Research On Mechanism Of Chemoresistance Promotion By Enhancing Aerobic Glycolysis In Gastric Cancer

Background:Gastric cancer is the second leading cause of cancer morbidity and mortality in China,which seriously threats people’s health.Chemotherapy is an important treatment for cancer.Cisplatin is a first-line chemotherapy drug for gastric cancer,and can cause multidrug resistance,restricting clinical efficacy.Previous studies have found that the tumor cells could enhance glycolysis to promote cisplatin resistance,however some kind of cisplatin-resistant tumors haved exhibited the opposite.Thus it is worthful to clarify the glycolytic phenotype of cisplatin-resistant gastric cancer cells and investigate the relationship between the cisplatin-resistance and glycolysis.The results of this study can provide a new way to reverse the cisplatin resistance.Objective:The aim of this study was to investigate glycolytic phenotype in natural and acquired cisplatin-resistant gastric cancer cells and to elucidate the effects glycolysis on drug resistance and the mechanism by which drug-resistant cells regulate glycolysis in gastric cancer cells.Method:In the early stage,the acquired cisplatin-resistant cell of BGC823/DDP was obtained after induction with the concentration gradient of cisplatin,while MGC803 was used as a natural cisplatin-resistant cell model,and BGC823 as sensitive model.We applied colorimetry to detect the substrate and the products of glycolysis in sensitve and resistant cells,including glucose consumption,pyruvic acid and lactic acid production.MTS,flow cytometry and Western Blot were used to detect the sensitivity of all cells to culture medium with low glucose or glycolytic inhibitor 2-DG and evaluate the effects of two treatments on drug resistance.The differential protein expression profiles of the sensitive and resistant cells were obtained by two dimensional electrophoresis combined with mass spectrometry,from which we screened out the glycolysis-related protein through the clusteranalysis of protein functions.Then we knocked down the special.protein by siRNA to detect the effect on glycolysis of resistant cells,including glucose consumption,pyruvic acid,lactic acid and ATP production.The MTS,flow cytometry assay and Western Blot were uesd to test cisplatin sensitivity in resistant cells after knocking down the special protein.In addition,the sensitivity of BGC823 to cisplatin was tested by overexpression of ENO1.RT-qPCR,Western blot assay and dual luciferase assay were used to study the mechanism by which cisplatin-resistant cells upregulated the protein.Immunohistochemistry was applied to analyse the protein expression in adjacent non-tumor tissues and tumor tissues and its relationship with clinical features.Results:Compared with the sensitive BGC823 cell,the resistant cells of MGC803 and BGC823/DDP exhibited enhanced glycolysis,which had greater glucose consumption,greater production of pyruvic acid and lactic acid.Resistant cells were more sensitive to low glucose culture medium or 2-DG.Inhibition of glycolysis reversed drug resistance.Two dimensional electrophoresis combined with mass spectrometry analysis revealed that glycolytic enzyme ENO1(Enolase 1)was highly expressed in BGC823/DDP cells,consistent results as evidenced by Western Blot.After konckdown of ENO1,the glucose consumption was decreased,as well as the production of pyruvic acid,lactic acid and ATP.Most of all,the drug-resistance also declined.BGC823 cells increased glycolysis and acquired cisplatin resistance by overexpression of ENO1.RT-qPCR showed that the expression level of ENO1 mRNA in drug resistant cells was slightly lower than that in sensitive cells.The results indicated that the transcriptional regulation was not involved in the upregulation of ENO1 in the cisplatin-resistant gastric cancer cells.Meanwhile,there was no significant difference in the half-life of ENO1 protein between the sensitive and resistant cells by using protein synthesis inhibitor of CHX.These results suggested that ENO1 might be posttranscriptionally regulated.It was found that ENO1 might be the target of miR-22 by using microRNA target prediction and RT-qPCR verification,and the expression of miR-22 was lower in resistant cells.Western Blot assay showed that when resistant cells were exogenously transfected with miR-22 mimic or sensitive cells with miR-22 inhibitor,the expression of ENO1 was down-regulated or up-regulated.Dual luciferase reporter assay suggested that miR-22 could bind to 3’-UTR of ENO1 mRNA and inhibit its function.The glycolysis of BGC823 cells was enhanced after transfection of miR-22 inhibitor while the glycolysis of BGC823/DDP cells was decreased after transfection of miR-22 mimic.Immunohistochemistry showed that ENO1 was highly expressed in gastric cancer compared with non-tumor tissues.ENO1 indicated a worse overall survival.Conclusion:In this study,we found that the cisplatin-resistant gastric cancer cells enhanced glycolysis by overexpression of ENOl,and relied more on this metabolic pathway when compared to sensitive cells.Inhibition of glycolysis could reverse drug resistance.The elevated ENO1 expression was attributed to the post transcriptional regulation of miR-22 in drug-resistant cells,therefore,miR-22 and ENO1 could be used as targets for the treatment of multidrug resistance.