U.S. patent application number 14/651972 was filed with the patent office on 2015-11-12 for anti-proliferative effects of palm vegetation liquor and extracts thereof in preventing pancreatic cancer.
This patent application is currently assigned to MALAYSIAN PALM OIL BOARD. The applicant listed for this patent is MALAYSIAN PALM OIL BOARD. Invention is credited to Smiti V. Gupta, Xiangming Ji, Pramod Khosla, Ravigadevi Sambanthamurthi, Yew Ai Tan.
Application Number | 20150320822 14/651972 |
Document ID | / |
Family ID | 50934711 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150320822 |
Kind Code |
A1 |
Gupta; Smiti V. ; et
al. |
November 12, 2015 |
Anti-Proliferative Effects of Palm Vegetation Liquor And Extracts
Thereof In Preventing Pancreatic Cancer
Abstract
Present invention relates to a composition to inhibit cancer
cell proliferation, wherein said composition comprises oil palm
extracts. The composition is useful for prevention of pancreatic
cancer by inhibiting clonogenicity, inducing apoptosis, regulating
gene expression, inducing anti-invasive effect, and induces cell
cycle arrest in S phase. Present invention also discloses a
composition that inhibits NF-.kappa.B activity and reduces cell
invasion, cell migration and metastasis. Present invention further
discloses the use of therapeutically effective amount of a
composition in inhibiting the growth of pancreatic cancer.
Inventors: |
Gupta; Smiti V.; (Oakland
Township, MI) ; Khosla; Pramod; (Troy, MI) ;
Ji; Xiangming; (Detroit, MI) ; Sambanthamurthi;
Ravigadevi; (Kajang, Selangor, MY) ; Tan; Yew Ai;
(Kajang, Selangor, MY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MALAYSIAN PALM OIL BOARD |
Kajang, Selangor |
|
MY |
|
|
Assignee: |
MALAYSIAN PALM OIL BOARD
Kajang, Selangor
MY
|
Family ID: |
50934711 |
Appl. No.: |
14/651972 |
Filed: |
December 3, 2013 |
PCT Filed: |
December 3, 2013 |
PCT NO: |
PCT/MY2013/000228 |
371 Date: |
June 12, 2015 |
Current U.S.
Class: |
424/727 |
Current CPC
Class: |
A61K 31/05 20130101;
A61K 36/889 20130101; A61K 45/06 20130101; A61K 9/0053 20130101;
A23V 2002/00 20130101; A61P 35/00 20180101; A23L 33/105
20160801 |
International
Class: |
A61K 36/889 20060101
A61K036/889; A23L 1/30 20060101 A23L001/30; A61K 45/06 20060101
A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2012 |
MY |
PI2012701146 |
Claims
1. A composition to inhibit pancreatic cancer cell proliferation
and clonogenicity, wherein said composition comprises plant
extracts.
2. The composition as claimed in claim 1, wherein said plant
extract is from oil palm.
3. The composition as claimed in claim 1, wherein said plant
extracts is from vegetation liquor.
4. The composition as claimed in claim 3, wherein said vegetation
liquor further comprising phenolics.
5. The composition as claimed in claim 1, wherein said composition
is useful for prevention of pancreatic cancer and diseases related
thereto.
6. The composition as claimed in claim 1, wherein said pancreatic
cancer is from PANC-1 cell line.
7. The composition as claimed in claim 1, wherein said pancreatic
cancer is from BxPC-3 cell line.
8. The composition as claimed in claim 1, wherein said composition
induces apoptosis.
9. The composition as claimed in claim 1, wherein said composition
regulates gene expression.
10. The composition as claimed in claim 8, wherein said apoptosis
is associated with inhibiting cell survival proteins.
11. The composition as claimed in claim 8, wherein said apoptosis
is associated with inhibiting anti-apoptotic protein.
12. The composition as claimed in claim 11, wherein said
anti-apoptotic protein is Survivin.
13. The composition as claimed in claim 9, wherein said gene
expression regulated is associated with Bcl-2 family.
14. The composition as claimed in claim 9, wherein said gene
expression is associated with down-regulating Bcl-XL
expression.
15. The composition as claimed in claim 8, wherein said apoptosis
is associated with increased expression of pro-apoptotic
proteins.
16. The composition as claimed in claim 15, wherein said
pro-apoptotic proteins are cleaved caspase 3, caspase 9 or Poly
(ADP-ribose) polymerase (PARP).
17. The composition as claimed in claim 1, wherein said composition
induces cell cycle arrest in S phase.
18. The composition as claimed in claim 1, wherein said composition
inhibits NF-.kappa.B activity.
19. The composition as claimed in claim 18, wherein said
NF-.kappa.B activity inhibition is via down-regulating p65 subunit
activity.
