Search

Background: Intrahepatic cholangiocarcinoma (ICC) incidence and mortality are increasing in the United States and worldwide. ICC etiologies involve chronic inflammation. We hypothesize that the nonsteroidal anti-inflammatory agent sulindac may prevent ICC by targeting cyclooxygenase-1 and -2 (COX-1, -2) as well as COX-independent pathways., Materials and Methods: ICC was induced with the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) in Syrian golden hamsters. Cholangiocarcinogenesis was accelerated by a choline-deficient diet and administration of DL-ethionine and L-methionine. Hamsters were gavaged twice daily for 10 wk with vehicle or sulindac 25, 50, or 75 mg/kg/dose. Harvested livers underwent gross and histopathological examinations. Tissues were analyzed by immunostaining, Western blot, and enzyme-linked immunosorbent assay (ELISA)., Results: ICC incidence and multiplicity were decreased in sulindac treatment groups versus control (P < 0.05). In addition, ICC and nontumor lesion sizes decreased in treatment versus control animals. Proliferative indices (Ki-67 immunostaining) decreased and apoptosis (ApopTag immunostaining) increased in treatment versus control (P < 0.05). No changes in COX-1 and -2 protein levels were detected by Western blot. Furthermore, prostaglandin E(2) (PGE(2)) levels were unchanged in treatment and control serum and liver tissues (P > 0.05), suggesting that the antitumor effects of sulindac are mediated by COX-independent mechanisms. Nuclear p65 (activated NF-kappaB) immunostaining decreased (P < 0.05), and protein levels of the NF-kB inhibitor IkappaB-alpha increased in treatment versus control groups. p65 ELISA of liver extracts confirmed decreased NF-kappaB binding activity in sulindac-treated versus control animals (P < 0.05)., Conclusion: Sulindac effectively prevents experimental cholangiocarcinogenesis, in part by inhibiting the NF-kappaB pathway.

The design of novel targeted or combination therapies may improve treatment options for pancreatic cancer. Two targets of recent interest are nuclear factor-kappaB (NF-kappaB) and cyclooxygenase (COX), known to be activated or overexpressed, respectively, in pancreatic cancer. We have previously shown that parthenolide, a proapoptotic drug associated with NF-kappaB inhibition, enhanced the growth suppression of pancreatic cancer cells by the COX inhibitor sulindac in vitro. In the present study, a bioavailable analogue of parthenolide, LC-1, and sulindac were evaluated in vivo using a xenograft model of human pancreatic cancer. Treatment groups included placebo, low-dose/high-dose LC-1 (20 and 40 mg/kg), low-dose/high-dose sulindac (20 and 60 mg/kg), and low-dose combination LC-1/sulindac (20 mg/kg each). In MiaPaCa-2 xenografts, tumor growth was inhibited by either high-dose sulindac or LC-1. In BxPC-3 xenografts, tumor size was significantly reduced by treatment with the low-dose LC-1/sulindac combination or high-dose sulindac alone (P < 0.05). Immunohistochemistry of BxPC-3 tumors revealed a significant decrease in Ki-67 and CD31 staining by high-dose sulindac, with no significant changes in COX-1/COX-2 levels or activity in any of the treatment groups. NF-kappaB DNA-binding activity was significantly decreased by high-dose LC-1. Cyclin D1 protein levels were reduced by the low-dose LC-1/sulindac combination or high-dose sulindac alone, correlating with BxPC-3 tumor suppression. These results suggest that LC-1 and sulindac may mediate their antitumor effects, in part, by altering cyclin D1 levels. Furthermore, this study provides preclinical evidence for the therapeutic efficacy of these agents.

Activation of the transcription factor nuclear factor-kappa B (NF-kappa B) has been implicated in pancreatic tumorigenesis. We evaluated the effect of a novel NF-kappa B inhibitor, parthenolide, a sesquiterpene lactone isolated from the herb feverfew, in three human pancreatic tumor cell lines (BxPC-3, PANC-1, and MIA PaCa-2). Parthenolide inhibited pancreatic cancer cell growth in a dose-dependent manner with substantial growth inhibition observed between 5 and 10 micromol/L parthenolide in all three cell lines. Parthenolide treatment also dose-dependently increased the amount of the NF-kappa B inhibitory protein, I kappa B-alpha, and decreased NF-kappa B DNA binding activity. We have previously shown that nonsteroidal anti-inflammatory drugs (NSAID) suppress the growth of pancreatic cancer cells. To determine whether inhibition of the NF-kappa B pathway by parthenolide could sensitize pancreatic cancer cells to NSAID inhibition, BxPC-3, PANC-1, and MIA PaCa-2 cells were treated with parthenolide and the NSAID sulindac, either alone or in combination. Treatment with the combination of parthenolide and sulindac inhibited cell growth synergistically in MIA PaCa-2 and BxPC-3 cells and additively in PANC-1 cells. In addition, treatment with the parthenolide/sulindac combination lowered the threshold for apoptosis. Increased levels of I kappa B-alpha protein were detected, especially in MIA PaCa-2 cells, after treatment with parthenolide and sulindac compared with each agent alone. Similarly, decreased NF-kappa B DNA binding and transcriptional activities were detected in cells treated with the combination compared with the single agents, demonstrating cooperative targeting of the NF-kappa B pathway. These data provide preclinical support for a combined chemotherapeutic approach with NF-kappa B inhibitors and NSAIDs for the treatment of pancreatic adenocarcinoma.

