12
Exogenous Expression of Bcl-X.sub.L or cFlip/FLAME-1 Protects Tumor
Cells from CD95-Induced Apoptosis
[0097] Bcl-X.sub.L and cFlip/FLAME-1 have both been demonstrated to
interfere with apoptotic signals initiated at the CD95 receptor,
although with different mechanisms. cFlip/FLAME-1 inactivates the
CD95 DISC by blocking the activation of caspase-8, whereas
overexpression of Bcl-X.sub.L, inhibits the mitochondrial apoptotic
pathways, which contribute to the execution phase of death receptor
signaling. To determine the capacity of Bcl-X.sub.L and
cFlip/FLAME-1 to inhibit CD95-induced apoptosis of tumor cells,
RT112, MDA-MB-231 and LNCaP cells stably overexpressing
Bcl-X.sub.L, and RT112 and LNCaP cells overexpressing cFlip/FLAME-1
were stimulated with anti-CD95 agonistic antibodies. Bcl-X.sub.L
was able to induce a significant protection of the three cell lines
from CD95-mediated death, indicating an important role of
mitochondrial events in CD95-mediated apoptosis of these cells
(FIG. 10A-C), while cFlip/FLAME-1 efficiently protected LNCaP and
RT112 cells from CD95-induced apoptosis at levels comparable to
those obtained with IL-4 treatment (FIGS. 10A and B). These results
support the hypothesis that the upregulation of Bcl-X.sub.L and
cFlip/FLAME-1 induced by IL-4 may contribute to an increased
resistance of tumors to apoptosis mediated by chemotherapeutic
drugs and by immune system effectors.
Example 13
In Vivo Production of IL-4 is Associated with Upregulation of
Bcl-X.sub.L and cFlip/FLAME-1 in Bladder and Prostate Cancer
[0098] To determine the role of IL-4 in tumor cell protection in
vivo, bladder, prostate and breast cancer specimens were analyzed
by immunohistochemistry in order to detect the presence of IL-4 and
the expression of IL-4-induced anti-apoptotic proteins. In line
with literature data, normal and neoplastic tissues consistently
showed the presence of IL-4 in all the different types of cancer
examined, while normal tissues were essentially negative (FIG.
12A-C). Serial section analysis indicated that IL-4 reactivity was
associated with the presence of a CD45.sup.+ immune infiltrate,
which likely represents the major source of IL-4 production in the
different tumors (FIG. 12A-C). Analysis of apoptosis-related
proteins showed that bladder, prostate and breast tumors display
high reactivity for both Bcl-X.sub.L and cFlip/FLAME-1, whose
expression in normal tissues was extremely low or undetectable
(FIG. 13A). As expected, the three types of tumor expressed the
IL-4 receptor, which was also present in normal tissues (FIG. 13A).
Moreover, the levels of Bcl-X.sub.L and cFlip/FLAME-1 observed in
vivo were compared with those of cells lines whose exogenous
expression of either Bcl-X.sub.L or cFlip/FLAME-1 conferred
protection from chemotherapeutic drugs and anti-CD95. The
expression of both Bcl-X.sub.L and cFlip/FLAME-1 was similar or
even higher in bladder and prostate cancer as compared with the
corresponding cell lines carrying the exogenous gene (FIGS. 13B and
C), while the in vivo expression of Bcl-X.sub.L in breast cancer
was slightly lower than that found in transduced MDA-MB-231 (FIG.
13D). Thus, in vivo production of IL-4 in bladder and prostate
cancer is associated with high expression of Bcl-X.sub.L and
cFlip/FLAME-1, which protect cancer cells from chemotherapy and
CD95-mediated apoptosis.
Example 14
In Vivo Production of IL-4 Protects Breast Cancer Cells from
Chemotherapy
[0099] To confirm that IL-4-induced upregulation of Bcl-X.sub.L is
able to protect epithelial breast cells from apoptosis, freshly
isolated normal breast epithelial cells were exposed to IL-4 and
analyzed Bcl-X.sub.L expression and sensitivity to a panel of
chemotherapeutic drugs commonly used to treat breast cancer. IL-4
treatment of normal breast epithelial cells considerably increased
the expression of Bcl-X.sub.L, which reached levels slightly lower
than those observed in freshly isolated neoplastic cells (FIG.
