Effect And Molecular Mechanism Of Metformin On Proliferation Of Renal Cell Carcinoma Under Glucose Deprivation

BAKGROUD:RCC is a common malignant tumor of the urinary system,which is a serious threat to human health.Recent kidney cancer genetic studies suggest that kidney cancer is essentially a metabolic-related disease [2].Studies have shown that the survival rate of patients with kidney cancer with diabetes is lower than that of patients with kidney cancer without diabetes [3].Over the years,the incidence of cancer in diabetic patients has been significantly higher than that of non-diabetic patients [4],and it is considered that diabetes is one of the important risk factors for cancer occurrence and death [5],which has caused related research fields at home and abroad.Scholars are highly concerned.A recent study by Cheng S et al [13] found that MF can significantly improve DFS and CSS in patients with localized renal cell carcinoma.However,other studies have shown that [14,15] diabetes is a poor prognosis in patients with renal cell carcinoma,and MF has no significant improvement in survival outcome in patients with T2 D complicated with renal cancer.The above results suggest that MF has different pharmacological effects in different sugar environments inside and outside the cell.As we all know,MF is a T2 D first-line hypoglycemic agent,and also an activator of AMPK.AMPK is a cellular energy receptor that promotes AMPK activation when cells lack energy.Tumor cells are often in a state of nutritional deficiencies,but they exhibit AMPK inhibition in a variety of tumor cells including kidney cancer,breast cancer,and lung cancer [20].A number of in vitro experiments have found that MF can inhibit the proliferation of prostate cancer cells,breast cancer cells,pancreatic cancer cells,melanoma cells,and renal cancer cells [8-12].Recent in vitro and in vivo studies have demonstrated that MF can activate the AMPK to regulate the m TOR pathway to activate anti-renal cell proliferation,prevent RCC clone formation,and induce cell cycle arrest [16].In the in vitro cell experiments of this study,it was found that MF can promote the proliferation of RCC cells and activate AMPK at the same time under low glucose conditions.Therefore,we analyzed that the cause of poor prognosis of MF in the treatment of RCC is related to the effect of AMPK on intracellular nutrient deficiency.In recent years,PKM2 has made substantial progress in the molecular regulation mechanism of tumorigenesis.It has been reported [19] that PKM2 nuclear translocation activates transcription factors and plays a role in the expression of?-catenin target gene c-Myc.Important role and promotes ?-catenin-mediated transcription of cyclin D1,promoting cell survival and proliferation.It further lays an important position for PKM2 in regulating tumor proliferation.Furthermore,we suggest that there is a similar mechanism of action for MF-induced RCC cell proliferation in glucose deprivation environments.At present,there are no relevant reports at home and abroad.OBJECTIVES:This study will investigate the effect of MF on the proliferation of RCC cells in glucose deprivation and elucidate the role of MF in promoting the proliferation of RCC cells under glucose deprivation and its molecular mechanism,and provide objective laboratory data for the development of new RCC clinical treatment strategies.To study the effect of MF on the proliferation of RCC cells in glucose deprivation,and to elucidate the role of MF in promoting the proliferation of renal cell carcinoma under glucose deprivation and its molecular mechanism,and to provide a theoretical basis for the development of a new strategy for clinicaltreatment of renal cell carcinoma.METHODS:The effects of MF on proliferation of RCC cells under normal or glucose deprivation conditions were firstly observed using renal cell carcinoma cell lines A498 and GRC-1: cell proliferation was detected by MTT assay and Brdu incorporation;tumor cell proliferation markers were detected by Western Blot assay.The expression level of Ki-67 was detected by Western Blot.The expression of AMPK and p AMPK was detected by staining.The effect of MF on apoptosis was observed by staining.Subsequently,the mechanism of MF promoting RCC cell proliferation under glucose deprivation was studied: si RNA knockdown AMPK expression or AMPK inhibitor in A498 cell was used to study the metabolism,proliferation and expression of ORF of RCC cells under glucose deprivation.AMPK expression level,?-catenin expression level and PKM2 expression level;si RNA knockdown ?-catenin expression in A498 cell after MF culture in glucose deprivation for RCC proliferation,expression of proliferation markers and PKM2 expression levels.Immunoprecipitation method was used to detect AMPK,PKM2 and ?-catenin interaction in MF-treated RCC cells under glucose deprivation;knockdown of PKM2 expression in A498 cells by si RNA technique or study of MF in PKM2 inhibitor SKN The effect of glucose deprivation on the proliferation of A498 cells and the expression of value-added markers.Finally,the in vivo experiments of animals were used to further study the anti-tumor effect of MF and SKN: the glucose deprivation was simulated by limiting eating and starvation.The8-week-old BALB/C was divided into 4 groups: control group,MF group,SKN group,MF+SKN.In the group,1×106 A498 cells were inoculated into nude mice,and the tumor size was recorded at 5,10,15 and 20 days after tumor inoculation,andthe general state was observed.On the 20 th day,the mice were photographed after taking the tumor,and the tumor size was measured;Western Blot method and immunohistochemical method were used to observe the expression level of Ki67 in tumor tissues.RESULT:1.Under