20. The composition as claimed in claim 19, wherein said
down-regulating is associated with decreasing vascular endothelial
growth factor (VEGF) gene expression.
21. The composition as claimed in claim 19, wherein said
down-regulating is associated with decreasing matrix
metalloproteinase 9 (MMP9) gene expression.
22. The composition as claimed in claim 1, wherein said composition
reduces cell invasion.
23. The composition as claimed in claim 1, wherein said composition
reduces cell migration and metastasis.
24. The composition as claimed in claim 1, further comprising
compounds with pharmaceutically acceptable carriers.
25. A method of using a therapeutically effective amount of a
composition to inhibit pancreatic cancer cell proliferation and
clonogenicity, wherein said composition comprises plant extracts,
and wherein the method is used in the preparation of a medicament
for preventing or inhibiting the growth of pancreatic cancer in an
individual by administering to an individual in need thereof.
26. The method as claimed in claim 25, wherein the composition is
accompanied with or without conventional chemotherapy or radiation
therapy or surgery,
27. The method as claimed in claim 25, wherein the composition is
administered orally or as a food supplement.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a composition of
anti-proliferative effects in cancer. More particularly, relates to
a palm-based composition that includes but is not limited to
phenolic compounds for use in preventing pancreatic cancer.
BACKGROUND OF THE INVENTION
[0002] The oil palm fruit (Elaeis guineensis) contains large
amounts of lipid or water soluble bioactive agents such as vitamin
E (tocopherols, tocotrienols), carotenoids and phytosterols. The
lipid-soluble tocotrienols are shown to have anti-tumor effects on
human cancer cells like prostate, breast, colon, melanoma, and lung
cancers. Water-soluble oil palm phenolics (OPP) have been
demonstrated to show potential anticancer properties.
Sambanthamurthi et al (2011) showed that phenolics inhibit tumour
progression in tumour-inoculated mice; however there is no
disclosure on tumour growth inhibition in pancreatic cancer using
palm vegetation liquor. Sekaran et al (2010) have demonstrated that
OPP can inhibit proliferation and induce apoptosis in different
cancer cell lines such as breast, lung, and skin cancers in vitro.
It was also found that OPP reduces tumor growth in vivo. However,
there is no study available to disclose the effect of oil palm
bioactive agents on pancreatic cancer cell growth. Therefore, it is
an object of present invention to utilize the extract from this oil
palm vegetation liquor in treating pancreatic cancer (PaCa).
[0003] Pancreatic cancer (PaCa) is one of the leading causes of
cancer death with poor survival rate due to its aggressiveness and
early metastases. Despite more focused research in this area
recently, the mortality rate for PaCa has remained fairly high. The
common treatments for PaCa patients are pancreatectomy, or surgical
resection of the organ, followed by chemo, targeted and/or
radiotherapies.
[0004] Gemcitabine is a purine analog which is used as a standard
clinical chemotherapeutic agent for advanced PaCa treatment;
however this agent has shown a poor tumor response rate with short
survival time. Erlotinib is a targeting epidermal growth factor
receptor tyrosine kinase which is used as an adjuvant to
gemcitabine by inhibiting cell growth signaling but again showing
marginally improved survival benefits in clinical trials.
[0005] Detrimental side effects of these chemotherapeutic agents
available targeting PaCa such as heart attack, stroke and death
have been documented. This results in limited advantage due to
their dose limiting toxicity to normal tissues. Thus, it is an
object of present invention to provide an alternative to enhance
chemotherapeutic activity for pancreatic cancer with minimal
adverse effects.
[0006] Another object of present invention is to curb the adverse
effects and chemoresistance suffered by most gemcitabine-treated
patients. It becomes crucial to develop a novel therapeutic agent
in order to improve prognosis with natural plant extracts.
[0007] It is yet another object of present invention to provide a
bioactive dietary agent used in combination with drug to achieve
the pleiotropic effects which lessen the toxicity and dose
requirements of therapeutics alone.
[0008] The nuclear factor .kappa.B (NF-.kappa.B) is widely used as
a regulator of genes that control cell proliferation and cell
survival, and it plays important roles in cancer cell
transformation, cell invasion, and apoptosis. P65 is a subunit of
NF-.kappa.B that interacts with promoter sequence of target genes
which in turn induces the expression of genes involving in
inflammatory, anti-apoptosis, and proliferation. This signaling
pathway has been targeted as a strategy for anti-cancer therapy in
PaCa patients. NF-.kappa.B is a common and constitutively expressed
transcription factor which is activated through a wide variety of
stimuli such as inflammation and oxidative stress. On stimulation,
NF-.kappa.B is translocated to the nucleus and binds to the
promoters of its target genes to begin transcription of specific
genes. Overexpression of NF-.kappa.B may results in apoptosis
resistance, angiogenesis, migration and invasion of cancer
cells.