14A). Accordingly, IL-4 significantly protected normal breast
epithelial cells from apoptosis induced by cisplatin, doxorubicin
and taxol (FIG. 14B). To confirm that in vivo exposure to IL-4 is
responsible for high Bcl-X.sub.L expression and resistance to
chemotherapy in breast cancer, Bcl-X.sub.L expression and
sensitivity to apoptosis of primary breast carcinoma cells
cultivated in the presence or absence of IL-4 was measured. Freshly
isolated breast cancer cells expressed high levels of Bcl-X.sub.L
and were scarcely sensitive to chemotherapeutic drugs (FIGS. 14C
and D). However, after 6 days of in vitro culture in the absence of
IL-4, breast cancer cells downregulated Bcl-X.sub.L and became
sensitive to chemotherapy-induced apoptosis, while in the presence
of IL-4 they maintained high Bcl-X.sub.L levels and low sensitivity
to chemotherapeutic drugs (FIGS. 14C and D). Thus, in vivo IL-4
production promotes the survival of breast cancer cells.
Materials and Methods (Referring to Examples 1 to 7 and FIGS. 1 to
6)
[0100] Specimens. Thyroid tissues affected by eight PTC (aged
28.+-.5), eight FTC (aged 44.+-.3) and four UTC (aged 65.+-.4.5),
were obtained at the time of thyroidectomy. Normal thyroid
specimens were obtained from the uninvolved, controlateral lobes of
thyroid glands with tumours. Histological diagnosis was based on
the identification of papillary elements, on the behavioural
characteristics of carcinoma cells (vascular and capsular invasion)
and nuclear atypia (shape and chromatin pattern). Transgenic mouse
hearts expressing human Bcl-2 and Bcl-X.sub.L, provided by G. L.
Condorelli (Thomas Jefferson University, Philadelphia, Pa.), were
used as positive controls.
[0101] Thyroid cell purification and culture. Thyroid tissues from
normal, PTC, FTC and UTC were digested for 2 hours with collagenase
(1.5 mg/ml) (Gibco BRL, Grand Island, N.Y.) and hyaluronidase (20
.mu.g/ml) (Sigma Chemical Co., St. Louis, Mo.) in DMEM. Thyrocytes
were purified from the digested tissues by hematopoietic cell
depletion with anti-CD45-coupled beads (Dynal, Wirral Merseyside,
U.K.) and 12 hours of flask adherence, which allowed removal of
other cells. After additional 12 hours of culture, thyroid cells
were allowed to grow in monolayer for the immunocytochemistry or
detached with trypsin+EDTA following exposure to cytokines or
chemotherapeutic agents for functional and protein analyses.
Thyrocytes were cultured in standard DMEM with 10% heat-inactivated
FBS (Hyclone Laboratories, Logan, UK) in the presence or absence of
human recombinant IL-4 (20 ng/ml), IL-10 (40 ng/ml) or IFN-.gamma.
(1000 IU/ml) (Euroclone, Paignton, UK) and cisplatin (300 ng/ml),
doxorubicin (5 .mu.M) and taxol (5 .mu.M) (Sigma) or TRAIL (Alexis,
San Diego, USA). For the IL-4 and IL-10 neutralization, thyroid
cancer cells were pretreated, for 48 hours, with anti-human IL-4
and IL-10 neutralizing antibodies (1 .mu.g/ml) (R&D systems,
MN, USA).
[0102] Cell death quantitation. Apoptotic events of neoplastic
thyrocytes were evaluated by DNA staining and flow cytometry
analysis. Thyroid cell pellets were resuspended in hypotonic
fluorochrome solution containing propidium iodide (50 .mu.g/ml), in
0.1% sodium citrate and 0.1% Triton X-100. The percentage of
hypodiploid nuclei was evaluated as previously described.
