Effects On Pancreatic Cancer Cells Chemotherapy Sensitivity Toward 5-FU By Inhibiting Hedgehog Signaling Pathway

Background and Objective:Pancreatic cancer is highly malignant tumor, the incidence rate in the world is rising, the average survival time after diagnosis is not more than 6 months, its diagnosis and treatment there are still many issues that must be resolved. And there is a very low radical surgical resection rate because most cases are already in the advanced stages when diagnosed. The role of chemotherapy has become more important due to its effect towards tumor lesion and metastasis, as well as remission of symptoms. However, Traditional chemotherapy is ineffective. Five-year survival rate is very low for those treated with chemotherapy. Chemo-resistance is currently a major obstacle in tumor chemotherapy. Most of patients present with unresectable disease, resistant to chemotherapy, and high relapse, making prognosis of Pancreatic carcinoma very disappointing.Hedgehog signaling pathway is one of the self renewal mechanism of cancer stem cell. It is composed of four parts, including HH ligand, two transmembrane protein-acceptor(PTCH),(Smo) compounds and transcriptive protein Gli in downstream. The self renewal ability of cancer cell is become stronger When the Hedgehog signaling pathway is in activation. Cyclopamine is the specificity inhibitor of Hedgehog signaling pathway and it could bound with protein Smo, so the pathway turn into inactivation and reduce the capability of self renewal. Nowadays, researches identified Hedgehog pathway expressed in many kinds of cancers. Hedgehog researches could bring more useful information for killing cancer cell in the further.Methods: 1. SW1990, T3M4 and PANC-1 pancreatic cancer cell lines are cultured in vitro, breeding the next generation.2. Three pancreatic carcinoma cell lines growth curve are analysis (MTT methed).3. Three cell lines were treated with 5-FU of different concentrations to screen suitable anti-drug concentration. And also MTT method 3 pancreatic cancer cells to determine 5-FU chemotherapy sensitivity of IC50 values.4. Different concentration of cyclopamine, which is HH signaling pathway inhibitor, are applied to treated the three pancreatic cancer cell lines. Experiment is divided into four groups:5-FU chemotherapy group, luM and 10uM cyclopamine combine with 5-FU group, tomatidine combine with 5-FU group. The following experiments are carryout:A. MTT test; B. Extracted proteins for Western Blot test; C. Multiple cancer stem cell marker CD133, CD44, CD24, EPCAM joint detection.RESULT:1,SW1990, PANC-1, T3M43 pancreatic cancer cell lines IC50 value of 5-FU chemotherapy were 31.97±5.21um,1.88±0.42mM,5.59±0.93uM. Of the three cell lines, T3M4 of the most sensitive to 5-FU chemotherapy, PANC-1 is not sensitive, and SW1990 is medium.2,Cell proliferation in 48 hours to 72 hours is slow, but after 48-72 hours, cell proliferation significantly accelerated into the logarithmic phase, and reach its peak after 5 days, and enter the plateau phase of cell proliferation. To T3M4 and SW1990 cell lines, after treated with cyclopamine, IC50 value of 5-FU chemotherapy is decreased dramaticaly (P<0.01), luM and 10uM cyclopamine significant difference between the statistical significance (P<0.05). But to PANC-1 cell line, the change is not significant. After the intervention with cyclopamine, Gli-1 which is HH pathway downstream protein decrease.3,after 5-FU chemotherapy treatment, cancer stem cell marker (CD44, CD24) increase significantly in SW1990, and decrease significantly after treated with cyclopamine and 5-FU (P<0.05). And CD133, EPCAM expression of differences among the groups is not significant (P> 0.05).Conclusion:1. In the three pancreatic cancer cell lines, T3M4 is the most sensitive to 5-FU chemotherapy, PANC-1 is not sensitive, and SW1990 is on the sensitivity of 5-FU medium.2. Cyclopamine intervention can significantly improve the T3M4 and SW1990 cells to 5-FU chemotherapy sensitivity, and its effect is proportional to the concentration. But to PANC-1 cells, the chemotherapy sensitivity increase is relatively slow.3. The 5-FU chemotherapy in cancer stem cell marker (CD44. CD24) expression ratio significantly increased, while CD 133 and EPCAM not obvious. the combine intervention of cyclopamine and 5-FU, CD44 and CD24 expression ratio significantly, while the CD133, EPCAM not obvious. Thus, CD24 and CD44 can serve as a pancreatic stem cell surface markers, However, EPCAM and CD 133 are not suitable.4.5-FU chemotherapy resistant cells, with some of the characteristics of cancer stem cells, can be inhibited by HH signaling pathway blockers.