[0009] Studies have shown that NF-.kappa.B is constitutively
activated in approximately 67% of pancreatic adenocarcinomas
compared to normal pancreatic tissue, and this is associated with
aggressive stage pancreatic cancer and drug resistance. Based on
the role of NF-.kappa.B in regulating carcinogenic activity in
pancreatic cancer and the anti-inflammatory behavior of palm
vegetation liquor, present invention serves the purpose to describe
the effect of palm vegetation liquor in pancreatic cancer cell
model, characterized in the potential down-regulation of the
NF-.kappa.B pathway.
[0010] Thus, it is yet another object of present invention to
provide a composition for anti-proliferation in pancreatic cancer
via regulation of NF-.kappa.B pathway.
[0011] It is a further object of the present invention to provide
an improved composition and method based on compounds extracted
from vegetation liquor i.e. aqueous stream of the palm oil milling
process for providing inhibitory effect against pancreatic
cancer.
[0012] Accordingly, present invention also relates to an extraction
process of bioactives from the oil palm vegetation liquor from the
milling process. The processing of oil palm produces large amounts
of vegetation liquor rich in phenolic compounds, shikimic acid,
fruit acids, fruit sugars and glycerol which can be further
enriched using conventional membrane filtration technology.
[0013] Further objects and advantages of the present invention may
become apparent upon referring to the preferred embodiments of the
present invention as shown in the accompanying drawings and as
described in the following description.
SUMMARY OF THE INVENTION
[0014] The invention relates to a composition to inhibit cancer
cell proliferation, wherein said composition comprises oil palm
phenolics derived from oil palm extracts and vegetation liquor. The
composition is useful for prevention of pancreatic cancer from
PANC-1 and BxPC-3 cell lines by inhibiting clonogenicity, inducing
apoptosis, regulating gene expression, anti-invasive effect, and
induces cell cycle arrest in S phase.
[0015] The apoptosis is associated with inducing caspase,
inhibiting cell survival proteins and inhibiting anti-apoptotic
protein. The anti-apoptotic protein disclosed herein is Survivin.
The composition also down-regulates Bcl-XL gene expression which is
from Bcl-2 family.
[0016] The composition induces apoptosis by increasing expression
of pro-apoptotic proteins such as cleaved caspase 3, caspase 9 or
Poly (ADP-ribose) polymerase (PARP).
[0017] Present invention also discloses a composition that inhibits
NF-.kappa.B activity via down-regulating p65 subunit activity.
Vascular endothelial growth factor (VEGF) and matrix
metalloproteinase 9 (MMP9) gene expressions are down-regulated to
reduce cell invasion, cell migration and metastasis. The
composition may be provided as compounds with pharmaceutically
acceptable carriers.
[0018] Present invention further discloses the use of
therapeutically effective amount of a composition in the
preparation of a medicament for preventing or inhibiting the growth
of pancreatic cancer in an individual by administering to an
individual in need thereof. The composition is accompanied with or
without conventional chemotherapy or radiation therapy or surgery,
or it may be administered orally or as a food supplement.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, appended claims, and accompanying drawings,
in which:
[0020] FIG. 1 shows the effect of phenolic-rich extract from oil
palm vegetation liquor designated palm juice (PJ) on pancreatic
cancer cell line PANC-1 and BxPC-3 survival and growth (A and B)
and clonogenicity (C and D);
[0021] FIG. 2 shows the apoptosis analysis by Histone Kit/DNA ELISA
method (A and B) and flow cytometry (C and D);
[0022] FIG. 3 shows the effect of PJ on gene expression;
[0023] FIG. 4 shows the response of PJ on cell cycle regulation in
PANC-1 (A) and BxPC-3 (B) cancer cell lines;
[0024] FIG. 5 shows the down regulation of NF-.kappa.B activity
following PJ treatment in PANC-1 and BxPC-3 cells; and
[0025] FIG. 6 shows the inhibition of cell invasion and migration
in PANC-1 and BxPC-3 cell lines.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The embodiments herein and the various features and
advantageous details thereof are explained more fully with
reference to the non-limiting embodiments that are illustrated in
the accompanying drawings and detailed in the following
description. Descriptions of well-known components and processing
techniques are omitted so as to not unnecessarily obscure the
embodiments herein. The examples used herein are intended merely to
facilitate an understanding of ways in which the embodiments herein
may be practiced and to further enable those of skill in the art to
practice the embodiments herein. Accordingly, the examples should
not be construed as limiting the scope of the embodiments
herein.