normal glucose concentration culture conditions,MF inhibited the proliferation of ARC and GRC-1 RCC cells;under glucose deprivation culture conditions,MF promoted the proliferation of A498 and GRC-1 RCC cells and up-regulated the tumor proliferation marker Ki-67.Expression,but no effect on cell apoptosis.2.Under normal glucose concentration and glucose deprivation culture conditions,Western Blot results showed that MF can promote AMPK phosphorylation,and glucose deprivation and MF synergistically enhanced AMPK phosphorylation.3.MF reduced ATP and lactate levels in RCC cells under glucose deprivation;increased ATP and lactate levels in RCC cells under glucose deprivation culture conditions(**,p<0.01).4.After silencing AMPK,the effect of MF-induced increase in ATP and lactate levels in RCC cells was inhibited under glucose deprivation culture conditions(*,p<0.05;**,p<0.01).Western Blot results showed that the effect of MF-induced upregulation of MN-induced tumor proliferation marker Ki-67 was inhibited under glucose deprivation culture conditions under glucose deprivation culture conditions;MTT and Brdu results showed that MF induced RCC cell proliferation was inhibited under glucose deprivation conditions.And q PCR results showed that the effect of MF-induced regulation of cell proliferation-related factors CCND1 and c-Myc wasup-regulated under glucose deprivation culture conditions(*,p<0.05;**,p<0.01).5.Under glucose deprivation culture conditions MF and AMPK combined with RCC cells for 48 h,MTT and Brdu results showed that AMPK inhibitors had no effect on MF-induced RCC cell proliferation under glucose deprivation culture conditions(*,p<0.05;**,p<0.01 However,the effect of MF-induced increase in ATP and lactate levels in RCC cells was inhibited(*,p<0.05;**,p<0.01).6.Under glucose deprivation culture conditions and MF treatment,the expression levels of cytoplasmic and nuclear proteins AMPK and ?-catenin were detected by Western Blot,and the expression levels of AMPK and ?-catenin in the nucleus increased.7.Western Blot results showed that knockdown of AMPK reduced MF-induced AMPK and ?-catenin nuclear translocation in RCC cells under glucose deprivation culture conditions;MF combined with AMPK inhibitors,MF-induced AMPK under glucose deprivation conditions there was no effect on the expression level of?-catenin.8.Western Blot results showed that knockdown of ?-catenin inhibited the upregulation of MF-induced tumor proliferation marker Ki-67 in glucose deprivation;MTT and Brdu results showed that knockdown of ?-catenin inhibited MF induction in glucose deprivation;.Cell proliferation;q PCR results showed that knockdown of?-catenin inhibited the up-regulation of MF-induced regulation of cell proliferation-associated factors CCND1 and c-Myc,which have synergistic effects.9.Western blot analysis of MF under glucose deprivation culture conditions showed that PKM2 nuclear translocation was promoted in A498 cells.After knocking out AMPK expression in A498 cells,the expression level of PKM2 in the nucleus was decreased.10.Under glucose deprivation culture conditions,MF promotes AMPK by recruiting PKM2 into the nucleus,promoting ?-catenin nuclear translocation and forming a complex with ?-catenin;knocking out AMPK or knocking out PKM2 makes MF promote AMPK,PKM2 and ?-catenin.The binding effect of ?-catenin disappeared;knockout of PKM2 reduced the nuclear translocation of ?-catenin under MF,but did not affect the nuclear translocation of AMPK under MF.11.The expression of PKM2 in A498 cells was knocked out.The results of Western Blot showed that the up-regulation of the expression of tumor proliferation marker Ki-67 induced by MF under glucose deprivation conditions was inhibited.The results of MTT and Brdu showed that the proliferation of MF induced by glucose deprivation was inhibited.Inhibition;q PCR results showed that MF-induced regulation of cell proliferation-associated factors CCND1 and c-Myc m RNA levels were inhibited under glucose deprivation.12,PKM2 inhibitor-Shikonin(SKN)and MF under glucose deprivation,MTT and Brdu results show that under the action of SKN,low glucose condition MF induced A498 cell proliferation inhibition;q PCR results show: Under the action of SKN,the effect of MF-induced up-regulation of the regulation of cell proliferation-related factors CCND1 and c-Myc was inhibited.13.Restricted eating,starvation and glucose deprivation status have a delayed effect on tumor growth in nude mice inoculated with RCC cells,but no inhibition.In vivo anti-tumor experiments showed that MF could not inhibit tumor growth under glucose deprivation conditions,but increased tumor volume(1150mm3 control800mm3);only SKN(600mm3)or combined with MF(350mm3)inhibited tumor growth;inhibited the combination of PKM2 and MF Inhibition of RCC proliferation was superior to inhibition of PKM2 alone.Western Blot + immunohistochemistryshowed that MF alone promoted tumor proliferation,and combined with SKN inhibited the expression of Ki-67.CONCLUSIONS:1.Under glucose deprivation conditions in RCC cells,MF promotes AMPK recruitment of PKM2 into the nucleus to promote ?-catenin nuclear translocation and form a complex with ?-catenin,thereby promoting RCC cell proliferation;2.The use of AMPK inhibitors did not affect the progress of the above process,indicating that MF induced proliferation of RCC cells under glucose deprivation conditions independent of AMPK kinase activity.3.Knockout of PKM2 can inhibit MF-induced RCC cell proliferation in glucose deprivation culture conditions in vitro,and inhibition of PKM2 can reverse MF-induced tumor cell proliferation in glucose deprivation state in vivo.4.Inhibition of PKM2 combined with MF inhibited RCC proliferation better than inhibition of PKM2 alone.