Par-4 Expressing Level Correlates Cisplatin Resistance In Pancreatic Cancer Cells

Pancreatic cancer is a highly malignant tumor of the serious harm to people‘s health.According to the world health organization‘s estimation,pancreatic cancer ranks 13 th in cancer incidence,and 8 th in mortality rate.it‘s a common tumor of our department.Since early pancreatic cancer had no obvious symptoms,usually diagnosed patients were in advanced stage.About 90% of the patients can't perform surgical eradication therapy.Overall,the five-year survival rate is very low,usually below 5%.The majority of patients need to receive chemotherapy,but the traditional chemical treatment for pancreatic cancer control provides little help.5 – Fluorouracil(5-FU)is commonly used in the past,but its reaction rate to pancreatic cancer is rarely more than 25%.Currently the first-line drug to invasive pancreatic cancer is gemcitabine only,and the recommended dose is 1000 mg/m2.But the reaction rate of the gemcitabine is still low,and it can‘t cure pancreatic cancer.The main effect of gemcitabine is to improve the quality of life,including its low toxici ty,relieving pain,and the survival time may be prolonged.Because there is not truly effective anti-cancer drugs available,FDA approved that gemcitabine could be applied in pancreatic cancer.In November 2005,the us food and drug administration approved the use of target drug erlotinib(tarceva)and gemcitabine combined as the first-line regimen to advanced pancreatic cance.although the survival time and the reaction rate is statistically significant,but the clinical benefit is not obvious.Cisplatin(CDDP)is a classic chemotherapy drug.Because the pancreatic cancer is likely to happen cisplatin resistance,therefore,cisplatin is not in pancreatic cancer chemotherapy.And the mechanism of the cisplatin resistance in pancreatic cancer is still not clear.The epithelial-mesenchymal transition(EMT)is a magical process that epithelial cells lose their cell features,and lose their adhesion,and become invasive and migratory,and then turn into mesenchymal cells.Recently,growing evidences have Suggested that EMT may play a great role in the development of chemoresistance in cancer therapy.In pancreatic cancer,Zeb-1 may induce EMT,and give rise to drugresistance in human pancreatic cancer cells.This suggests that EMT contributes to drug resistance in pancreatic cancer.So the important EMT molecular mechanism,the drug-resistant mechanism of pancreatic cancer cells,researching high-effective therapy,and exploring potential therapeutic targets,reversing the drug-resistant mechanism,has important significance.Prostate apoptosis response-4(Par-4)is highly expressed in the prostate and was initially identified in prostate cancer cells that were induced to undergo apoptosis.Par-4 in a series of human cancer tissues have been found downregulated including pancreatic cancer.Par-4 downregulation has been proposed to be a critical event in tumorigenesis.Ectopic Par-4 high expression will lead to most of the cancer cell apoptosis,but does not cause immortalized cells and normal cell apoptosis.Besides,Par-4 can increase sensitivity of cancer cells to apoptotic agents such as doxorubicin,TNF-?,and TNF-related apoptosis-inducing ligand.Research also shows that Par-4 increased sensitivity to the 5-FU in colon cancer cell.Specially in pancreatic cancer,Par-4 plays a critical role in determining the sensitivity of pancreatic cancer cells to small-molecule inhibitors of Bcl-2 family proteins-induced apoptosis.Nevertheless,whether Par-4 is involved in CDDP resistance in cancer cells remains to be defined.This study was to reveal relationship between Par-4 and CDDP resistance in pancreatic cancer cells.1? To establish cisplatin induced CDDP resistant pancreatic cancer cell line BXPC-3/CDDPThe CDDP concentration in medium starts from 0.1?g/ml,and rises to 1.0 ?g/ml gradually.BXPC-3 / CDDP cell line.Compared with BXPC-3 cell,the cells are more slender,scattered,like mesenchymal cell.We use the MTT test to detect the drug resistance in cells.Results show BXPC-3 / CDDP cells compared with its parent BXPC-3 is more drug resistant.The establish of pancreatic cancer cells line BXPC-3/CDDP which of cisplatin resistance successes.2? Detect BXPC-3 and BXPC-3 / CDDP ?s EMT markers by Western blot.Including snail,twist1,E-cadherin,N – cadherin.Evaluate the ability of invasion and metastasis of the two cells.Results show that,compared with BXPC-3 cells,BXPC-3 /CDDP cells‘ snail expression level rises,level of twist1 expression rises,level of E-cadherin drops,N-cadherin level rise.Transwell experiments showed: BXPC-3 / CDDP compared with its parent cell BXPC-3 invasiveness rises.The above results show that: in BXPC-3 / CDDP EMT happened.3? Western blot detects Par-4 protein levels,and q RT-PCR detects Par-4 mRNA level.The results suggest that,compared with BXPC-3,BXPC-3 / CDDP‘s Par-4 level is lower both in protein and mRNA expression.4? Over express par-4 in BXPC-3/CDDP cells by transfected pcDNA3.1-par-4.Western blot detects EMT relevant markers E-cadherin,N-cadherin.Transwell assay detects invasiveness after transfection.MTT assay detects drug resistance.Reasults showed that after transfected pcDNA3.1-Par-4,BXPC-3/CDDP cells‘ E-cadherin level rose,N-cadherin level dropped,invasiveness dropped,CDDP resistance ability droppe d.The above results show that: after transfected pcDNA3.1-Par-4 in BXPC-3 / CDDP,MET(mesenchymal to epithelial transition)happened.5? Adopt the strategy of RNA interference in BXPC-3,reduce the Par-4 expression.After transfected Par-4 si RNA,Western blot detects EMT relevant markers E-cadherin,N-cadherin.Transwell assay detects cell invasiveness.Reasults showed that after transfected Par-4 si RNA,BXPC-3 cells‘ E-cadherin level dropped,N-cadherin level rose,invasiveness rose,CDDP resistance ability rose.The above results show that: after transfected Par-4 si RNA in BXPC-3,EMT happened.6? Adopt the strategy of RNA interference in BXPC-3,and add PI3k/Akt blocker LY294002.After transfected Par-4 si RNA,and added PI3k/Akt blocker LY294002,EMT relevant markers E-cadherin,N-cadherin is detected by Western blot.Transwell assay detects cell invasiveness.Reasults showed that compared with the group transfected Par-4 siRNA but without LY294002,the group transfected Par-4 siRNA,and added PI3k/Akt blocker LY294002,had a higher level of E-cadherin,lower level of N-cadherin,and invasiveness dropped,CDDP resistance ability dropped.The above results show that: after transfected Par-4 si RNA in BXPC-3,and added PI3k/Akt blocker LY294002,the EMT process was reversed.Another western blot test confirmed that after transfected Par-4 siRNA,the pAkt/total Akt ratio in BXPC-3 showed a trend of rising.7? Xenograft studiesIn vivo studies,xenograft BXPC-3 tumors were sensitive to CDDP treatment.Treatment with CDDP alone had little effect on the growth of Par-4 siRNA-transfeced BXPC-3 tumors in nude mice and the survival rate compared with control.Inhibition of PI3K/Akt pathway using LY294002 reversed CDDP resistance in Par-4 siRNA-transfected BXPC-3 tumors.Above all,we know thatPar-4 downregulation confers CDDP resistance via PI3K/Akt pathway-dependent EMT in BXPC-3 cells.