[0027] Present invention provides palm juice (PJ) which constitutes
a water soluble phenolic-rich extract from the palm fruit (Elaeis
guineensis) and/or its vegetation liquor and has documented high
antioxidant, anti-inflammatory, and anti-carcinogenic activity in
breast cancer cells.
[0028] Present invention also discloses anti-tumor effects of the
phenolic-rich extract by treating two pancreatic cancer cell lines
(PANC-1 and BxPC-3) with different doses of palm juice (PJ). PJ was
observed to induce anti-proliferative, apoptotic and anti-invasive
effects using the
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium) (MTS) assay, cytoplasmic histone-DNA fragment
quantification and matrigel invasive assays in a dose dependent
manner. Real-time PCR has confirmed the anti-invasive effects
induced by PJ through a decrease in the gene expressions of MMP-9
and VEGF. Flow cytometer is used to demonstrate that cells arrest
in S phase in cell cycle analysis. Western blot analysis has also
been conducted to show that apoptosis induced by PJ was associated
with down regulation of expression in Survivin and Bcl-XL gene and
up regulation of expression in cleaved caspase 3, caspase 9 and
PARP gene. These results demonstrate the anti-tumor activity of PJ
in pancreatic cancer cells, providing initial evidence towards its
potential therapeutic uses.
BEST MODE FOR CARRYING OUT THE INVENTION
[0029] The method of preparation and use of the present invention
is further illustrated by the following experimental examples. It
should be understood that these experimental examples, while
indicating preferred embodiments of the invention, are given by way
for better elucidation only. A person skilled in the art can
ascertain the essential characteristics and embodiments of this
invention, therefore various changes may be provided to adapt to
various usages and conditions.
Materials and Methods
Cell Culture, Reagents and Antibodies:
[0030] Human PaCa cell lines, including PANC-1 and BxPC-3 were
grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with
10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in
5% CO.sub.2. Palm juice (PJ) extracted from oil palm was prepared
at a stock concentration of 1500 mg/ml GE (gallic acid
equivalents). Protease inhibitor cocktail, primary antibodies for
Poly (ADP-ribose) polymerase (PARP), .beta.-actin and cell lysis
buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na.sub.2EDTA, 1 mM
EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
beta-glycerophosphate, 1 mM Na.sub.3VO.sub.4, 1 .mu.g/ml
leupeptin), primary antibodies against cleaved caspase 3 and
cleaved caspase 9, Survivin, Bcl-X.sub.L and secondary antibodies
were obtained from commercially available supply.
Cell Viability Studies by MTS Assay
[0031] The PANC-1 and BxPC-3 cells (5.times.10.sup.3) were seeded
and incubated overnight in 96-well culture plates. The medium was
then removed and replaced with a fresh medium containing 20, 30, 40
or 50 .mu.l of PJ (1500 mg/mL GE) per ml of cell prior to 72 h of
incubation. After that, 20 .mu.l of MTS assay reagent was added to
each well and incubated for 2 h at 37.degree. C. in a humidified,
5% CO.sub.2 atmosphere. The reading was then recorded in absorbance
at 490 nm using plate reader. This assay is repeated by replacing
PJ with dimethyl sulfoxide (DMSO) as the vehicle control. Each
variant of the experiment was performed in triplicate.
Histone/DNA ELISA for Detection of Apoptosis
[0032] The Cell Death Detection Enzyme-linked immunosorbent assay
(ELISA) Kit was used to detect apoptosis in PaCa cells. One million
cells were seeded and incubated overnight in six-well plates before
treating with PJ or control for 72 h. The cells were then lysed and
the cytoplasmic histone/DNA fragments were extracted and incubated
in microtiter plate modules coated with anti-histone antibody.
Peroxidase-conjugated anti-DNA antibody were used to detect the
immobilized histone/DNA fragment before color development with
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
substrate for peroxidase. The spectrophotometric absorbance of the
samples was determined by using plate reader at wavelength of 405
nm.
Clonogenic Assay
[0033] One million cells were seeded and incubated overnight in a
100 mm dish. The cells were cultured with 20, 30, 40 or 50 .mu.l of
PJ or control for 72 h prior to viable cell counting and plating
with about 5,000 cells per plate. The cells were then incubated for
21 days at 37.degree. C. in a 5% CO.sub.2 incubator. All the
colonies were fixed in 4% paraformaldehyde and stained with 2%
crystal violet.
Flow Cytometry and Cell Cycle Analysis
[0034] Cells were seeded and incubated overnight in 100 mm dish
before subjecting all the cells for starvation for 24 h. The cells
were released into control or PJ containing media for 72 h of
incubation. Subsequently, cells were collected and fixed with
ice-cold 70% (v/v) ethanol for 24 h. After subjecting cells for
centrifugation at 3000.times.g for 5 min, the cell pellet was
washed with PBS (pH 7.4) and resuspended in PBS containing
propidium iodide (50 Hg/mL), and DNase-free RNase (1 .mu.g/mL).
