LASS2 Reduces Chemoresistance Of Bladder Cancer Cells By Inhibiting Endoplasmic Reticulum Stress-mediated Autophagy

Objectives:1.To investigate the effects of tumor suppressor gene LASS2 on chemosensitivity and chemoresistance of bladder cancer cells2.To investigate the difference of autophagy levels between adriamycin(ADM)-resistant cell lines and ADM-sensitive cell lines of bladder cancer under the intervention of ADM,the effect of LASS2 on autophagy levels in various types of bladder cancer cells,and the effect of autophagy on the apoptosis of bladder cancer resistant cells under the intervention of ADM.3.To investigate the difference of endoplasmic reticulum stress(ERs)levels in ADM-resistant cell lines and ADM-sensitive cell lines of bladder cancer under the intervention of ADM,and the effect of LASS2 on ERs and their related apoptosis in bladder cancer resistant cells.To elucidate the possible mechanism that LASS2 reverses chemoresistance of bladder cancer cells by regulating autophagy.Methods:1.Screening and constructing ADM-resistant bladder cancer cell line Biu87-R.Immunofluorescence staining and quantitative real-time PCR(qRT-PCR)were used to verify the chemoresistance of Biu87-R cells to ADM.After transfection of the LASS2 overexpression plasmid,the effects of LASS2 overexpression on the chemosensitivity and chemoresistance of bladder cancer cells were detected by CCK-8 assays,cell clone formation assays,and matrigel invasion assays.2.The effect of LASS2 overexpression on the number of autophagy vacuoles in various types of bladder cancer cells under the intervention of ADM was detected by transmission electron microscopy.After transfected of GFP-LC3 plasmid,the effect of LASS2 overexpression on the distribution density of autophagy fluorescent puncta in various types of bladder cancer cells were detected by immunofluorescence technology.The effect of LASS2 overexpression on the expression of autophagy marker proteins in various types of bladder cancer cells was detected by Western blot assays.The effect of inhibition of autophagy on the apoptosis of bladder cancer resistant cells under ADM intervention was detected by flow cytometry.3.Immunofluorescence staining and Western blot were used to detect the effects of LASS2 on ERs marker proteins GRP78 and PDI in various types of bladder cancer cells under the intervention of ADM.The effects of LASS2 on ADM-induced ERs-related apoptosis in Biu87-R cells were assayed by flow cytometry and Western blot.The ERs inducer Tunicamycin(Tun)was used to intervene Biu87-R to detect the effects of ERs-mediated autophagy in Biu87-R cells.Finally,we examined the mechanism pathway proteins involved in ERs-induced autophagy in Biu87-R cells by Western blot assays and explored the effects of LASS2 on the expression levels of mechanism pathway-related proteins.Results:1.The cell viability of bladder cancer cell lines T24,Biu87,EJ,and RT4 gradually decreased with the increase of ADM concentration.Among them,T24 cells had the lowest sensitivity to ADM,while Biu87 cells had the highest sensitivity to ADM,and the invasion ability of the two cell lines under ADM intervention was significantly inhibited after transfected of the LASS2 overexpression plasmid.ADM-resistant bladder cancer cell line Biu87-R has higher IC50 values for ADM,cisplatin,5-FU,and cyclophosphamide,and the expression level of multidrug resistance-related protein P-gp is also significantly increased.In contrast,the expression level of LASS2 protein in Biu87-R cells was significantly lower than that of Biu87,and overexpression of LASS2 could significantly reduce the P-gp expression level and IC50 value for ADM in Biu87-R cells.Besides,overexpression of LASS2 could significantly enhance the inhibitory effect of ADM on the proliferation and clone formation of Biu87-R cells.2.ADM could significantly increase the number of autophagy vacuoles,the density of GFP-LC3 green fluorescent puncta,and enhance the conversion of cytosolic LC3(LC3-I)to membrane LC3(LC3-II)in bladder cancer T24 cells and the degradation of P62.And the number of autophagy vacuoles,the density of GFP-LC3 green fluorescent puncta,the conversion of LC3 and the degradation of P62 in Biu87-R cells were significantly higher than that of Biu87 cells under ADM intervention.Overexpression of LASS2 could significantly inhibit the number of autophagy vacuoles,the density of GFP-LC3 green fluorescent puncta,the conversion of LC3,and the degradation of P62 in T24 cells,and could effectively reverse the above hallmark events of autophagy in ADM-resistant cells Biu87-R.Furthermore,after treatment with autophagy inhibitor 3-MA,Biu87-R significantly reduced the chemoresistance to ADM.3.The fluorescence intensity of GRP78 in bladder cancer T24 cells was significantly enhanced under ADM intervention.Western blot also confirmed that ADM could significantly enhance the expression levels of GRP78 and PD1 in T24 cells,and their expression level under ADM in Biu87-R cells was significantly higher than that of Biu87 cells.Overexpression of LASS2 could significantly reverse the induction of GRP78 and PDI expression by ADM in T24 cells and Biu87-R cells.In contrast,the apoptosis of Biu87-R cell under ADM was significantly lower than that of Biu87 cells,and overexpression of LASS2 significantly enhances the apoptosis induced by ADM in Biu87-R,and increases the cleaved PARP and caspase-12 and other ERs-related apoptotic protein expression levels.Intervention with the ERs inducer Tun could significantly increase the levels of ERs and autophagy that are inhibited by LASS2 in Biu87-R cells,and could also significantly reduce the apoptosis of ADM-resistant cells caused by LASS2 overexpression.In addition,the expression levels of PERK/eIF2? signaling pathway-related proteins such as PERK,ATF4,p-PERK,and p-eIF2? in Biu87-R cells under ADM intervention were significantly increased,and the expression of these proteins was significantly reduced after LASS2 overexpression.Conclusions:1.The expression level of LASS2 was significantly negatively correlated with chemoresistance of bladder cancer cells,Overexpression of LASS2 could significantly reduce the chemoresistance of ADM-resistant cells and enhance the antitumor effect of ADM on drug-resistant cells of bladder cancer.2.The level of autophagy in chemoresistant cells of bladder cancer was higher than the non-resistant cells under the intervention of ADM.Overexpression of LASS2 could significantly inhibit the level of autophagy in ADM-resistant cells of bladder cancer,and inhibition of autophagy could significantly reduce the resistance of bladder cancer cells to ADM.3.ADM could activate ERs in bladder cancer cells,while overexpression of LASS2 could significantly inhibit ADM-induced ERs and increase apoptosis in ADM-resistance cells.In addition,ERs can induce autophagy by promoting the activation of the PERK/eIF2? signaling pathway in ADM-resistance cells of bladder cancer,while LASS2 overexpression could reverse ERs-mediated autophagy in ADM-resistance cells by inhibiting the activation of the PERK/eIF2? pathway.