An Experimental Study On Treatment Of Pancreatic Carcinoma With Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

PART ⅠTHE EXPRESSION OF TRAIL RECEPTOR INPANCREATIC CANCERObjective Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a newly discovered member of tumor necrosis factor superfamily. There are four distinct receptors that can interact with TRAIL: death receptor(DR4 and DR5) and decoy receptor(DcRl and DcR2). TRAIL-induced apoptosis can be triggered by TRAIL binding to DR4 and DR5, which have similar cytoplasmic domains called death domain. In contrast, apoptotic signaling can be blocked by interacting with DcR1, which lacks the intracellular domain; with DcR2, which contais a truncated non-functional death domain. DR4 and DR5 are expressed broadly and often concomitantly with TRAIL, whereas the distributions of DcRl and DcR2 are relatively restricted. The competitive binding of TRAIL between these two groups of receptor is thought to influence TRAIL-induced apoptotic signaling in different tissues. This study investigated the expressions of TRAIL receptors in human pancreatic cancer.Methods The expression of TRAIL receptors mRNA were detected by semi-quantitive reverse transcriptase polymerase chain reaction method in tissues of pancreatic cancer and normal pancrease.Results DR4 and DR5 mRNA expressed in all tumor and normal pancrease tissues. DcR1 and DcR2 mRNA were detected in all normal pancreatic tissues, but the positive expression of DcRl in pancreatic cancer tissues was 56.3%(18/32), DcR2 was 62.5%(20/32). The expression of DR4 and DR5 in the pancreatic cancer tissues was significantly higher than that in the normal pancreatic tissues(p<0.05). The expression of DcRl and DcR2 mRNA was unique in all tissues. The expression of DR5 was correlated with cell differentiation and tumor stage. Higher cell differentiation and lower tumor stage determined higher level of DR5 mRNA. The expression of DR4, DcRl and DcR2 did not correlate with cell differentiation, tumor stage or lymphonodus metastasis.Conclusion (1) TRAIL receptors expression is prevalent in pancreatic cancer tissues with diffenent receptor types existing. High death receptor expression may play an important role in TRAIL inducing pancreatic cancer apoptosis. (2) The expression of DR5 is positively correlated with the differential level of pancreatic cancer cell and nagetively correlated with tumor stage. (3) The expression of DR4s DcRl and DcR2 are not a suitable index as the differential level of pancreatic cancer cell.PART IISTUDY OF APOPTOSIS INDUCED BY TRAIL IN PANCREATIC CANCER CELLSObjective Pancreatic cancer is a fairly common yet dismal disease with low response to chemotherapy,radiotherapy and immunotherapy. Therefore, new treatment modalities are important to effectively control and palliate pancreatic cancer disease. TRAIL induces rapid apoptosis in a wide variety of tumor cell lines,but seems to have little or no detected cytotoxic effect on normal cells in vitro and in vivo. In this study, we detect the expression of TRAIL recepors in different pancreatic cancer cell lines, investigate the effect of TRAIL on the growth of human pancreatic cancer cell lines and illustrate its possible mechanism.Methods The expression of TRAIL receptor mRNA was assayed by semi-quantitive RT-PCR in pancreatic cancer cell lines of Canpan-2 and BxPC-3. The pancreatic cancer cells were exposured to different concentrations of TRAIL, the cytotoxicity was detected by MTT assay, the rates of apoptosis were examined by flow cytometry. The change of cell morphology were obsered by phase-congtrast microscopy. DNA ladder assay was employed for the apoptosis.Using TRAIL along or TRAIL together with caspase inhibitor zVAD-fmk to treat canpan-2 cell, and the cell growth inhibition rate was investigated by MTT assay.Results The expression of death receptor 4(DR4)-. death receptor 5(DR5) > decoy receptor (DcRl) and decoy receptor 2(DcR2) was detected in pancreatic cancer cell lines. High death receptor expression in pancreatic cancer