Alternatively, freshly purified thyrocytes were plated in
96-bottomed plates in triplicate at 15,000 cells/well and cultured.
The number of viable cells was detected by CellTiter Aqueous Assay
Kit (Promega Corporation, WI, USA) adding 20 .mu.l of solution
reagent directly to culture wells, incubating for 1 hours at
37.degree. C. and recording absorbance at 490 nm.
[0103] Immunostaining procedure. Immunohistochemical stainings were
performed on paraffin embedded thyroid sections 5 .mu.m in
thickness. Deparaffinised sections were pre-treated with 3%
hydrogen peroxide fro 10 min at room temperature to inhibit
endogenous peroxidase. Then slides were incubated for 10 min with
Tris Bufferd Saline (TBS) containing 3% bovine serum albumin (BSA)
to block the unspecific staining. Following elimination of excess
serum, sections were exposed for 1 hour to specific antibodies
against Bcl-x.sub.L (H-5, mouse IgG.sub.1, Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.), Bcl-2 (124, mouse IgG1,
Dako) IL-4 (B-S4 mouse IgG1, Caltag Laboratories, Burlingame,
Calif.), IL-10 (B-N10 mouse IgG2a, Caltag), IFN-.gamma. (B27, mouse
IgG1, Caltag), TRAIL-R1 to R4 (Alexis, San Diego, USA) or isotype
matched controls at appropriate dilutions. Prior to immunostaining
for Bcl-2 and Bcl-xl, dewaxed sections were treated for 10 min in
microwave oven in 0.1 M citrate buffer. After two washes in TBS,
sections were treated with biotinylated anti-rabbit or anti-mouse
immunoglobulins, washed in TBS and incubated with streptavidin
peroxidase (Dako LSAB 2 Kit, Dako Corporation Carpinteria Calif.,
USA). Staining was detected using 3-amino-9-ethylcarbazole (AEC) as
a colorimetric substrate. Counterstaining of tissue sections was
performed using aqueous hematoxylin.
[0104] Protein isolation and Western Blotting. Cell pellets were
resuspended in ice-cold NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5,
150 mM NaCl, 1 mM EGTA, 1% NP-40) containing 1 mM PMSF, leupeptin
(1 .mu.g/ml), pepstatin (1 .mu.g/ml) and aprotinin (1 .mu.g/ml).
Each lysate (30 .mu.g) was fractioned on 12% SDS-polyacrylamide
gels and blotted to nitrocellulose (Hybond, Amersham, Little
Chalfont Buckinghamshire England, UK). Membrane was blocked for 1 h
with nonfat dry milk in TBS containing 0.05% Tween 20 and
successively incubated for 2 h with Abs specific to actin (Ab-1,
mouse IgM, Calbiochem, Darmstadt, Germany), Bcl-2 (124, mouse IgG1,
Upstate Biotechnology Inc.), Bcl-x.sub.L (H-5, mouse IgG1, Santa
Cruz Biotechnology), IL-4 (3007.11, mouse IgG1, R&D Systems,
Inc., Minneapolis, USA), IL-10 (23738.111, mouse IgG2b, R&D
Systems), IFN-.gamma. (25718.111, mouse IgG2a, R&D Systems).
After washing, the blots were incubated for 1 hour with
HRP-conjugated anti-mouse Abs (Amersham) and visualized using an
enhanced chemiluminescence detection system (SuperSignal West Dura
Extended duration Sustrate, Pierce, Ill., USA). rhIL-4, rhIL-10 and
rHIFN-.gamma. (Euroclone) were used as positive control.
