Effects Of Cisplatin And Paclitaxel Resistance On The Malignant Phenotype Of Esophageal Cancer Cells And Its Mechanism

Objective:1.In vitro establishment of human esophageal cancer cisplatin-resistant cells KYSE150/CDDP and paclitaxel-resistant cells KYSE150/PTX.2.To observe the effect of cisplatin and paclitaxel resistance on the malignant phenotype of human esophageal cancer cells.3.To explore the molecular mechanisms affecting cisplatin and paclitaxel resistance.Methods:1.Using low-dose intermittent induction + increasing concentration in vitro,using cisplatin and paclitaxel as induction drugs,establishing human cisplatin-resistant KYSE150/CDDP cell line,human esophageal cancer paclitaxel-resistant KYSE150/PTX cell line,observe the morphology of drug-resistant cells during the construction process.Then the MTT method was used to measure the drug resistance index of KYSE150/CDDP cells and KYSE150/PTX cells and whether there was cross-resistance.2.Using MTT assay,cell colony formation assay,DAPI staining,adhesion assay and scratch healing assay,in vitro tubule formation assay to observe cisplatin and paclitaxel resistance to esophageal cancer cell proliferation,apoptosis,adhesion,migration,and the effects of angiogenesis.3.The expression levels of Caspase-3,E-cadherin and MMP-2,VEGF and IQGAP1 in KYSE150/CDDP cells,KYSE150/PTX cells were detected by Western blot.4.To investigate whether the IQGAP1 gene interferes with the chemosensitivity of KYSE150/CDDP and KYSE150/PTX cells.Firstly,the IQGAP1 gene short hairpin RNA interference plasmid was constructed and identified,then KYSE150/CDDP cells,KYSE150/PTX cells were stably transfected,and the expression level of IQGAP1 protein was identified by Western blot.Finally,it was detected by MTT,DAPI staining,scratch and adhesion test.The effect of IQGAP1 on chemosensitivity,proliferation,apoptosis and migration ability of drug-resistant cells,and the changes of MMP-2 protein and ERK pathway were detected by Western blot.Results:1.Over 10 months,establish human cisplatin-resistant KYSE150/CDDP cell line and human esophageal cancer paclitaxel-resistant KYSE150/PTX cell line,observe the morphology of drug-resistant cells under microscope,and find that KYSE150 cells are stable to CDDP and PTX.No changes in cell morphology were observed after drug resistance.2.Compared with the parental KYSE150 cells,the results of MTT and cell clone formation showed that the growth rate of cisplatin and paclitaxel-resistant cell lines was slowed down and the proliferation ability was also decreased.The results of DAPI staining indicated that cisplatin and paclitaxel-resistant cells were present.The anti-apoptosis ability was enhanced;the results of scratch healing experiments showed that the scratch healing ability of KYSE150 cells was increased after cisplatin and paclitaxel resistance for24 h.The adhesion test results showed that the intercellular aggregation ability of the two groups of resistant cells was weakened and separated.The ability to enhance,suggesting that cisplatin and paclitaxel resistance decreased cell adhesion and migration ability;in vitro tubule formation experiments showed that angiogenic ability of cells after cisplatin and paclitaxel resistance was enhanced.3.Western blot results showed that compared with the parental KYSE150 cells,the expression levels of Proaspase-3,MMP-2,VEGF and IQGAP1 in cisplatin and paclitaxel-resistant cells increased,while the expression of E-cadherin decreased.4.The sequencing of the short hairpin RNA interference plasmid of IQGAP1 gene was confirmed by sequencing.The proportions of cells with GFP were(76±5.780)% and(83±3.851)%,respectively.RT-PCR and Western blot also showed that the constructed IQGAP1 interference vector could significantly inhibit the expression of IQGAP1 gene in KYSE150 cells.The stable cell line and the negative control cell line of IQGAP1 interference were constructed by G418 screening and named K/D/sh RNA and K/D/Control,K/P/sh RNA and K/P/Control,Western blot results showed that IQGAP1 protein expression level in K/P/sh RNA cells was significantly lower than that in K/P/Control cells,suggesting that the stable drug resistance cell lines of IQGAP1 interference were successfully constructed;interfering with the expression of IQGAP1 gene reduced drug tolerance of drug-resistant cells,decreased anti-apoptotic ability,enhanced intercellular adhesion and decreased migration ability.Compared to K/D/Control cells,MMP-2,p-ERK levels were decreased in K/D/sh RNA cells.Conclusions:1.Successfully established human cisplatin-resistant KYSE150/CDDP cell line and human esophageal cancer paclitaxel-resistant KYSE150/PTX cell line,and obtained cisplatin and paclitaxel resistance,cell proliferation ability,anti-apoptosis,migration and Increased angiogenic capacity and decreased intercellular adhesion,suggesting that these changes may be an important reason for the clinical effects of cisplatin and paclitaxel resistance.2.The results of Western blots confirmed that IQGAP1,Procaspase-3,E-cadherin,MMP-2 and VEGF genes are involved in the formation of cisplatin and paclitaxel resistance mechanisms in esophageal cancer.3.The IQGAP1 gene interference vector was successfully constructed,and the recombinant plasmid was used to construct a stable cell line that interfered with IQGAP1 gene expression in KYSE150/CDDP,KYSE150/PTX cells.After IQGAP1 interference,the drug sensitivity of drug-resistant cells and intercellular adhesion were increased,proliferative capacity,and decreased anti-apoptosis,decreased the migration ability of drug-resistant cells by down-regulating MMP-2 levels,and IQGAP1 interference may reverse the resistance of esophageal cancer cisplatin by ERK pathway activation.