GSTM Isozyme-selective Inhibitor Sensitize Resistant Cancer Cells To Cisplatin In Vitro And In Vivo

Malignant tumor is one of the most serious diseases that endanger human health.In Europe and America,the mortality of malignant tumor is near to that of cardiovascular disease.Chemotherapy is a common method for the treatment of malignant tumor.Cisplatin is a first-line drug in the treatment of many kinds of cancer,such as lung cancer,ovarian cancer,etc..Cisplatin resistance often leads to failure of chemotherapy.In the mechanisms of cisplatin resistance,the mechanism of drug resistance mediated by overexpression of GST is widespread in drug resistant tumor cells.In order to solve the difficult problem of cisplatin resistance to chemotherapy,To design low toxicity and high efficiency GST inhibitor is urgently.In the preliminary study,we found that the highly selective GSTM inhibitor EBEA,a highly selective GSTM inhibitor,has a significant role in enhancing the sensitivity of ovarian cancer resistant cells to COC1/DDP and SKOV3/DDP.This research is based on detecting the homologous series of compounds of resistant cells to cisplatin sensitizing effect.It is found that EDEA has significantly increased sensitivity to ovarian cancer SKOV3/DDP cells and lung cancer A549/DDP.We found EDEA combined with cisplatin has inhibitory effect on the growth of transplanted tumor and EDEA has potential anti-tumor effect on ovarian cancer cell line COC1/DDP.The following is the method used in this study,as well as the results of conclusions:CCK-8 cell proliferation and apoptosis assay was used to detect EDEA toxicity on normal cell HEK293 and LO2,low toxic dose was 1uM.We also detected the tumor cell lines SKOV3 and SKOV3/DDP,COC1 and COC1/DDP,A549,A549/DDP,under the low toxic dose.Determination sensitization of EDEA to SKOV3/DDP,A549/DDP.That 2 uM EDEA can sensitivitize SKOV3/DDP,A549/DDP about 3 times.Flow cytometry was used to detect the apoptosis rate of all treatment groups of different drug resistant cells.There was increased rate in combine group compared with control group.The expression of Caspase-3,Bcl-xL,Bak,Bax,Bcl-2 were detected by Western-blot.After treated,apoptosis was promoted in the combine group.Western blot showed inhibitor with low concentration of DDP treated resistant cells,Bcl-2 family apoptotic protein expression was up-regulated and anti-apoptosis protein expression was down regulated.Subcutaneous inject tumor cells on nude mice to establish transplanted tumor model.Effect of EDEA combined with cisplatin in the treatment of tumor were observed.SKOV3/DDP and A549/DDP cells were cultured and injected in the back of BALB/C nude mice by 1 × 107/site.The animal model of human drug resistant ovarian cancer was established,and the human non small cell lung cancer model was established.Then tumor model was randomly divided into 4 groups,solvent group treated with 0.2% Tween and 1% DMSO in saline,DDP group received cisplatin,EDEA group given ethacrynic acid ethylenediamine,combined group also given cisplatin and ethacrynic acid ethylenediamine,intraperitoneal injection,every 3/4 days administration,8 times in total.Observed the growth of tumor,the average inhibitory rate of tumor,and different treatment groups of mice weight,appetite,mental state,we measured tumor volume change and draw the tumor growth curve.On the end of treatment,we weighed the tumor and made HE staining.The apoptosis rate of tumor cell was detected by TUNEL;immunohistochemistry was used to detect the expression of CD31 and Ki67.It was found that the volume of SKOV3/DDP model were obvious difference between EDEA combined DDP group and control group after the third administration.A549/DDP model in the treatment of the sixth after the combined group compared with the control group.TUNEL staining of tumor tissue showed that the apoptosis cells in combination group were significantly more than those in the control group,p<0.05.Ki67 staining showed that the proliferation of cells in the combined group was significantly less than that in the control group,p<0.05.This study detected cell viability,animal level,protein level to verify EDEA against cisplatin resistant-cells,A549/DDP,COC1/DDP,SKOV3/DDP.It is found that low concentration of EDEA combined with low concentration of cisplatin can play a significant sensitizing effect on drug resistant cell line SKOV3/DDP,A549/DDP.EDEA combined cisplatin can inhibit the growth of transplanted tumor model.Further validation of the resistant strains of the sensitizing effect of EDEA.Although EDEA has no sensitization effect on COC1/DDP,but in a low dose of normal cells under low toxicity,can lead a large number of cells to apoptosis,and whether with the use of cisplatin or not.Speculated that EDEA has potential anti-tumor effect on COC1/DDP.The mechanism of EDEA and the mechanism of anti tumor action are closely related to the cell pathway of Bcl2 family members in the initiation of apoptotic pathway.