[0105] Production of retroviral particles and infection of
thyrocytes. Bcl-2 and Bcl-x.sub.L cDNAs were cloned in PINCO
vector. The amphotropic Phoenix packaging cell line was transiently
transfected with PINCO using the calcium-phosphate/chloroquine
method. Infection was performed by culturing 5.times.10.sup.5
thyrocytes in 1 ml of 0.45 mM filtered supernatant containing viral
particles. Then, cells were centrifuged for 45 min at 1800 rpm and
placed back in the CO.sub.2 incubator for 2 hours. Three infection
cycles were performed before the thyrocytes were placed back in
supplemented medium. Sorted and enriched positive cells were plated
and exposed to cisplatinum, doxorubicin and taxol for evaluation of
cell death.
Materials and Methods (Referring to Examples 8 to 14 and FIGS. 7 to
14)
Cell Culture and Reagents
[0106] Human LNCaP prostate cancer cell line and human MDA-MB-231
breast cancer cell line were obtained from the American Type
Culture Collection (ATCC, Manassas, Va.). Human RT112 bladder
carcinoma cell line was kindly provided by Dr. M. Cippitelli
(Regina Elena Cancer Institute, Rome, Italy). Cells were grown in
RPMI 1640 (Life Technologies Inc., Grand Island, N.Y.) containing
10% heat inactivated fetal bovine serum supplemented with
L-Glutamine 2 mM and 100 units/ml Penicillin-Streptomycin. Cells
were kept in 5% CO.sub.2 atmosphere and routinely passaged when
80-85% confluent. Human recombinant IL-2 and IL-4 were purchased
from PeproTech Inc. (Rocky Hill, N.J.). Camptothecin, cisplatin,
daunorubicine, etoposide and vincristine were purchased from
Sigma-Aldrich Inc. (Sain Louis, Mo.) and resuspended in DMSO
(cisplatin, camptothecin and etoposide) or in H.sub.2O
(daunorubicine and vincristine). Anti-CD95 agonistic antibody
(clone CH11) was purchased from Upstate Biotechnology (Lake Placid,
N.Y.). Monoclonal antibody anti-Bcl-X.sub.L (H5, mouse) was from
Santa Cruz (Santa Cruz, Calif.), antibody anti-cFlip/FLAME-1 (NF6,
mouse) was kindly provided by Dr. P. H. Krammer (German Cancer
Reseach Center, Heidelberg, Germany). Antibody anti .beta.-actin
(goat) was purchased from Oncogene (Boston, Mass.):
[0107] Normal and cancer tissues from breast, prostate and bladder
specimens were digested for 3 hours with collagenase (1,8 mg/ml)
(Gibco BRL, Grand Island, N.Y.) and hyaluronidase (25 .mu.g/ml)
(Sigma Chemical Co., St. Louis, Mo.) in DMEM. After 12 hours of
culture, normal and cancer cells were detached with trypsin+EDTA
following exposure to cytokines or chemotherapeutic agents for
functional and protein analyses. Cells were cultured in standard
DMEM with 10% heat-inactivated FBS (Hyclone Laboratories, Logan,
UK) in the presence or absence of human recombinant cytokines.
Cell Viability Assay
[0108] Cell viability was determined using the CellTiter 96.RTM.
AQ.sub.ueous One Solution Cell Proliferation Assay (Promega,
Madison, Wis.), according to the manufacturer's instructions. The
assay is based on reduction of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
-2H-tetrazolium, inner salt (MTS) to a colored formazan product
that is measured spectrophotometrically. Cells were seeded in
96-well tissue culture plates and incubated at 37.degree. C. in a
5% CO.sub.2 incubator overnight. The next day IL-2 or IL-4 were
added at a concentration of 20 ng/ml. After two days cells were
treated for 10 hours with chemotherapeutic drugs or anti-CD95, then
20 .mu.l of MTS were added to each well. After 3 hours of
incubation at 37.degree. C. with the MTS reagent, the plates were
read on a Multilabel Counter (Victor2, Wallac, Perkin-Elmer Inc.,
Norwalk, Conn.) and dye absorbance was measured at 490 nm.
[0109] Immunostaining procedure. Immunohistochemical analyses were
performed on 7 .mu.m thick paraffin embedded sections.
