| Esophageal carcinoma(EC), one of the most common malignancies in China,has rather high incidence and mortality in Linzhou city (formerly Linxian county) andthe nearby Huixian county of Henan province. To treat this cancer, chemotherapy isoften considered as one major treatment of choice. The phenomena of multidrugresistance (MDR), however, hiders the effective therapy and is the most commoncause to the chemotherapy-based treatment failure. Numerous clinical data reveal thatthe multidrug resistance in tumor is related to death of more than 90% of tumorpatients. Multidrug resistance is defined that the cancer cells, after exposed to onedrugs, may produce cross-resistance to other drugs whose structure and function areunlike. The ability of drug resistance comes from the selective pressure that the cancercells have to face to and it is one of the major reasons which result in the recrudesceof tumor. Diverse mechanisms might contribute to the MDR of cancer cells; thegenerally accepted mechanism is that the pump proteins in the membrane activelyexpel the cytotoxic drugs from cells, maintaining the drug level below a cell-killingthreshold. There are also other mechanisms play their roles in drug resistance: such as,the enhanced detoxification ability (e.g. Glutathione s-Transferase) and the enhancedability to repair damaged DNA (e.g. DNA polymeraseβ), the quantity or activityalteration of drug target proteins (e.g. topoisomeraseⅡ) and the alteration of activityand expression of protein kinase C. Usually MDR of one kind of tumor cell may be mediated by several different mechanisms and MDR of different tumor cells may becaused by different mechanisms.Therefore, establishing a drug-resistant cell line in vitro, screening drug-resistantassociated genes and searching for the key molecular alteration on gene expressionlevel all have important theoretical and practical senses to us in comprehensiveunderstanding drug-resistance mechanism and identifying target molecules whichcould reverse the drug resistance.The present study was designed to induce a drug-resistant cell lineEC9706/cDDP from a parent human esophageal carcinoma cell line EC9706. Afterthat, human esophageal carcinoma drug-resistance associated genes were screenedwith RT-PCR. Then, GST-πand polβwere selected for further research on theirrelativity to drug resistance, polβand GST-πgene expressions were regulated andeffects on the drug-resistance of esophageal carcinoma cell were observed. Eukaryoticexpression vectors of these two genes were constructed and transfected into EC9706cell lines respectively, and then the biological effects of the different transfected cellswere observed, siRNA lentivirus expression vectors targeting to the two genes werealso constructed respectively, and then infect EC9706/cDDP cell line. The biologicaleffects of the different infected cells were observed.This project includes four parts:Part OneEstablishment of a Cisplatin-induced Human Esophageal Carcinoma DrugResistant Cell Line EC9706/cDDP and Screening of drug resistance-related genesMethods: The drug-resistant cell line EC9706/cDDP was established in culture bypulse exposing EC9706 parent cells to moderate concentration ofcis-diamminedichloroplatinum (cDDP) over a period of 9 months. Morphologicstudies were performed with light microscopy. Methyl thiazolyl tetrazoliumcytotoxicity assay (MTT) was adopted to determine the drug sensitivities to cDDP,5-fluorouracil (5-FU), vincristine (VCR) and paclitaxel. Influence of freezing anddrug withdraw on the resistance were studied. Cell growth curve was paintedafterward, the doubling time and adherent efficiency were accounted. Comparison ofthese data was performed. Flow cytometry (FCM) was used to determine cell cycle. P-gp activity was evaluated by Rh123 influx and effiux assay. The expressions ofpolβ, mdrl, MRP, LRP, DHFR and GST-πmRNA were analyzed by RT-PCR.Results: Nine months were taken to establish drug resistant cell lines EC9706/cDDP.No obvious morphologic changes were observed between resistant and parent sellline. Sensitivity to cDDP was decreased and the resistance index (RI) to cDDP was15.7. At the same time, EC9706/cDDP had cross-resistance to 5-FU, VCR andpaclitaxel which the cells never exposed to. The RIs were 3.2, 2.8 and 1.8respectively. When EC9706/cDDP was