The purpose of this study was to determine whether resveratrol, a stilbene (see chemical structure, ) might have a role in the treatment of PaCa either alone or in combination with gemcitabine and if so, through what mechanism. We used 4 different well-characterized human PaCa cell lines derived from various types of pancreatic cancer e.g., AsPC-1 is derived from pancreatic adenocarcinoma; MIA PaCa-2 and Panc-28 are pancreatic carcinoma while Panc-1 represents pancreatic duct cell carcinoma. To facilitate the monitoring of tumor growth in animals, one of these cell lines, MIA PaCa-2 was stably transfected with luciferase reporter and used in the orthotopic transplant model in mice.
Resveratrol inhibits growth, synergizes the effect of gemcitabine and down regulates constitutive NF-κB activation and NF-κB-regulated gene products
We first investigated the effect of resveratrol on the proliferation of 4 different PaCa cell lines. Resveratrol inhibited the growth of all 4 human pancreatic cancer cells (AsPC-1, MIA PaCa-2, Panc-1 and Panc-28) in a dose- and time-dependent manner ().
Whether resveratrol can potentiate the effect of gemcitabine against these 4 cell lines was also examined. We employed an esterase staining assay (live/dead assay) to establish whether resveratrol can potentiate the apoptosis induced by gemcitabine. As shown in , the dose of resveratrol (10 μM) or gemcitabine (100 nM) that had minimum effect on apoptosis alone produced synergistic apoptosis when combined.
How resveratrol potentiates the effects of gemcitabine was also investigated. NF-κB has been shown to be constitutively expressed in these cell lines and causes resistance to apoptosis. Whether resveratrol causes down regulation of constitutive NF-κB activation in MIA PaCa-2 cells was examined by using a DNA binding assay. Results show that the treatment with resveratrol inhibited NF-κB activation in a dose-dependent manner ().
Figure 2 (a) EMSA results showing that resveratrol suppresses the constitutive activation of NF-κB in a dose-dependent manner. The MIA PaCa-2 (1 ×106) cells were treated with indicated concentration of resveratrol for 4 hr, nuclear extracts were (more ...)
Whether resveratrol downregulates the NF-κB-regulated gene products was also examined. We found that resveratrol suppressed the constitutive expression of anti apoptotic (bcl-2, bcl-xL), proliferative (COX-2, cyclin D1), metastatic (MMP-9) and angiogenic (VEGF) protein expression in a dose-dependent manner in MIA PaCa-2 cells (). Resveratrol also induces the cleavage of PARP in MIA PaCa-2 cells ().
Resveratrol inhibits the growth of orthotropic implanted PaCa in nude mice
We examined the therapeutic potential of resveratrol and gemcitabine either alone or in combination on the growth of orthotopically implanted human pancreatic cells in nude mice. The experimental protocol is depicted in . We decided to use MIA PaCa-2 cells for in vivo studies because first, we found MIA PaCa-2 relatively more sensitive from rest of cells and second, MIA PaCa-2 is stably transfected with luciferase. MIA PaCa-2 cells were implanted in the pancreas tails of nude mice. After a week, based on the initial IVIS image we randomized animals into 4 groups and started the treatment per the experimental protocol. The treatment was continued for 4 weeks and animals were sacrificed 6 weeks after tumor cell injection. The IVIS imaging was done on every week after the tumor implantation ( left panel).
Figure 3 Resveratrol potentiates the effect of gemcitabine to inhibit the growth of pancreatic cancer in nude mice. (a) Schematic representation of experimental protocol described in Material and Methods. Group I was given with Vehicle (100 μL, p.o., daily) (more ...)
The bioluminescence imaging results showed a gradual increase in tumor volume in the control group (, right panel) as compared with rest of the groups. The tumor volume in the combination of resveratrol and gemcitabine group was significantly lower than resveratrol alone group (p < 0.01) or gemcitabine alone group (p < 0.05). Gemcitabine alone treatment was shown to be effective as resveratrol (p < 0.001 when compared to control; p > 0.05 when compared to resveratrol alone group) (, right panel).