Deparaffinized sections were treated for 10 min. in microwave oven
in 0.1 M citrate buffer. Following elimination of excess serum,
sections were exposed for 1 hour to specific antibodies against
Bcl-x.sub.L (H-5, mouse IgG.sub.1, Santa Cruz Biotechnology, Inc.,
Santa Cruz, Calif.), CD45RO (UCHL1, mouse IgG2a, Dako Corporation
Carpintera Calif., USA) IL-4 (B-S4 mouse IgG1, Caltag Laboratories,
Burlingame, Calif.), cFLIP (rabbit polyclonal IgG, Upstate
Biotechnology, Lake Placid, N.Y.), IL-4R(C-20, rabbit polyclonal
IgG, Santa Cruz, Calif.) or isotype matched controls at appropriate
dilutions. After two washes in TBS, sections were treated with
biotinylated anti-rabbit or anti-mouse immunoglobulins, washed in
TBS and incubated with streptavidin peroxidase (Dako LSAB 2 Kit).
Staining was detected using 3-amino-9-ethylcarbazole (AEC) as a
colorimetric substrate. Counterstaining of cells and tissue
sections was performed using aqueous hematoxylin.
Western Blotting
[0110] Cell pellets were washed twice with cold PBS and lysed on
ice for 30 minutes with 1% NP40 lysis buffer (20 mM Tris-HCl pH
7.2, 200 mM NaCl, 1% NP40) in the presence of 1 mM
phenylmethylsulphonyl fluoride (PMSF) and 2 .mu.g/ml each of
aprotinin, leupeptin and pepstatin. Cell debris was removed by
centrifugation at 13,000 rpm for 10 minutes at 4.degree. C. Lysate
concentration was determined using the Bio-Rad protein assay
(Bio-Rad Laboratories, Richmond, Calif.). Aliquots of cell extracts
containing 30 .mu.g of total protein were resolved on 10% or 12%
SDS-PAGE and transferred to a Hybond-C extra nitrocellulose
membrane (Amersham Pharmacia Biotech, Piscataway, N.J.). Filters
were blocked for 1 hour at room temperature in 5% nonfat-dry milk
dissolved in TBS-T (10 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.2%
Tween 20) and then incubated in 1% BSA/TBS-T containing a dilution
of primary antibody (1:200 anti Bcl-X.sub.L, 1:10 anti c-Flip and
1:10000 anti .beta.-actin) for 3 hours (Bcl-X.sub.L and
.beta.-actin) or overnight (cFlip). After washing in TBS-T buffer,
filters were incubated for 45 minutes in 5% nonfat-dry milk
dissolved in TBS-T containing a 1:4000 dilution of corresponding
peroxidase-conjugated secondary antibody (Amersham). Proteins were
visualized with the enhanced chemiluminescence technique (Super
Signal West Pico Pierce, Rockford, Ill.).
Production of Retroviral Particles and Infection of Cell Lines
[0111] cFlip and Bcl-X.sub.L cDNAs were cloned into the PINCO
retroviral vector carrying the Green Fluorescent Protein (GFP) as a
reporter gene (25). The amphotropic packaging cell line Phoenix was
transfected with the PINCO-1/Bcl-X.sub.L, PINCO-1/cFlip plasmids or
with the empty vector by standard calcium-phosphate-chloroquine
method. Culture supernatants containing viral particles were
collected after 48 hours, filtered (0.45 .mu.m) and added to
3.times.10.sup.5 cells plated on 6-well plates. For one cycle of
infection, cells were centrifuged at 1800 rpm for 45 minutes at
32.degree. C. and kept in the incubator for 75 minutes.
[0112] Cells were subjected to two infection cycles each day for
two consecutive days and then placed in standard medium.
GFP-positive cells were analyzed by flow cytometry 24 hours after
the last infection cycle (FACScan, Becton Dickinson, San Jose,
Calif.).