stored with cDDP at -196℃for 3 month andthen was recovered, its characterization of anti-tumor drug resistance was stillmaintained. But if it was cultured in RPMI-1640 without cDDP for a period of 1month, the resistant index will decrease. Compared to the parent cells, EC9706/cDDPhad a prolonged doubling time (29.79±0.48h vs 25.79±0.45h)(P＜0.05), lowergrowth rate. The drug-resistance cell line also has significantly increased number of Sand Gl-phase cells and clono-genicity (P＜0.05). There is no statistic significancebetween the differences of Rhl23 influx and effiux of these two cell lines (P＞0.05).Compared with EC9706 parent cells, the expressions of polβ, DHFR and GST-πmRNA in EC9706/cDDP cell line increased obviously (P＜0.05) . However, theexpressions of TopoⅡdecreased(P＜0.05)and there were no changes of expressionofmdrl, MRP (P＞0.05).Part TwoConstruction of GST-π, polβeukaryotic expression vectors and research on thebiological effects of EC9706 cells transfected by the recombinantsMethods:1. Whole cDNA primer of GST-πwith sticky ends was designed and mRNA wasextracted from EC9706 cell line. And thus the whole GST-πgene of EC9706 cell wasobtained by RT-PCR and inserted into T vector. Recombinant was sequenced. Thensubclone of GST-πgene recombined with plRES2-AcGFP 1 vector was constructed.2. Whole cDNA primer of polβwith sticky ends was designed. Open reading frame ofpolβwas amplified from the wild type polβrecombinant plasmid pcDNA3.1-polβ,and inserted into T vector. Then subclone of polβgene recombined withpIRES2-AcGFP1 vector was constructed. 3. The recombined vectors were transfected into EC9706 cell lines with lipofectaminemethod respectively; and screened with G418 for two weeks. Stably overexpressiveEC9706 cell lines carrying wild type GST-πor polβwere obtained. Semi-quantitativeRT-PCR and western blot were performed to detect the expression level of GST-πandpoll3 in transfected EC9706 cell lines. Morphologic studies were performed with lightmicroscopy. Drug sensitivity to cDDP was determined by MTT. Double-layer softagar assay was used to determine the malignancy of cells.Results:1. Eukaryotic expression vector containing GST-πgene (plRES2-AcGFP1-GST-π)and vector containing wild type polβgene (plRES2-AcGFP1-polβ) were constructedsuccessfully. They were transfected into EC9706 respectively and green fluorescencecan be detected on the transfected cells under fluorescence microscope. The EC9706cells which overexpress GST-πor wild type polβgene was isolated by screening withG418. There is no morphologic difference between ante- and post-transfection.2.The relative expression level of GST-πmRNA in cells (2.34±0.12)transfected withplRES2-AcGFP1-GST-πwas higher than that in cells (0.94±0.14)without transfection(P＜0.05). Expression of GST-πprotein was also increased. RI of transfected cells tocDDP was 2.36.3. The relative expression level of polβmRNA in cells (1.21±0.15)transfected withplRES2-AcGFP 1-polβwas higher than that in cells (0.52±0.13)without transfection(P＜0.05). Expression of polβprotein was also increased. RI of transfected cells tocDDP was 1.78. The result of soft agar growth test indicated that the clono-genicity ofEc-9706-polβcell line is 57%, compare to EC9706 (24%) and EC9706-KC (26%),there is no statistic significance(P＜0.05).Part ThreeConstruction of lentivirus of siRNA targeting to GST-π, polβand its reversal ofdrug-resistanceMethods: Two pairs of single strand DNA template, which code short hairpin RNA(shRNA) of GST-π, polβ, were designed and synthesized respectively. They wereannealed and cloned into lentiviral expression vector pRNAT-U6.2/Lenti which contained U6 promoter and green fluorescent protein (GFP). Recombinants wereconfirmed by PCR and sequenced. Four lentviral vector of siRNA targeting to GST-π,poll3 were constructed: GSTsil,GSTsi2,polsil,polsi2. 