| Objective:Cardamonin is a chalcone of Chinese herb Alpiniae katsumadai with anti-tumor and anti-inflammatory activities,but its effects and mechanism on gastric cancer still remains to be determined.The purpose of this study is to clarify the inhibitory effect of cardamomin on gastric cancer in vivo and in vitro,and to explore the molecular mechanism of cardamonin in inhibiting gastric cancer based on glycolysis and mitochondrial function.Method:1.CCK-8 was used to detect cell viability of MGC-803 and SGC-7901;Annexin VFITC/PI staining was used to detect the apoptosis of gastric cancer cells;Ed U was used to detect the proliferation of gastric cancer cells;Western blot(WB)was used to detect the expression of proliferation and apoptosis-related proteins in gastric cancer cells.2.The anti-tumor effect of cardamonin was determined by xenograft in athymic mice and the weight and tumor volume were recorded every three days.The effect of cardamonin on proliferation and apoptosis of tumor tissue was examined by HE,immunohistochemistry,TUNEL staining and WB.3.Differential genes between control group and cardamonin treatment group were detected by RNA-seq and analyzed by GO annotation.These differential genes were performed to predict the possible molecular functions of cardamonin on the gene products of MGC-803 cells from the three aspects of cell location,biological process and molecular function.Furthermore,the potential signal pathway through which cardamonin exerts antitumor efficacy was predicted by KEGG enrichment analysis.4.The expression of differential genes related to energy metabolism screened by RNAseq was examined by WB.The effect of cardamonin on energy metabolism of gastric cancer cells was investigated by glucose uptake assay,intracellular and extracellular lactate content assay,seahorse glycolysis rate assay and mitochondrial stress test.Furthermore,the substrate oxidation stress test was used to analyze the effects of three mitochondrial substrates(glucose/pyruvate,glutamine and long-chain fatty acids)on mitochondrial function before and after administration of gastric cancer cells.5.The tissue samples of 159 gastric adenocarcinoma patients were statistically analyzed for clinicopathological characteristics,and the expression level of GLUT4 in gastric adenocarcinoma tissue was detected by immunohistochemistry.The SPSS 25.0software package was used to analyze the relationship between above factors,clinicopathological characteristics and survival prognosis.The statistical package R was applied to perform genetic analysis and mapping.6.Gastric cancer cells were transfected with c-Myc and GLUT4 overexpression plasmids to detect the effects of cardamonin on gastric cancer cell viability,c-Myc,GLUT4 protein expression,glycolysis rate and mitochondrial aerobic phosphorylation.The above experiments were used to explore the inhibitory effect of cardamonin on the growth of gastric cancer and investigate whether the inhibition of glycolysis rate and mitochondrial aerobic phosphorylation in gastric cancer cells by cardamonin were mediated by the c-Myc/GLUT4 axis.7.WB was used to detect the effect of cardamonin on the expression of c-Myc/GLUT4 axis energy metabolism-related proteins in nude mice gastric cancer MGC-803 transplanted tumor tissue.Results:1.Cardamonin showed significant inhibitory effect on gastric cancer cell lines MGC-803 and SGC-7901.Further study revealed that cardamonin could inhibit the expression of Ki67 protein in gastric cancer cells,reduce the rate of Ed U positive cells,increase the apoptosis rate of the two gastric cancer cells,down-regulate the expression of antiapoptotic protein Bcl-2 and PARP,and up-regulate the expression of proteins like cleaved caspase-9,cleaved caspase-3 and cleaved PARP.All of the results suggest that the apoptosis-promoting effect of cardamonin was mediated by the mitochondrial pathway in gastric cancer cells.2.Compared with the control group,3 mg/kg,6 mg/kg and 24 mg/kg cardamonin groups could significantly inhibit the growth of tumor tissues in nude mice;3 mg/kg cardamonin inhibited the rate of Ki67 positive cells and the expression of Ki67 protein level in tumor tissues,and increased the necrosis rate,apoptosis rate and the protein expression of pro-apoptotic genes cleaved caspase-3 and cleaved PARP in tumor tissues.3.There were 1309 differential genes between the two groups for GO function annotation,and the result showed that the biological processes of cardamonin on MGC-803 cells included biological regulation and metabolic processes,etc.KEGG pathway enrichment analysis obtained two significant metabolic pathways,namely aspartic acid/alanine/glutamine metabolism and glucose metabolism,suggest that the inhibitory effect of cardamonin on gastric cancer may be closely related to its regulation of the energy metabolism.4.Cardamonin could significantly inhibit the glucose uptake,lactic acid production and efflux,and reduce the glycolysis rate and mitochondrial oxidative phosphorylation of MGC-803 and SGC-7901 cells.The results of substrate oxidative stress assay indicated that the mitochondrial oxidative phosphorylation substrates of MGC-803 and SGC-7901 cells were mainly glucose/pyruvate and glutamine.Cardamonin could further reduce the mitochondrial oxidative phosphorylation substrates glucose/pyruvate and glutamine in MGC-803 and SGC-7901 cells.5.We divided all 159 gastric adenocarcinoma patients into high GLUT4 expression(n= 93)and low GLUT4 expression(n = 66)and found that patients with high GLUT4 expression had higher T-stage(P = 0.020)and lymph node metastasis(P = 0.004),later TNM stage(P = 0.001)and higher Ki67 expression(P = 0.001).In addition,KaplanMeier analysis showed a significant correlation between high GLUT4 expression and reduced patient survival(P = 0.0025),leading to the conclusion that GLUT4 expression was associated with poor prognosis in patients with gastric adenocarcinoma.6.c-Myc or GLUT4 overexpression could antagonize the inhibitory effect of cardamonin on the viability of gastric cancer cells(MGC-803 and SGC-7901 cells),and significantly up-regulate the glycolysis rate and mitochondrial aerobic phosphorylation level of gastric cancer cells(MGC-803 and SGC-7901 cells).c-Myc or GLUT4 overexpression plus cardamonin group could antagonize the inhibitory effect of cardamonin on the glycolysis rate and mitochondrial aerobic phosphorylation level of gastric cancer cells(MGC-803 and SGC-7901 cells).It is suggested that cardamonin may inhibit the glycolysis and mitochondrial aerobic phosphorylation of MGC-803 and SGC-7901 cells through c-Myc/GLUT4 axis,and ultimately inhibit the growth of gastric cancer cells.7.Cardamonin was able to inhibit the expression of c-Myc/GLUT4 axis energy metabolism-related proteins in tumor tissues.Conclusion:In this study,we found that GLUT4 expression was associated with poor prognosis in patients with gastric adenocarcinoma,and GLUT4 expression was positively correlated with Ki67 expression.Cardamonin could inhibit the growth of gastric cancer in vivo and in vitro,promote apoptosis and inhibit the proliferation of gastric cancer cells.The mechanism may be related to the inhibition of c-Myc/GLUT4 axis-mediated energy metabolism reprogramming,ultimately exerting gastric cancer inhibitory effect through dual inhibition of glycolysis and mitochondrial function... |
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