We found that resveratrol alone when given orally at 40 mg kg−1 significantly inhibited the growth of the tumor (p < 0.001 when compared to control) (). Gemcitabine alone was as effective as resveratrol (p < 0.001 when compared to control; p > 0.05 when compared to resveratrol alone group); and the combination of the two agents were more effective in reducing the tumor burden. The tumor volume in the combination of resveratrol and gemcitabine group was significantly lower than resveratrol alone group (p < 0.01) or gemcitabine alone group (p < 0.01) ().
Resveratrol inhibits CD31 and Ki-67 expression
While Ki-67-positive index is used as a marker for cell proliferation, the CD31 index is a marker for micro vessel density. Whether resveratrol and gemcitabine modulate these markers, was examined. shows that both resveratrol (p < 0.05) and gemcitabine (p < 0.05) alone significantly down regulated the expression of Ki-67 in PaCa tissue and the combination of the 2 was most effective (p < 0.01). Similarly when examined for CD31, we found that both agents significantly reduced the CD31 expression as compared to control group () and two together were most effective (p < 0.01 when compared to gemcitabine alone) ().
Figure 4 Resveratrol enhances the effect of gemcitabine against tumor cell proliferation and angiogenesis in pancreatic cancer. (a) Immunohistochemical analysis of proliferation marker Ki-67 indicates the inhibition of pancreatic cancer cell proliferation in resveratrol (more ...)
Resveratrol inhibited the constitutive NF-κB activation and NF-κB-regulated gene products in PaCa
We evaluated the effect of resveratrol and gemcitabine on NF-κB levels in pancreatic tumor tissues. shows that resveratrol either alone or in combination with gemcitabine was quite effective in suppressing the constitutive expression of NF-κB in pancreatic cancer tissue. Gemcitabine alone had no significant effect on constitutive NF-κB in PaCa tissue.
Figure 5 Effects of resveratrol and gemcitabine against expression of NF-κB and NF-κB-regulated gene products in pancreatic cancer tissue samples. (a) Detection of NF-κB by DNA binding assay (EMSA) in orthotopic tumor tissue samples showed (more ...)
PaCa showed overexpression of proliferative (c-myc, cyclin D1, COX-2), survival (bcl-2, bcl-xL, survivin, XIAP), invasion (ICAM-1, MMP-9, CXCR4) and angiogenesis (VEGF) gene products, all under the control of NF-κB. Whether resveratrol and gemcitabine can modulate the expression of these NF-κB-regulated gene products, was examined by western blot analysis. We found that resveratrol alone significantly down regulated the expression of cyclin D1, COX-2, ICAM-1, MMP-9 and survivin (). It did not significantly affect CXCR4, bcl-2, bcl-xL or VEGF expression. Treatment with both resveratrol and gemcitabine was needed to down regulate CXCR4, bcl-2, bcl-xL and VEGF expression. Thus the combination of gemcitabine and resveratrol was effective in down regulating the overexpression of proliferative (c-myc, cyclin D1, COX-2), survival (bcl-2, bcl-xL, survivin, XIAP), invasion (ICAM-1, MMP-9, CXCR4) and angiogenesis (VEGF) gene products.
Whether modulation of nuclear NF-κB, COX-2, VEGF and MMP-9 can also be detected by immunohistochemical methods was examined. As shown in these gene products were substantially down regulated in resveratrol-treated samples. The down regulation was less impressive with gemcitabine alone. The immunohistochemical data supports the data obtained from western blot.
Figure 6 Immuno histochemical analysis of nuclear p65, COX-2, MMP-9 and VEGF showed the inhibition of NF-κB, COX-2, MMP-9 and VEGF by either resveratrol alone or in combination with gemcitabine. Percentage, positive staining for the given biomarker. The (more ...)
These results collectively indicate that resveratrol suppress the activation of NF-κB thereby inhibiting the expression of genes involved in proliferation, survival, invasion and angiogenesis.