Statistical Analysis
[0113] The percentage of apoptotic cells was derived from the
percentage of viable cells that was directly calculated from the
values of the MTS assay. The percentage of protection from
apoptosis was determined as:
% protection=100%-X
X was calculated with the formule:
(C1-C2):100=(S1-S2):X
where: C1 is the viability of untreated control cells, C2 is the
viability of control cells treated with the apoptotic stimulus, S1
and S2 are the viability of cells preincubated with cytokines or
transduced with anti-apoptotic genes, untreated (S1) or treated
(S2) with the apoptotic stimuli.
[0114] Paired t-test was used to analyze the statistical
significance of the experimental results. P values less than 0.05
were considered significant. Data are presented as mean
values.+-.one standard deviation of the mean.
DESCRIPTION OF THE DRAWING
[0115] FIG. 1. Resistance to apoptotic cell death induced by
chemotherapeutic drugs in thyroid cancer cells. Percentage of
apoptotic cells in freshly purified thyrocytes from normal thyroid
gland, PTC, FTC and UTC, exposed for 6, 12 and 24 h to cisplatinum
(300 ng/ml), doxorubicin (5 .mu.M) and taxol (5 .mu.M). (Data are
mean.+-.s.d. of four independent experiments).
[0116] FIG. 2. Anti-apoptotic molecules expression on thyroid
cancer. (a, b) Immunohistochemical analysis of Bcl-X.sub.L and
Bcl-2 on paraffin embedded normal thyroid gland, PTC, FTC and UTC
sections revealed by AEC (red staining). (b, c) Immunoblot analysis
of Bcl-x.sub.L and Bcl-2 in freshly purified thyrocytes lysates
from normal, PTC, FTC and UTC. Bcl-xl and Bcl-2 transgenic hearts
were used as positive controls (+control). Loading controls were
done by detecting .beta.-actin in the same membrane blot (one of
representative experiment of four is shown).
[0117] FIG. 3. Protection from chemotherapy-induced cell death in
thyrocytes transduced with Bcl-x.sub.L and Bcl-2. Immunoblot
analysis of (a) Bcl-x.sub.L and (b) Bcl-2 expression on flow
cytometry sorted thyrocytes transduced with empty vector (Vector),
Bcl-x.sub.L and Bcl-2. Loading control was assessed by .beta.-actin
staining. (c) Percentage of apoptosis in normal thyrocytes
transduced as in a and b following exposure to chemotherapeutic
drugs. (d) GFP-positive cells stained with ethidium bromide and
observed by immunofluorescence microscope. One representative
experiments of three performed is shown.
[0118] FIG. 4. IL4 and IL-10 expression on thyroid cancer cells.
(a) Immunohistochemical analysis of IL-4, IL-10 and IFN-.gamma. on
paraffin embedded normal thyroid gland, PTC, FTC and UTC sections
(red staining). (b) Immunostaining for IL-4, IL-10 and IFN-.gamma.
of purified thyrocytes from all histological variants of thyroid
epithelial carcinoma. (c) Western analysis of IL-4, IL-10 and
IFN-.gamma. in freshly purified cancer thyrocytes. rhIL-4, rhIL-10
and rhIFN-.gamma. (20 ng/lane) were used as positive control. These
experiments are representative of results from three independent
experiments each using cultures from different patient
specimens.
[0119] FIG. 5. IL-4 and IL-10 rescue normal thyrocytes from
chemotherapy-induced apoptotic cell death. (a) Percentage of
apoptotic events of purified normal thyroid cells pre-treated for
48 h with control medium (left panel), rhIL-4 (20 ng/ml) or rhIL-10
(40 ng/ml) and then cultured with cisplatinum, doxorubicin and
taxol for 12 additional hours. (b) Immunoblot analysis of normal
thyrocytes cultured with IL-4 or IL-10 as in a or rhIFN-.gamma.
(1000 IU/ml).
[0120] FIG. 6. Neutralizing antibodies against IL-4 and IL-10
sensitize thyroid carcinoma cells to chemotherapy. (a) Kinetics of
viable cells on carcinoma thyrocytes cultivated with medium alone
or with anti-IL-4 or with anti-IL-10 or with anti-IL-4+anti-IL-10.