293FT cells werecotransfected with lentiviral vector and ViraPowerTM Packaging Mix. The titer ofvirus was tested according to the expression level of GFP. EC9706/cDDP cells wereinfected by the lentivirus respectively. Semi-quantitative RT-PCR and western blotwere performed to detect the expression level of GST-πand polβin infectedEC9706/cDDP cell lines. Drug sensitivity to cDDP was determined by MTT.Interference effect was observed by immunofluorescence assay.Results:1. Lentiviral vector of siRNA targeting to GST-π, polβ(GSTsi1,GSTsi2,polsi1,polsi2) were constructed. The titer of virus was up to 1.8-2.3×107 CFU/ml. Thesevectors have high infective rate to cells and the transfected cells emitted greenfluorescence under fluorescence microscope.2.The relative expression level of GST-n mRNA in cells infected by GSTsi1 andGSTsi2 were 0.27±0.03, 0.12±0.02 respectively, lower than that in controlcells(0.91±0.03)(P＜0.05). Expression of GST-πprotein was also decreased. RI ofinfected cells to cDDP was 9.59. In immunofluorescence assay, the number ofinfected cells that emitted red fluorescence under fluorescence microscope is lowerthan than that of control cells.3.The relative expression level of polβmRNA in cells infected by polsil and polsi2were 0.09±0.02,0.28±0.05 respectively, lower than that in control cells(0.62±0.03)(P＜0.05). Expression of polβprotein also decreased. RI of infected cells to cDDPwas 13.9. In immunofluorescence assay, the number of infected cells that emitted redfluorescence under fluorescence microscope is lower than than that of control cells.Part FourNude Mice ExperimentMethods:1. EC9706 and EC9706/cDDP cells were subcutaneously injected into athymic nudemice to set up the animal models of grafted tumor. When the tumors were about 5mm in diameter, the animals were treated with chemotherapy.2.Four nude mice with EC9706 cells xenografts were injected with cDDP (200μg/ml,0.1 ml every 3 days) into their tumors.3. Sixteen nude mice with EC9706/cDDP cells xenografts were divided into 4 groupsrandomly. They are PBS group, cDDP (200μg/ml) group, cDDP plus GSTsi2 group(lentivirus targeting to GST-n and 200μg/ml cDDP, mix evenly), cDDP plus polsilgroup (lentivirus targeting to poll3 and 200μg/ml cDDP, mix evenly). The animals ineach group were treated with PBS, cDDP and cDDP plus lentivirus respectively.(0.1ml/3days/4). Tumor's volumes were measured regularly.4. The mice were killed after chemotherapy, xenografts were extracted and volumeswere measured.Results:1. EC9706 and EC9706/cDDP all have oncogenic activity in nude mice. They inducedthe formation of endermic grafted tumors about 8d post-transplantation. All animalgenerated tumor. The tumor size reached up to 4～5mm in diameter after 12d. Thetime which tumor formed in nude mice treated with EC9706 or EC9706/cDDP is7.75±0.96d or 8.00±0.82d, there is no statistic significance between them (P＞0.05).2. By the end of treatment with cDDP, the tumor volume of nude mice with EC9706cells xenografts and nude mice with EC9706/cDDP cells xenografts were610.00±35.59 mm3 and 990.00±99.33 mm3,respectively (P＜0.05).3. The tumor volume of nude mice with EC9706/cDDP cells xenografts treated withPBS, cDDP(200μg/ml)were 1124.00±25.13 mm3, 990.00±99.33 mm3,respectively. Nosignificance can be observed(P＞0.05). The tumor volume of the group of cDDP plusGSTsi2 was 747.50±72.30 mm3 after treatment, it is lower than that of groups of PBSand cDDP(P＜0.05); while the tumor volume of the group of cDDP plus polsil was937.50±118.70 mm3,and had no significant difference with groups of PBS and cDDP(P＞0.05).Conclusion:1. The newly established stable cell line EC9706/cDDP possessed typical multidrugresistant characters and its drug-resistant phenotype is steady. It provides basis for thefurther study and could be used as an ideal model for the research on mechanism andreversal of drug resistance. Abnormal expression of GST-π, DHFR, polβand TopoⅡ genes played a major role in the development of MDR. Expression of mdr-1 and MRPwere not involved.2. Overexpression of GST-πgene results in decrease in sensitivity of cells to cDDP.Silencing of GST-πby RNAi can partly reverse the drug resistance of EC9706/cDDPcell.3. Overexpression of polβgene produces the condition which sensitivity of cells tocDDP decreased and the malignant phenotype increased. Silencing of polβby RNAican partly reverse the drug resistance of EC9706/cDDP cell.4. Nude mice model with EC9706 cells xenografts or EC9706/cDDP cells xenograftswas set up. It may provide an ideal type for the study of MDR in vivo.5. Injecting cDDP plus GSTsi2 to xenografts derived from drug resistant cells couldinhibit its growth; while injecting cDDP plus polsil to xenografts could not inhibit itsgrowth.
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