To Explore The Effect Of Antimony On The Progression Of Bladder Cancer And Its Mechanism

ObjectiveTo explore whether heavy metal antimony can promote the progression of bladder cancer,and further explore the potential molecular mechanism of its regulation.To provide further evidence for the prevention and treatment of bladder cancerMethodsSerum of bladder cancer patients and non-cancer patients were collected to detect antimony content in serum by mass spectrometry.At the same time,the relationship between antimony content and the absence of disease recurrence in patients with bladder cancer was analyzed by statistical methods;EJ cells were treated with potassium tartrate,potassium antimony tartrate and complete medium,respectively.The effect of antimony on the progression of bladder cancer was examined by MTT test,scratch test and Transwell test;In the experiment of subcutaneous tumor transplantation in nude mice,the effect of antimony on the proliferation of bladder cancer was determined by observing the size of the tumor;The stable Mt-Keima EJ cell line was constructed,and the effect of antimony on mitophagy of EJ cells was examined by confocal microscopy;ROS kit,JC-1 kit,ATP kit,ADP/ATP ratio kit and mitochondrial respiratory enzyme complex Ⅰ/Ⅱ/Ⅲ/Ⅳ kit were used to detect the mitochondrial damage caused by antimony;Flow cytometry was used to detect the effect of low concentration antimony on EJ cell apoptosis;The expression differences of PINK1 and PARK2(Parkin)in normal bladder tissues and bladder cancer tissues were predicted in TCGA database,and the effects of low concentration of antimony on the expression of PINK1 and PARK2(Parkin)in bladder cancer EJ cells were detected by RT-qPCR and Western blot;EJ cells were treated with complete medium,potassium antimony tartrate,mitochondrial autophagy agonist(CCCP)and CCCP+potassium antimony tartrate,respectively.Western blot was used to detect the effect of antimony on classical mitophagy pathway(PINK1/Parkin pathway),and MTT assay was used to further examine the effect of mitophagy on bladder cancer;Serum of bladder cancer patients and non-cancer patients were collected to detect the differential contents of ceramide and S1P by ELISA assay;At the same time,the contents of ceramide and S1P in cells and the serum of nude mice were determined by ELISA assay,and the effect of antimony on the metabolism of sphingolipid(ceramide to SIP)was analyzed;The differential expression of GPC5 and ACER2 genes in bladder cancer and normal bladder tissue was predicted in TCGA database,and the relationship between GPC5 and ACER2 in bladder cancer was predicted;EJ cells with stable overexpression of GPC5 gene were constructed.The effects of antimony on the progression of bladder cancer by regulating GPC5 gene were examined by MTT assay,scratch assay and Transwell assay,and the effects of antimony on the apoptosis of EJ cells by regulating GPC5 gene were detected by flow cytometry assay;The effect of antimony on sphingolipid metabolism(i.e.,ceramide to SIP conversion)by affecting GPC5 gene was detected by ELISA assay;RT-qPCR and Western blot were used to determine whether antimony exposure promoted the progression of bladder cancer through the activation of GPC5-ACER2-ERK1/2 pathway.ResultsThe results of mass spectrometry showed that the content of antimony in serum of patients with bladder cancer was significantly higher than that of non-cancer patients,and antimony content was negatively correlated with disease-free survival rate of patients with bladder cancer;In vitro,the concentration(0.8umol/L)of antimony exposure promoted the proliferation,migration and invasion of bladder cancer EJ cells;In vivo experiments,low concentration of antimony can significantly promote the proliferation of xenograft tumor in nude mice;Exposure to low concentration antimony can significantly inhibit the occurrence of mitophagy in EJ cells;By examining ROS,ATP content,ADP/ATP ratio,and mitochondrial respiratory enzyme complex Ⅰ/Ⅱ/Ⅲ/Ⅳ activity,we determined that low concentration antimony exposure causes mitochondrial damage through ROS-dependent oxidative stress;The results of flow cytometry showed that antimony exposure could inhibit the apoptosis of EJ cells;TCGA database showed that the expression levels of PINK 1 and PARK2(Parkin)genes in bladder cancer tissues were lower than those in normal tissues,which was consistent with the database results.RT-qPCR and Western blot experiments showed that low concentration antimony exposure could significantly inhibit the activation of PINK1/Parkin pathway in bladder cancer EJ cells;Mitophagy agonist CCCP was used to demonstrate that mitophagy mediated by PINK1/Parkin pathway could inhibit the proliferation of EJ cells induced by antimony exposure to a certain extent by MTT assay.ELISA results showed that the content of ceramide of bladder cancer patients in serum was significantly lower than that of non-cancer patients,while SIP was significantly higher than that of non-cancer patients;At the same time,using ELISA assay,we found that low concentration antimony exposure can inhibit the conversion of ceramides to SIP in bladder cancer EJ cells,and can also inhibit the conversion of ceramides to SIP in serum of nude mice;The results of TCGA database showed that the expression of GPC5 gene in bladder cancer tissues was significantly lower than that in normal bladder tissues,and the expression of ACER2 gene in bladder cancer tissues was significantly higher than that in normal bladder tissues.In bladder cancer tissues,the Pearson results of GPC5 gene and ACER2 gene were negatively correlated;The up-regulation of GPC5 gene can significantly reverse the biological progress of antimony-induced bladder cancer EJ cells to a certain extent,and can also reverse the antimony-induced apoptosis inhibition effect;By ELISA assay,it was found that the up-regulation of GPC5 could significantly reverse the effect of antimony induced ceramide conversion to S1P;At the mRNA and protein levels,we found that antimony could inhibit the expression of GPC5 gene in bladder cancer EJ cells,thereby upregulating the ACER2 gene to promote sphingolipid metabolism,and finally activating the ERK1/2 pathway to promote the progression of bladder cancer.ConclusionsLow concentration of heavy antimony can promote the progression of bladder cancer.On the one hand,heavy antimony can promote the progression of bladder cancer by inhibiting PINK1/Parkin-mediated mitophagy.On the other hand,heavy antimony promotes sphingolipid metabolism by inhibiting the expression of GPC5 gene and upregulating the expression of ACER2 gene,thus activating the ERK1/2 pathway and promoting the progression of bladder cancer.We believe that heavy antimony is a carcinogenic factor of bladder cancer,and appropriate interference with mitophagy and sphingolipid metabolism is helpful for the prevention and treatment of patients with bladder cancer caused by heavy antimony.