Percentage of viable purified thyroid carcinoma cells pre-treated
for 48 h with control medium, anti-IL-4 (1 .mu.g/ml) or anti-IL-10
(1 .mu.g/ml) or anti-IL-4+anti IL-10 and then cultured with
chemotherapeutic drugs for 24 additional hours (right panel). (Mean
of one of representative experiment of four is shown). (b)
Percentage of viable PTC, FTC and UTC cells pre-treated for 48 h
with anti-IL-4+anti IL-10 and then cultured with cisplatinum,
doxorubicin and taxol for 12 and 24 hours.
[0121] FIG. 7. IL-4 protects RT112 bladder carcinoma cells from
chemotherapy- and anti CD95-induced apoptosis.
(A) Percentage of viable RT112 bladder carcinoma cells pre-treated
for two days with increasing doses of IL-4 (5, 10, 20, 50 and 100
ng/ml) which were maintained also during incubation with
chemotherapeutic drugs and exposed to 50 ng/ml camptothecin of 7
.mu.M etoposide. The treatment with 20 ng/ml IL-4 for two days is
able to protect cells from chemotherapy-induced apoptosis. The
protective effect is maintained at higher concentrations of IL-4
than 20 ng/ml. Cell viability was determined after 24 hours using
the MTS assay as described in Materials and Methods and the
percentage of protection from cell death was calculated as
described in Materials and Methods. The results shown are the
mean.+-.s.d. of four independent experiments. (B) Time course of
RT112 bladder carcinoma cells following exposure with IL-4 are
protected from etoposide-induced cell death from day 2 to day 4,
until IL-4 is maintained in the culture medium. Cells, pretreated
up to 3 days with IL-4 or IL-2, were exposed to 7 .mu.M etoposide
for 2 days, washed and placed in fresh medium without cytokines
until day 6. When the cytokine is removed by washing the percentage
of protection is downmodulated. (C) Immunoblot analysis of
Bcl-X.sub.L and cFlip/FLAME-1 expression levels in RT112. Untreated
(0), or treated cells were exposed for two days to 5, 10, 20, 50 or
100 ng/ml IL-4. One representative experiment of two performed is
shown. (D) Immunoblot analysis of Bcl-X.sub.L and cFlip/FLAME-1
expression levels in RT112 untreated (day 0) or treated from day 1
to day 3 with IL-4 and washed and placed in fresh medium without
IL-4 from day 3 to day 6. (E) IL-4 prevents anti CD95-induced
apoptosis. Percentage of viable RT112 cells pretreated for two days
with IL-4 or IL-2 and kept in the presence of cytokines throughout
the experiment. Successively, cells were incubated with 30 ng/ml
anti-CD95 agonistic antibody in standard medium, Control cells (-)
were stimulated with anti-CD95 in the absence of cytokine
pre-treatment. The results shown are the mean.+-.s.d. of four
independent experiments. * Indicates P<0.05 vs control; **
P<0.01 vs IL-2; *** P<0.001 vs control or IL-2.
[0122] FIG. 3. IL-4 prevents apoptosis induced by chemotherapeutic
drugs. Apoptosis percentage of LNCaP prostate cancer cells (A) and
MDA-MB-231 breast cancer cells (B) pretreated for two days with
IL-4 or IL-2. LNCaP cells were treated with 300 ng/ml cisplatin
(left) or 1 .mu.M vincristine (right) and MDA-MB-231 cells were
treated with 300 ng/ml cisplatin (left) or 5 .mu.M daunorubicin
(right). Control cells (-) were stimulated with chemotherapeutic
drugs in the absence of cytokine pretreatment. The results shown
are the mean.+-.s.d. of four independent experiments.
*** Indicates P<0.001 vs control and IL-2.
[0123] IL-4 protects carcinoma cells from CD95-induced apoptosis.
