The Establishment Of Drug Resistance Human Esophageal Squamous Carcinoma Models And The Studies On Anti-drug-resistance Activities And Mechanisms Of Action Of JP

Multidrug resistance is one of the major impediments for effective chemotherapyin cancer patients as cancer cells may become cross-resistant to a broad spectrum ofchemotherapeutic agents after a single drug treatment. Multidrug resistance greatlylimited the effect of the chemotherapy drug and resulted in the failure ofchemotherapy and the recurrence of tumor. Therefore, it is essential to investigate themultifunctional mechanisms and overcoming strategies of multidrug resistance.This project includes two parts: Part one is about the establishment of apaclitaxel resistant human esophageal squamous carcinoma cell line EC109/Taxoland in vivo xenograft model and the exploration of their possible mechanisms ofresistance; Part two is to evaluate the anti-resistance activity of JP, and to explore itspossible mechanisms.Part OneThe establishment of paclitaxel-resistant EC109/Taxol in vitro cell line and invivo xenograft model and the research on the mechanisms of resistanceMethods: The resistant cell line was established in vitro by intermittent exposurehuman esophageal squamous carcinoma cell line (EC109) to a high concentration ofPTX with time-stepwise increment over a period of6months. The multidrugresistance of EC109/Taxol to anticancer agents was evaluated by3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. Themorphological features were observed by invert microscope. Apoptosis was measuredby Flow cytometry (FCM) and Hoechst33258fluorescence staining. Cell growth,cell doubling time and colony formation of EC109and EC109/Taxol cells werecompared. FCM was used to determine the distribution of the cell cycle too. Theprotein levels of Bcl-2, Bax, Procaspase-3and P-gp were detected by western blotting.P-gp activity was evaluated by Rh123accumulation and efflux assay. EC109andEC109/Taxol cells were subcutaneously injected into athymic nude mice to set up theanimal models of grafted tumor. In vivo resistance characterization was investigated. Results: EC109/Taxol cells were of67.175fold resistance to PTX and2.500fold to5-fluorouracil (5-FU),1.191fold to cisplatin (CDDP),3.491fold to epirubicin (EPI).Compared with parent cells, the resistant cells were smaller, mixing with giant cellsin different sizes. FCM and Hoechst33258fluorescence staining confirmed thatEC109cells treated with PTX showed significantly higher percentage of apoptoticcell than EC109/Taxol cells. The proliferation rate of EC109/Taxol cells wassignificantly slower than that of the parental cells, with increased cell doubling time.Simultaneously, the EC109/Taxol cells had less potential to form colonies. Thepercentages of cells in G0/G1and S phases were significantly increased inEC109/Taxol in comparison with those in EC109, while the percentage of(G2/M)-phase cells was decreased. Bcl-2, Procaspase-3and P-gp protein expressionswere efficiently higher in the EC109/Taxol than in the EC109, but Bax was lower inthe EC109/Taxol than in the EC109. Mean fluorescence intensity in EC109/Taxolwas significantly declined by Rh123assay. We successfully established aPTX-resistant EC109/Taxol nude mice model. EC109/Taxol cells did not changePTX resistance in vivo.Part TwoJP up-regulates p53and induces apoptosis in human multidrug-resistantEC109/Taxol cellsMethods: The MTT assay was used to detect the cell proliferation after JP acting onEC109and EC109/Taxol cells, while the colony assay is used to identifyEC109/Taxol proliferative capacity. We can study the influence that JP produced oncell growth inhibition and in its anti-drug activity by cell growth curve; Flowcytometry was taken to detect how JP affects EC109/Taxol cycle distribution. Andthe apoptosis changes that caused by JP is detected by Hoechst33258staining andflow cytometry. To detect the protein expression levels of EC109/Taxol, weconducted western bolt, thus explore the mechanism of JP anti-tumor drug resistanceactivity. Results: JP significantly inhibited the proliferation of SPC-A-1/CDDP cells,induced apoptosis, arrested the cell cycle phase at G2/M phase. In additional, JPaffected the proliferation of EC109/Taxol cells in a dose-and time-dependent manner. Western blot detected that JP can up-regulating the expression of p53andCleaved-caspase-9of EC109/Taxol, and down-regulating the expression ofProcaspase-3, Procaspase-9and Bcl-2of EC109/Taxol in a concentration-dependentmanner.Conclusions:1、 This is the first report on the establishment of EC109/Taxol cell line with higherresistance and a PTX-resistant EC109/Taxol nude mice model. Bcl-2, Bax,Procaspase-3and P-gp are involved in the resistance of cell lines to PTX, which areinvaluable tools to study the resistance of anticancer drugs and to find the methods toovercome the resistance.2、 JP can induce apoptosis in multidrug-resistant human EC109/Taxol cells bydown-regulating the expression of Procaspase-3, Procaspase-9and Bcl-2andup-regulating the expression of p53and Cleaved-caspase-9. The research provides anew way to treat drug-resistant tumors.