Regulation Of Indomethacin,Single Or In Combination With Erlotinib In The Growth Of Pancreatic Carcinoma

Aims: To examine the effects of indomethacin(IND),a cyclooxygenase inhibitor,single or in combination with erlotinib(ERL),an epidermal growth factor receptor(EGFR) blocker on cell growth in human pancreatic carcinoma and to determine its effects on cell growth,cell cycle progression ,proliferation and apoptosis and on angiogenic regulation of pancreatic cancer cells,so as to elucidate the antitumor activity of IND and (or) ERL on pancreatic cancer cells and its possible mechanisms.Methods: 1.Pancreatic cancer cells were treated with ERL alone or in combination with IND with different concentration and time to evaluate the antiproliferative effects by cell viability using MTT assay. 2.The influence on cancer cell apoptosis,cell cycle progression before or after treatment was detected by transmission electronmicroscopy,terminal deoxynucleotidyl transferase-mediated nick end labeling assay(TUNEL) and flow cytometric analysis.3.RT-PCR was used to investigate the expression of COX-2 and EGFR and some apoptosis-associated protein such as bcl-2,bax,bcl-xl,bak mRNA and to detect the expression of COX-2 and EGFR protein using western blotting in BxPC-3 cancer cells. 4.Pancreatic cancer xenograft tumor model was established in BALB/C nude by inoculating BxPC-3 cells subcutaneously. The weights of the resected subcutaneous tumors, tumor growth curve and suppression rates were observed after four weeks treatment.5.Hematoxylin and eosin (HE) stain was used to observe tumor morphology. The COX-2 and EGFR protein expression in xenograft tissues was determined by immunohistochemical staining.The microvessel density(MVD)was also detected using immunohistochemistry labeled with factorâ…§antibody . VEGF mRNA expression of xenografts was evaluated by RT-PCR after ERL and(or) IND orally administered.Cancer cell apoptosis of xenografts was detected by TUNEL.6.The effects of agents on angiogenesis in vitro was evaluated by tube formation assay. Results: 1.The proliferation of pancreatic cancer cells was significantly inhibited by IND and ERL in a dose and time dependant manner, and the combination of both had a more potent inhibitory effect.2.Transmission electronmicroscopy, TUNEL and flow cytometry demonstrated a significant increase of apoptosis cells proportion and Apoptotic Index (AI) after treatment and cell cycle G0/G1 arrest was higer in therapy groups compared with the control. The combination of the two agents resulted in a synergistic effect on cancer cell apoptosis and cell cycle arrest. 3.Treatment of the cell line with either IND or ERL could dowen-regulate the expression of COX-2 and EGFR mRNA, and combined treatment of IND with ERL resulted in a synergistic effect. COX-2 and EGFR protein was also significantly decreased treated with two agents using western blot analysis.RT-PCR showed the expression of the anti-apoptotic genes,such as bcl-2,bcl-xl mRNA was inhibited by two agents ,while pro-apoptotic genes,bax mRNA had no change in IND group but a little increased in ERL and the combind group. 4.A dramatic delay of tumor progression was observed with a more prolonged time in the therapy groups compared with the control group in a nude mice xenograft model in vivo .The inhibition rate was 74.5%,67% and 82.2% in ERL,IND and the combind group ,respectively ,after 4 weeks therapy. 5.RT-PCR and immunohistochemical staining showed the expression of COX-2,EGFR mRNA and protein was markedly down-regulated in treated mice than those in the control group.The cancer cell apoptosis proportion and AI of xenograft tumor tissues was higher in therapy groups by TUNEL assay. The expression of bax mRNA in xenograft tumor was up-regulated after treated with ERL or ERL combined with IND. 6.The tube formation assay revealed that the Human Umbilical Vein Endothelial Cells were present without closed hollow tubes in IND or ERL therapy groups.The combined treatment resulted in profound suppression of HUVEC tube formation .Immunohistochemistry staining with factorâ…§antibody showed the microvascular density (MVD) was dramatically decreased in therapy groups compared with that in the control. The expression of VEGF mRNA was significantly inhibited after treatment with two agents either in BxPC-3 cancer cell line in vitro or in xenograft tumor tissues in vivo.Conclusions: ERL and(or) IND have a negative regulatory effect on the growth of pancreatic carcinoma both in vitro and in vivo, and the combination of the two agents exerts a synergistically or additively inhibitory effect.The study reminds us that the COX-2 and EGFR signal transduction pathways interact at several levels.The mechanisms of the agents on pancreatic carcinoma involved in the induce of the cancer cell apoptosis,the inhibitory of the cell proliferation,cancer cell cycle G0/G1 arrest,imbalance of the pro-apoptotic genes and the anti-apoptotic factors,down-regulation of the pro-angiogenic factors VEGF,decrease of the pancreatic tumor neovascularization.The COX-2 and EGFR pathways inhibitor regimen may provide a potential and promising strategy for pancreatic cancer chemotherapy.