Apoptosis evaluation of LNCaP (C) and MDA-MB-231 (D) cells in the
presence of IL-4 or IL-2 and incubated with 30 ng/ml anti-CD95
agonistic antibody. Control cells (-) were stimulated with
anti-CD95 in the absence of cytokine pretreatment. The results
shown are the mean.+-.s.d. of four independent experiments.
*Indicates P<0.05 vs control; ** P<0.01 vs IL-2; ***
P<0.001 vs control or IL-2.
[0124] FIG. 9. IL-4 upregulates Bcl-X.sub.L and cFlip/FLAME-1
expression levels. Immunoblot analysis of Bcl-X.sub.L and
cFlip/FLAME-1 levels in LNCaP (A) and MDA-MB-231 (B). One
representative experiment of four performed is shown.
[0125] Comparison of Bcl-X.sub.L and cFlip/FLAME-1 expression in
RT112 (C), LNCaP (D) and MDA-MB-231 (E) treated with IL-4 versus
the exogenous expression of Bcl-X.sub.L and cFlip/FLAME-1 in
retrovirally transduced cancer cells. Data show increase in
Bcl-X.sub.L and cFlip/FLAME-1 in IL-4 treated tumor cell lines as
compared to IL-2 treated cell lines (filled bars) versus
gene-transduced cell lines as compared to empty vector-transduced
cell lines (open bars).
[0126] Cell populations 100% positive for GFP expression were
analyzed 48 hours after the last infection cycle.
[0127] FIG. 10. Exogenous expression of Bcl-X.sub.L or
cFlip/FLAME-1 prevents tumor cells chemotherapy-induced apoptosis.
Cell lines stably overexpressing Bcl-X.sub.L or cFlip/FLAME-1 were
treated with chemotherapeutic drugs as described in FIG. 1 and FIG.
2. RT112 cells were treated with camptothecin (A) or etoposide (B),
LNCaP cells treated with cisplatin (C) or vincristine (D), and
MDA-MB-231 cells treated with cisplatin (E) or daunorubicin (F).
The results shown are the mean.+-.s.d. of five independent
experiments.
[0128] FIG. 11. Exogenous expression of Bcl-X.sub.L or
cFlip/FLAME-1 protects tumor cells from anti-CD95-induced
apoptosis. RT112 (A), LNCaP (B) and MDA-MB-231 (C) cell lines
stably overexpressing Bcl-X.sub.L or cFlip/FLAME-1 treated with
anti-CD95 agonistic antibody. The results shown are the
mean.+-.s.d. of five independent experiments.
[0129] FIG. 12. Immunohistochemical analysis of CD45 and IL-4 on
paraffin embedded normal or bladder (A), prostate (B) and breast
(C) cancer sections revealed by AEC (red staining).
[0130] FIG. 13. (A) Immunohistochemical analysis of Bcl-X.sub.L,
cFlip/FLAME-1 or IL-4R on paraffin sections of normal or bladder,
prostate and breast cancer specimens (red color).
[0131] Exogenous expression of Bcl-X.sub.L and cFlip/FLAME-1 in
retrovirally transduced cancer cells, RT112 (B), LNCaP (C) and
MDA-MB-231 (D) in comparison with primary tumor cells. Immunoblot
analysis of cancer cells transduced with empty vector (vector),
with Bcl-X.sub.L or cFlip/FLAME-1 (gene) and primary cancer cells
(cancer). One representative experiment of three performed for each
cell line is shown.
[0132] FIG. 14. (A) Immunoblot analysis of Bcl-X.sub.L
expressionlevels in primary cancer cell untreated (C) or treated
with IL-2 or IL-4. (B) Percentage of cell death in untreated and
IL-4 pretreated cells and exposed to cisplatin, doxorubicin or
taxol. Control cells were stimulated with chemotherapeutic drugs in
the absence of cytokines pretreatment. (C) Immunoblot analysis of
Bcl-X.sub.L in primary cancer cells preincubated for 6 days with or
without IL-4. (D) Percentage of apoptotic cell death in primary
tumor cells incubated with IL-4 and exposed to cisplatin,
doxorubicin and taxol.
* * * * *