Objectives: The MAP kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway is critical for cell growth and survival. In the current study, we examined the effect of inhibiting the MEK-ERK pathway in pancreatic tumor cells with the MEK-specific inhibitor U0126. In addition, we investigated whether the MEK-ERK pathway influenced the response of pancreatic cancer cells to nonsteroidal antiinflammatory drugs (NSAIDs)., Methods: Cell growth was monitored by a colorimetric proliferation assay and cell counts. Cell cycle analysis was performed using flow cytometry. Apoptosis was measured using a DNA fragmentation ELISA. Protein expression was detected by Western blot., Conclusion: Treatment with U0126 dose dependently inhibited the growth of 3 human pancreatic carcinoma cell lines (BxPC-3, PANC-1, and MIA PaCa-2). U0126 treatment resulted in cell-cycle alterations but did not induce apoptosis. Growth inhibitory concentrations of NSAIDs unexpectedly increased ERK phosphorylation in BxPC-3 and MIA PaCa-2 cells. We therefore evaluated the effect of treating pancreatic tumor cells with the combination of the NSAID sulindac and U0126. Treatment with U0126 complemented sulindac-induced growth inhibition in BxPC-3 and PANC-1 cells. The expression of several cell cycle (p21, p27, cyclin D1) and apoptotic (survivin, Bcl-xL) regulatory proteins was altered after U0126 and/or sulindac treatment. Our findings suggest that inhibition of the MEK-ERK signaling pathway may sensitize pancreatic tumor cells to NSAID therapy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) may be effective treatment for pancreatic cancer. We have previously demonstrated that NSAIDs suppress pancreatic cell growth in vitro by inhibiting cell cycle progression but have little effect on apoptosis. In fact, we have shown that NSAIDs, in some instances, increase Akt phosphorylation in human pancreatic carcinoma cells suggesting activation of the phosphatidylinositol 3'-kinase (PI3K)-Akt survival (antiapoptotic) pathway. We subsequently examined the effects of treating the human pancreatic cancer cell lines BxPC-3 and PaCa-2 with a specific inhibitor of the PI3K/Akt pathway, LY294002, in the presence or absence of the NSAID sulindac. The growth effects of sulindac (250 to 500 micromol/L) and/or LY294002 (1 to 100 micromol/L) were determined by a colorimetric proliferation assay and cell counts. The combination of low-dose LY294002 (10 micromol/L) and sulindac enhanced the growth inhibitory effects of sulindac in BxPC-3 and PaCa-2 cells. Treatment of both cell lines with the LY294002/sulindac combination altered the cell cycle distribution as determined by flow cytometry and also lowered the apoptotic threshold as measured with an enzyme-linked immunosorbent assay to detect DNA fragmentation. These effects were associated with changes in the expression and/or phosphorylation level of proteins and kinases that regulate cell cycle progression and apoptosis. Taken together, our results suggest that inhibition of the PI3K/Akt signaling pathway may sensitize pancreatic tumor cells to therapy with NSAIDs such as sulindac.

Cyclooxygenase-2 (COX-2) has been implicated in the development of gastrointestinal malignancies. The aim of the present study was to determine COX-2 expression/ activity throughout stages of experimental and human pancreatic neoplasia. COX-2 immunohistochemistry was performed in pancreata of hamsters subjected to the carcinogen N-nitrosobis-(2-oxopropyl) amine (BOP) and in human pancreatic tumors. COX-2 activity was determined by prostaglandin E2 assay in tumor versus matched normal pancreatic tissues. The activity of the COX inhibitor sulindac was tested in the PC-1 hamster pancreatic cancer model. COX-2 expression was elevated in all pancreatic intraepithelial neoplasias (PanINs) and adenocarcinomas. In BOP-treated hamsters, there were significant progressive elevations in COX-2 expression throughout pancreatic tumorigenesis. In human samples, peak COX-2 expression occurred in PanIN2 lesions and remained moderately elevated in PanIN3 and adenocarcinoma tissues. COX-2 activity was significantly elevated in hamster and human pancreatic cancers compared to pair-matched normal pancreas. Furthermore, hamster pancreatic tumor engraftment/formation in the PC-1 hamster pancreatic cancer model was reduced 4.9-fold by oral administration of sulindac. Increased COX-2 expression is an early event in pancreatic carcinogeneses. The BOP-induced hamster carcinogenesis model is a representative model used to study the role of COX-2 in well-differentiated pancreatic tumorigenesis. COX inhibitors may have a role in preventing tumor engraftment/formation. [ABSTRACT FROM AUTHOR]