The Mechanism Research Of IDH1 Mediating The Inhibition Of Withaferin A On The Skin Carcinogenesis In Mouse JB6 P+ Cells

Objection:JB6 P+ cells,a well-established model for tumor promotion,were employed to explore the mechanism of IDH1 mediating the inhibition of Withaferin A on the skin carcinogenesis in the molecular biology and cell levels,which might provide clues to discover new drugs and molecular targets for chemoprevention.Methods:1.To explore the mechanism of Withaferin A(WA)maintaining the stability of IDH1 in the early stage of skin carcinogenesis.The JB6 P+ cells treated with tumor promoter TPA or/and WA were applied to detect the expression of IDH1,which was used to observe the impact on IDH1.The experiments were conducted in transcription,translation and post-translation stages respectively to record the mechanism of WA stabilizing IDH1.Primarily,the IDH1 mRNA levels were detected.Then,the cells were pretreated with cycloheximide(CHX),a protein synthesis inhibitor,and the expression of IDH1 protein was measured.Subsequently,the activities of proteasome 20 S and the expression of IDH1 pretreated with proteasome inhibitor MG132 were examined.Eventually,the levels of IDH1 ubiquitination was measured by the immunoprecipitation.2.To investigate the mechanism of IDH1 as a tumor suppressor gene restraining the skin carcinogenesis.The overexpression vector pcDNA3.1-IDH1 and short interfering RNA(siRNA)were employed to over express and silence IDH1 in JB6 P+ cells respectively.The concentration of ?-KG which is the product of IDH1 and the mitochondrial complex I activity,combined with the expression and activity of lactate dehydrogenase related to glycolysis,were detected simultaneously to observe the effect of IDH1 on mitochondrial function and aerobic glycolysis.3.To observe the regulatory mechanism of IDH1 involved in WA suppressing tumorigenesis.JB6 P+ cells were treated with TPA or/and WA,and the expression of HIF-1? and its target gene Glut1,combined with the activity of VEGF and PHD which is the downstream of IDH1,were measured to observe the influnce of WA on HIF-1? pathway.The expression levels of LDH were measured to observe the impact of WA on glycolysis.The IDH1 was over-expressed and silenced in JB6 P+ cells respectively,and the parameters mentioned above were detected to observe the interaction between IDH1 and HIF-1? pathway.4.The experimental results were statistically analyzed by one-way analysis of variance(ANOVA),followed by Student-Newman-Keuls used for multi-group comparisons.Results:1.WA stabilized IDH1 by inhibiting the ubiquitin-proteasome pathway.1.1 WA inhibited TPA-induced the downregulation of the expression and activity of IDH1(P < 0.05).1.2 The IDH1 mRNA levles were no difference in groups(P > 0.05).1.3 WA inhibited TPA-induced the downregulation of IDH1 before and after CHX pretreatment(P < 0.05).1.4 WA suppressed TPA-induced increases in proteasome 20 S activities(P < 0.05).The effect of TPA or/and WA on IDH1 protein was decreased by MG132 pretreatment,and its protein expression restored to normal levels(P > 0.05).1.5 The IDH1 protein and its ubiquitination levels were detected before and after immunoprecipitation.The data mirrored that WA restrained TPA-induced the downregulation of IDH1.However,TPA-induced the upregulation of IDH1 ubiquitination was reversed by WA(P < 0.05).2.IDH1,as a possible tumor suppressor,inhibited aerobic glycolysis and maintained mitochondrial functions.Overexpression of IDH1 facilitated the accumulation of its product ?-KG and improved the mitochondrial complex I activity.And the expression and activity of LDH were abated(P < 0.05).The downregulation of IDH1 attenuated the product of ?-KG and the mitochondrial complex I activity(P < 0.05),but promoted the upregulation of LDH.3.The inhibition of WA on HIF-1? pathway and glycolysis was mediated by IDH1.3.1 WA decreased TPA-induced the upregulation of Glut1 and HIF-1? expression,combined with the enhancement of VEGF activity.And WA reversed TPA-induced the downregulation of activity of PHD(P < 0.05).3.2 Overexpression of IDH1 resulted in a decrease of HIF-1? and Glut1 expression,combined with the activity of VEGF(P < 0.05),which was blocked by the silence of IDH1.3.3 WA prevented the upregulation of LDH induced by TPA(P < 0.05).Conclusions:1.WA mantained TPA-induced the downregulation of IDH1 expression by inhibiting the ubiquitin-proteasome pathway.2.IDH1 functioned as a possible tumor suppressor by inhibiting aerobic glycolysis and maintaining mitochondrial functions.3.WA as a potential chemopreventive agent inhibited tumor promotion,starting from stabilizing IDH1 to inactivating HIF-1? signaling and glycolysis.