cells was differed from low decoy receptor expression with statistical significance.The growth of human pancreatic cancer cell could be significantly inhibited by TRAIL with concentration and time dependence. At the concentration of 100ng/ml,the growth inhibition rate of TRAIL to Canpan-2 cell were (29.25±0.54)°/ 481k 72h respectively, when the concentration was lOOOng/ml, the growth inhibition rate to canpan-2 cell were (55.26±1.37)%> (62.49±1.42)%, (71.34±2.04)% respectively. At the concentration of lOOng/ml, the growth inhibition rate of TRAIL to BxPC-3 cell at the acting time of 241k 481k 72h were (33.08±1.32)%, (47.62±1.60)%, (56.08±1.36)% respectively, when the concentration was lOOOng/ml, the growth inhibition rate to BxPC-3 cell were (59.35±1.19)%, (67.17±2.03)%, (78.52±1.61)% respectively. There was not significant difference of growth inhibition rate between different cell lines. There were characteristic ladder bands after Canpan-2 was treated by TRAIL( 1 OOng/ml) for 24h.The apoptosis rate investigated by flow cytometer was (59.35±1.19)%, (67.17±2.03)%^ (78.52±1.61)% respectively at concentration of 50ng/ml ^ 1 OOng/ml ^ 500ng/ml. When the acting time was 24h ^ 481k 72h, the apoptosis rate was (59.35±1.19)%, (67.17±2.03)%, (78.52±1.61)% respectively. The difference was significant. The typical apoptic cells were observed by phase-congtrast microscopy. There were characteristic ladder bands after Canpan-2 was treated by TRAIL (1 OOng/ml) for 24h. The growth inhibition rate of Canpan-2 treated with TRAIL andzVAD-fmk was (6.26±0.42)%, and (46.54±1.26)% used TRAIL along. There was a significant difference between them.Conclusion (1) TRAIL receptor expression is prevalent in pancreatic cancer cells with different receptor types existing. (2) TRAIL could significantly inhibit the growth of pancreatic cancer cells by inducing apoptosis,and show dose-effect and time-effect relationship. (3) TRAIL-induced apoptosis in Canpan-2 cell mediated through caspase activation.PART IIISYNERGISTIC ACTIVITY OF TRAIL AND CHEMO-THERAPEUTIC AGENTS IN PANCREATIC CANCER CELLObjective TRAIL could not induce apoptosis in all of tumor cells. It is important to investigate how to overcome the resistance to TRAIL and to explore the new methods to increase the sensitivity to TRAIL. When some tumor cell were not sensitive to TRAIL,some factor or chemotherapeutic agents could transverse its resistance. This study examined the synergy of TRAIL and 5-Fu or gemcitabine in both cytotoxic and apoptic assays on pancreatic cancer cell lines, and explored its mechanism.Methods Canpan-2 were treated with different concentration TRAIL along, 5-Fu along, gemcitabine along,and TRAIL together with 5-Fu or gemcitabine, the cell growth inhibition rate and apoptosis rate was investigated by MTT assay and FCM after 24h respectively. The change of cell morphology were obsered by phase-congtrast microscopy. The expression level of DR4x DR5^ DcRl-. DcR2 mRNA were evaluated by semi-quantitive RT-PCR before or after treatment by 5-Fu. Expression of caspase-8, Bcl-2 and Bax were quantified by immunocytochemistry.Results Chemotherapeutic agents, such as 5-Fu and gemcitabine, dramatically augmented TRAIL induced cytotoxic function. There were significant differences in the cytotoxicity between the use of single agent and in combination with TRAIL. Theapoptosis rate by FCM was paralleled with the inhibitory rate by MTT. The structure feature of apoptosis cell such as apoptotic body was found by phase-congtrast microscopy. Those means apoptosis was the main manner of synergitic effect. No significant difference was found between expression levels of DR^ DR5^ DcRl s DcR2 mRNA before and after treatment by 5-Fu in Canpan-2 cells.5-Fu could increase the expression of caspase-8 and Bax.Conclusion (1) The combination of human TRAIL and chemotherapeutic agents, such as 5-Fu and gemcitabine, exert a synergistic effect on pancreatic cancer cell. (2) The synergistic effect is related to the increase of expression of caspase-8 and Bax induced by 5-Fu.