Samples were then incubated at room temperature for 2 h, and DNA
content was determined using a flow cytometer.
Annexin V-FITC Method for Apoptosis Analysis
[0035] Annexin V-Fluorescein isothiocyanate (FITC) apoptosis
detection kit was used to measure the apoptotic cells. PANC-1 and
BxPC-3 cells were incubated in the presence or absence of PJ for 48
h before subjecting cells for trypsinisation, washing with ice-cold
PBS and resuspension in 1.times. binding buffer at a concentration
of 10.sup.5 cells per ml in a total volume of 100 .mu.l. The cells
were then added with 51 .mu.l of Annexin V-FITC and 5 .mu.l of PI
(Propidium Iodide) and were kept in the dark for 20 min at room
temperature. Finally, each tube was added with 400 .mu.l of
1.times. binding buffer and the number of apoptotic cells was
analyzed using flow cytometer.
Wound Healing Assay
[0036] PANC-1 and BxPC-3 were seeded and incubated overnight in a
six well plate at a concentration of 4.times.10.sup.5 cells per
well. After incubation, culture media were removed and a scratch
wound across each well was made using fine tips. In order to ensure
no loosely held cells were attached near to wound areas, the wound
areas were washed three times with PBS. Subsequently, the cells
were cultured in presence or absence of PJ and the wound images
were taken as 0 h. After 20 h, wound healing pictures were taken
under microscope.
Cell Invasive Assay
[0037] A biocoat matrigel invasion kit was used to evaluate the
tumor invasive ability according to the manufacturer's protocol.
Around 5.times.10.sup.4 cells of PANC-1 and BxPC-3 with basal media
was transferred in each 6-well upper chamber in the presence or
absence of PJ, while 3 ml of culture medium with 10% FBS was added
into each lower chamber of 24-well plate. The cells in the upper
chamber were removed using a cotton stick after 20 h of incubation.
Then the cells that invaded through the Matrigel matrix membrane
were stained with 4 .mu.g/mL Calcein AM in Hanks buffered saline at
37.degree. C. for 1 h. Each of experimental conditions was
performed in triplicates. Subsequently, fluorescence of the invaded
cells was read using a microplate reader at excitation/emission
wavelengths of 530/590 nm. A fluorescent microscope was used to
capture images of these fluorescently labeled invasive cells.
Protein Extraction and Western Blotting
[0038] PANC-1 and BxPC-3 cell lines were treated with or without of
PJ for 72 h to evaluate the effects of treatment on PARP, cleaved
caspase 3, cleaved caspase 9, Survivin, Bcl-X.sub.L, and
.beta.-actin expressions. Cells were lysed in cold lysis buffer for
30 mins on ice and the protein concentrations were determined using
a protein assay kit. The samples were loaded on 10%
SDS-polyacrylamide gels and subjected to electrophoresis. After
electrophoresis, the gel was electrophoretically transferred to a
nitrocellulose membrane using transfer buffer (25 mM Tris, 190 mM
glycine, 20% methanol) in a transfer apparatus. The membranes were
incubated for 1 h at room temperature with 5% nonfat dried milk in
Ix tris-buffered saline (TBS) buffer containing 0.1% polysorbate 20
(TBS-T) before incubating overnight at 4.degree. C. with primary
antibodies. The membranes were subjected to washing for 3 times
with TBS-T and incubated with the secondary antibodies containing
2% bovine serum albumin (BSA) for 2 h at room temperature before
measuring signal intensity using a chemiluminescent imager.
Real-Time Quantitative PCR for Gene Expression Analysis
[0039] Total RNA was isolated according to the kit manufacturer's
protocols. First strand cDNA synthesis was performed on 2 .mu.g of
total RNA from each sample using TaqMan reverse transcription
reagents kit in a total volume of 20 .mu.l. Reverse transcription
reaction were performed at 25.degree. C. for 10 min, followed by
48.degree. C. for 30 min and 95.degree. C. for 5 min. Real-time PCR
analysis were performed and sequences of the primers sets used for
this analysis are as follows: MMP-9, forward primer (5'-CGG AGT GAG
TTG AAC CAG-3') and reverse primer (5'-GTC CCA GTG GGG ATT TAC-3');
VEGF, forward primer (5'-GCC TTG CCT TGC TGC TCT AC-3') and reverse
primer (5'-TTC TGC CCTCCT CCT TCT GC-3'); GAPDH, forward primer
(5'-CAG TGA GCT TCC CGT TCAG-3') and reverse primer (5'-ACC CAG AAG
ACT GTG GAT GG-3').
