Study On The Synergistic Inhibitory Effect Of YAP Inhibitor Verteporfin Combined With Cisplatin On Esophageal Squamous Cell Carcinoma Cells

Objective:1.To investigate the effects of Yes-associated protein 1（YAP1）inhibitors Verteporfin（VP）and cisplatin（CDDP）on the survival rate of esophageal squamous cell carcinoma（ESCC）cells,and to explore whether the combined application of VP and CDDP has a synergistic effect on ESCC cells;2.To explore the effects of VP and CDDP alone and in combination on the malignant phenotype of ESCC cells;3.To explore the molecular mechanism of VP combined with CDDP in ESCC.Methods:1.The effect of different concentrations of VP and CDDP on cell viability after treatment for 24 h,48 h and 72 h was detected by thiazolyl blue tetrazolium bromide（MTT）colorimetric method,and IC₅₀was calculated using Graph Pad Prism 9;2.According to the IC₅₀ of the two drugs,the cells were treated at three concentrations for each drug,and the cell survival rate was determined by MTT method.According to the cell viability rate after the combination of drugs,the online data analysis tool Synergy Finder 2.0 and Compu Syn software were used to calculate synergy score and combination index（CI）,determine the synergistic effect of VP and CDDP combination,and furtherly determine the drug combination concentration in subsequent experiments;3.VP and CDDP as single agents or in combination treated KYSE150 and KYSE30 cells,and the morphological changes of cells were observed using an inverted microscope;Propidium lodide（PI）staining was used to detect cell death;The level of reactive oxygen species（ROS）in cells was detected by dihydroethidium（DHE）probes;4′,6-diamidino-2-phenylindole（DAPI）staining was used to detect apoptosis of cells;Dansylcadaverine（MDC）staining to detect autophagy in cells;The cell adhesion ability of ESCC was detected by cell aggregation experiment and cell detach experiment.Wound healing assays were used to detect the migration capacity of cells;4.Western blot experiments were used to detect the effects of VP and CDDP as single agents or in combination on apoptosis-related proteins PARP-1 and Procaspase-3,metastasis-related molecule MMP-2 and AKT signaling pathway.Results:1.Different concentrations of VP or CDDP led to decreased viability of KYSE150 and KYSE30 cells,with dose-and time-dependent;2.Compared with the drug alone,the combination of VP and CDDP significantly inhibited the survival of cells,and the combination of VP and CDDP had a synergistic effect calculated by Synergy Finder 2.0 and Compu Syn;3.Compared with the control group,the cells in VP and CDDP single agent treatments began to become rounded,and the cells in the CDDP treatment appeared hypertrophy.While compared with the control group,the combination treatment led to the cell number decreases more obviously,the cell hypertrophy was more obviously.After PI staining,it was found that cell death increased in the single drug group compared with the control group,and the number of cell deaths in the combination group was higher than that in the drug group alone.Compared with the control group,ROS levels increased in the group with single agents,and the ROS level in the combination group resulted in higher ROS levels in cells than in single treatments.After DAPI staining,it was found that there were very few apoptotic cells in the control group,the apoptosis rate was increased in the drug group alone,and the nucleus aggregation appeared,while the apoptosis rate was higher in the combination group than in single treatments,and the cells appeared nuclear aggregation and nuclear fragmentation.MDC staining showed that autophagy was decreased in VP group,autophagy was enhanced in CDDP group and autophagic vacuoles appeared,and autophagy was decreased in combination group.The cell aggregation experiment and cell detach experiment showed that the adhesion ability of cells in the drug group alone was enhanced compared with that in the control group,and the homogeneous adhesion of cells in the combination group was significantly stronger than that in the drug group alone.Wound healing was assessed after 48 h and displayed that the scratch width in the drug group alone was slightly wider than that of the control group,and the scratch width in the combination group was significantly larger than that of the drug group alone;4.Compared with the drug alone,the combined application of VP and CDDP led to a significant increase in the expression of apoptotic protein PARP-1 and Procaspase-3 were significantly reduced.The expression of MMP-2 of metastasis-related molecule decreased.Compared with the control group and drug alone,the expression level of p-AKT in the combination group was significantly down-regulated,while the expression of total protein AKT did not change significantly.Conclusions:1.VP and CDDP alone can inhibit the survival of ESCC cells;2.The combined application of VP and CDDP has a synergistic inhibitory effect on ESCC cells;3.VP in combination with CDDP can reduce the proliferation capacity of ESCC cells compared to single treatments,increase cell death,increase ROS levels,apoptosis,and inhibit autophagy,enhance cell-cell adhesion and reduce cell migration;4.VP in combination with CDDP promotes apoptosis of ESCC cells by activation of apoptotic proteins PARP-1 and caspase-3;Weaken the migration and invasion capacity of ESCC cells by reducing the expression of MMP-2 in cells;The combination of VP and CDDP maybe show synergistic effect through the AKT signaling pathway.