[0040] All these primers were verified by virtual PCR, and primer
concentrations were optimized to avoid primer dimer formation.
Real-time PCR amplifications were performed using 2.times.SYBR
Green PCR Master Mix. Two microliters of RT reaction were used for
a total volume of 25 .mu.l quantitative PCR reactions. The thermal
profile for SYBR real-time PCR was 95.degree. C. 10 min followed by
50 cycles of 95.degree. C. 15 s and 60.degree. C. 1 m. Data were
analyzed according to the comparative fold increase or decrease in
gene expression determined by quantitation of normalized
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression in each
sample.
Microwell Colorimetric NF-.kappa.B Assay
[0041] In order to evaluate the binding activity of NF-.kappa.B, a
transcription factor ELISA kit for P65 was used according to the
manufacturer's protocol. One million of PANC-1 and BxPC-3 cells
were seeded and incubated overnight in 100 mm dish before treating
with PJ or control for 72 h and nuclear protein extraction from
each sample. Then, 2 .mu.g of each sample were incubated in
microplate coated with anti-p65 DNA sequence. Peroxidase-conjugated
anti-DNA antibody was used to detect the p65-DNA binding complex
before color development with ABTS substrate for peroxidase. The
chemiluminescence and volume of the samples were recorded and
analyzed.
Data Analysis
[0042] Results are expressed as means.+-.standard error of the mean
(SEM) and statistical comparisons between groups were done using
one-way ANOVA. Values of p<0.05 were considered to be
statistically significant and individual p-values are reported in
the figures.
Results
Effects of PJ on Cell Growth/Survival of PaCa Cells
[0043] The cytotoxic potential of PJ on pancreatic cancer cell
lines was evaluated by treating PANC-1 and BxPC-3 cells with
different concentrations of PJ and culture medium followed by the
MTS and clonogenic assays as shown in FIG. 1. FIG. 1 shows the
effect of PJ on pancreatic cancer cell line PANC-1 and BxPC-3
survival and growth (A and B) and clonogenicity (C and D).
[0044] Both cancer cell lines were treated with increasing
concentrations of PJ. Results are presented as mean.+-.SEM of three
assay replicates. In reference to FIG. 1, * indicates P<0.05 and
** indicates P<0.01 versus respective DMSO treated controls. The
number of cells counted in the control treatment was considered
100% and the number of cells in PJ treated cells was calculated in
relative to this control.
[0045] In PANC-1 cell line, treatment with 20, 30, 40 and 50
.mu.l/ml of PJ for 72 h resulted in 35%, 51%, 75% and 91% of cell
growth inhibition in relative to control, respectively. Similarly,
treatment on BxPC-3 cell line with 20, 30, 40 and 50 .mu.l/ml of PJ
for 72 h resulted in 12%, 30%, 54% and 75% of cell growth
inhibition in relative to control respectively. These results
indicate that the efficiency of PJ as an inhibitor of pancreatic
cancer cell growth.
[0046] Clonogenic assay as depicted in FIGS. 1C and 1D confirms the
effects of PJ on cell growth by revealing the treatment of PANC-1
and BxPC-3 with increasing concentrations of PJ (30 and 40
.mu.l/ml) resulted in a greater reduction in the number of
colonies, as indicated by reduced crystal violet staining. As shown
in FIG. 1, the results from the clonogenic assay were consistent
with the MTS data where PJ significantly inhibited pancreatic
cancer cell growth. Summarizing both assays, it is found that PJ
inhibited cell proliferation and clonogenicity in both cell lines
where PANC-1 cells were more sensitive to the effects of PJ than
BxPC-3 cells for all concentrations tested.
Induction of Apoptosis by PJ
[0047] FIG. 2 depicted the analysis of apoptotic ability of PJ with
Histone/DNA ELISA method (A and B) and flow cytometry (C and D).
Cells were treated with increasing concentration of PJ or treated
with DMSO as vehicle control prior to incubation for 72 h. The
incubation is followed by staining with Annexin V and propidium
iodide for flow cytometry analysis. The flow cytometric cell
distribution of cells is illustrated in dot plots (FIGS. 2C and
2D). The values represent .+-.SEM of triplicate samples. *
indicates P<0.05 and ** indicates P<0.01 versus DMSO-treated
control groups.
[0048] FIGS. 2A and 2B show PJ induced apoptosis in PANC-1 (FIG.
2A) and BxPC-3 (FIG. 2B) cell lines in a dose dependent manner.
FIGS. 2C and 2D indicates quantitation of apoptotic cells, as
detected by Annexin V staining after treatment with 20 and 30
.mu.l/ml of PJ, thus confirming the apoptosis-inducing effect of PJ
in both cell lines. The result showed that PJ treatment is
statistically significant (**P<0.01) increasing in percentage of
apoptotic cells in both pancreatic cancer cell lines at IC.sub.50
dosage of PJ.
Analysis of Cell Cycle Distribution after Treatment with PJ
[0049] The cell cycle distributions of PANC-1 and BxPC-3 cells
treated with varying concentrations of PJ were analyzed to
determine possible underlying mechanism of cell proliferation
suppression as depicted in FIG. 3A (PANC-1) and FIG. 3B (BxPC-3).
The distribution of populations in different cell cycle phases was
quantified using flow cytometer after 72 h of incubation.
[0050] FIGS. 3A and 3B indicate that both cell lines were arrested
in the S phase by PJ in a dose-depended manner. For PANC-1 cell
(FIG. 3A), about 28% cells are arrested in S phase in treatment
group (40 .mu.l/ml) as compared to 25% in control cells. A similar
observation was made in BxPC-3 cells (FIG. 3B) with 52% of cells
arrested in S phase in treatment group (50 .mu.l/ml) as compared to
21% in control cells.
[0051] Cell cycle distribution of cancer cells treated with
increasing concentrations of PJ are shown along with the
percentages of the cell cycle stages, G0-G1, G2-M, and S phase.
These findings reveal that PJ induced cell cycle arrest in the S
phase for both cancer cell lines.
PJ Both Up-Regulates Pro-Apoptotic Genes and Down-Regulates
Anti-Apoptotic Genes
[0052] Western blotting represents the expression of PARP cleavage,
caspases, Bcl-xL and Survivin in PANC-1 and BxPC-3 cancer cell
lines after 72 h of treatment with increasing PJ concentration to
further elaborate the molecular mechanisms involve in PJ-induced
apoptosis of pancreatic cancer cells. The .beta.-actin protein was
utilized as the protein loading control for this experiment.
[0053] Specifically, the expression of proteins involved in the
induction of cleaved caspase dependent apoptosis (caspase 3,
caspase 9), anti-apoptotic Bcl-2 family (Bcl-xL) and anti-apoptotic
protein--Survivin were analyzed. FIG. 4 illustrates that PJ
increases expression of pro-apoptotic proteins, cleaved caspase 3
and 9 and PARP in both cancer cell lines, while decreases
production of anti-apoptotic proteins such as Bcl-xL and Survivin
in both PANC-1 and BxPC-3 cell lines.
[0054] This analysis demonstrates that PJ induces apoptosis in both
cancer cell lines through a dual-mechanistic approach: activation
of apoptosis inducing caspases and inhibition of cell survival
proteins.
Inhibition of NF-.kappa.B Activity with PJ
[0055] Microwell colorimetric NF-.kappa.B assay was performed to
characterize the effect of PJ on NF-.kappa.B activity. FIG. 5 shows
the down regulation of NF-.kappa.B activity as increasing
concentration of PJ used in treatment on PANC-1 and BxPC-3 cells.
FIGS. 5A and 5B shows the analysis of p65 activity in PANC-1 and
BxPC-3. P65 activation decreased in a similar dose-dependent manner
following PJ treatment in both cell lines. Both PANC-1 and BxPC-3
cell lines exhibited significantly lower NF-.kappa.B (p65) activity
at higher concentrations of PJ used in treatment.
[0056] FIGS. 5C and 5D shows the effect of PJ on VEGF and MMP-9
expression in relative to DMSO treated control. VEGF and MMP-9
expression was significantly reduced in treatment with PJ at
dose-dependent manner. The effect on MMP-9 was more pronounced in
both cancer cell lines as observed in FIGS. 5C and 5D. Vascular
endothelial growth factor (VEGF) and matrix metalloproteinase 9
(MMP9) being the downstream genes of NF-.kappa.B, are responsible
for cancer cell invasion and migration expression. The expressions
of VEGF and MMP9 were measured by real time PCR after reverse
transcriptase PCR. These results strongly suggest that PJ inhibited
NF-kB activity and its target genes expressions.
[0057] The results are presented as mean.+-.SEM of three assay
replicates. * indicates P<0.05 and ** indicates P<0.01 versus
respective DMSO treated controls.
Inhibition of Cell Migration and Invasion by PJ
[0058] The cell invasion and wound healing assays were used to
analyze the effect of PJ on invasion and migration on the
pancreatic cancer cell lines. FIG. 6 show PJ inhibits migration and
cell invasion in PANC-1 and BxPC-1 cell lines in dose-dependent
manner. Cells were cultured in varying concentrations of PJ for 20
h and were photographed after staining with Calcein AM. Cells
cultured in the absence of PJ are used as control. FIGS. 6A and 6B
illustrates the fluorescence of the invaded cells in PANC-1 and
BxPC-1 cell lines respectively. PJ decreased cell invasion in a
dose dependent manner, as semi-quantified by the fluorescence index
reading. Results are presented as mean.+-.SEM of three assay
replicates. * indicates P<0.05 and ** indicates P<0.01 versus
respective DMSO treated controls.
[0059] The wound healing assay shows the cell migration in PANC-1
and BxPC-1 cell lines as depicted in FIGS. 6C and 6D respectively.
When the cells reached confluence, a single wound was scratched
across each well. After 20 h incubation with various concentrations
of PJ, wound closure areas were visualized using a microscope.
These results support the cell invasion findings that increasing PJ
concentrations prevents cell migration in an increased
dose-dependent manner. Thus, this demonstrates that PJ effectively
retards migration and metastasis in PANC-1 and BxPC-3.
[0060] Present invention discloses PJ which inhibits proliferation,
growth, migration, and invasion by inducing apoptosis, cell cycle
arrest, and down-regulating NF-.kappa.B activity in both cell
lines.
[0061] This statement is supported by various experiments such as
MTS and clonogenic assays wherein PJ inhibited cell proliferation
and clonogenicity in both cell lines. Besides that, PJ was tested
to be effective in inducing apoptosis in a dose-dependent manner
for PANC-1 and BxPC-3. Furthermore, the flow cytometry analysis on
cell cycle distribution revealed that PJ induces cell cycle arrest
in the S phase for both PANC-1 and BxPC-3 cells. Together, these
data suggest PJ inhibits cell growth, induces apoptosis and induces
cell cycle arrest in these cancer cell lines and hence could prove
to be an effective antitumor agent.
[0062] Western blot analysis evaluated the expression of caspases
and PARP protein. The experiment data also demonstrated that PJ
simultaneously caused a decrease in the expressions of the
anti-apoptotic proteins such as Bcl-xL and Survivin in both PANC-1
and BxPC-3, and also increase in the expressions of pro-apoptotic
proteins such as cleaved caspase 3, cleaved caspase 9 and cleaved
PARP. Hence, present invention discloses that the apoptotic
capacity of PJ is induced through a dual mechanism, demonstrating
that PJ can indeed be a powerful nutraceutical against cancer.
[0063] NF-.kappa.B plays important roles in many cellular processes
including cell proliferation, anti-apoptosis invasion, and
angiogenesis all of which are crucial for cancer development and
progression. By using the chemilluminance NF-.kappa.B ELISA kit, we
demonstrated that P65 activity significantly decreased as PJ
concentration increased in both cell lines as shown in FIGS. 5A and
5B. Since VEGF and MMP9 are known to be involved in downstream
signaling of NF-.kappa.B responsible for cell migration and
invasion, relative expressions of VEGF and MMP9 for both cell lines
were evaluated. The study shows the expression of both VEGF and
MMP9 was significantly decreased in response to increased PJ
concentrations in both PANC-1 and BxPC-3 cell lines as shown in
FIGS. 5C and 5D.
[0064] The extracts from oil palm disclosed in present invention
have also exhibited properties to inhibit cell invasion and
migration. The current studies show that PJ caused a dose-dependent
reduction in cell invasion and migration in both PANC-1 and BxPC-3
cells as shown in FIG. 6. This further supports PJ's candidacy in
treating cancer.
[0065] Thus, present invention has disclosed the extracts from oil
palm to work as a multi-faceted chemotherapeutic agent against
pancreatic cancer. Benefits of its consumption may potentially
involve inhibition of cell proliferation and growth, and induction
of cell cycle arrest and apoptosis. The mechanisms through which
these actions occur have also been partially elucidated in this
study, the most important of which is the evident down-regulation
of the NF-.kappa.B pathway.
[0066] The composition as disclosed in present invention may be
provided as compounds with pharmaceutically acceptable carriers.
Present invention further discloses the use of therapeutically
effective amount of a composition in the preparation of a
medicament for preventing or inhibiting the growth of pancreatic
cancer in an individual by administering to an individual in need
thereof. The composition is accompanied with or without
conventional chemotherapy or radiation therapy or surgery, or it
may be administered orally or as a food supplement.
[0067] It is understood by a person skilled in the art that the
methods for experiments and studies are described as
exemplifications herein and thus the results are not intended,
however, to limit or restrict the scope of the invention in any way
and should not be construed as providing conditions, parameters,
agents, chemicals or starting materials which must be utilized
exclusively in order to practice the present invention. It is
therefore understood that the invention may be practiced, within
the scope of the appended claims, with equivalent methods for the
experiments than as specifically described and stated in
claims.
* * * * *