| Objective: Diallyl disulfide(DADS),a sulfuric compound derived from garlic.However,the mechanisms of action underlying this compound’s anticancer activity have not been fully elucidated.The aim o f this study was to identify the mechanism by which AMP-activate d protein kinase alpha1(AMPKα1)contributes to the anti-tumor effects of DADS.Different concentrations of DADS could affect MGC803 gastric cancer cell energy metabolism,at the same time through further investigated the mechanism that diallyl disulfide could suppress aerobic glycolysis by down-regulating AMPKα1 in gastric cancercells.Methods: In this study,MGC-803 gastric cancer cells were cultured in vitro.With different concentrations of DADS(10,20,40mg/L) treatment of gastric cancer cells,the levels of glucose concentration and lactate production level in the medium were respectively examined with using a Glucose Colorimetric Assay Kit II and a Lactate Assay Kit,respectively.Cell lysates were analyzed by western blot using antibodies specific for AMPKα1 and LDHA,and the mRNA levels of AMPKα1 were determined by real-time PCR using specific primers.Screening of sh-AMPKα1 best pieces,using a technique called RNA interference,silenced AMPKα1 expression of MGC-803 cells,the experiment was divided into five groups(mock,scramble,sh-AMPKα1 group,sh-AMPKα1+DADS and DADS group),The sh RNA-AMPKα1 was transfected into MGC-803 cells using lipofectamine 2000.And then to detect DADS to silenced AMPKα1 after MGC-803 cells of glucose and lactic acid concentration,at the same time observation AMPKα1m RNA,AMPKα1 and LDHA protein expression changes.Results:1.DADS inhibits glucose uptake and lactate production in gastric cancer cells.Inhibition in of anaerobic glycolysis was observed in diallyl disulfide-t reated MGC-803 cells,with including an increased glucose concentration i n the medium(P<0.05),decreased lactate production in the medium(P<0.05)and down-regulation of AMPKα1 and LDHA expression.It was best inhibitory effect when 20mg/L DADS on glucose uptake and lactate production.2.DADS regulates the expression of AMPKα1 and LDHA protein in gastric cancer cells.Compared with the blank group,the expression of AMPKα1m RNA was down regulated(P<0.05),and the expression level of AMPKα1 and LDHA protein.3.AMPKα1 is the target gene of DADS.The results showed that the levels of glucose concentration in medium of the sh-AMPKα1+DADS group were up-regulated and it was not signif cantly regulated compared with sh-AMPKα1 group(P>0.05),and the levels of lactate were up-regulated and it was not significantly regulated compared with sh-AMPKα1 group(P>0.05).Futhermore,the AMPKα1 mRNA expression in the sh-AMPKα1+DADS group was of no significantly difference compared with the sh-AMPKα1 group(P>0.05),but were down-r egulated compared with scrambled oligonucleotide-treated cells(P<0.05).Futhermore,compared with the mock group,the glucose concentration increased,the lactate concentration decreased in medium and AMPKα1 mRNA decreased of the DADS group,the difference was statistically significant(P<0.05).Compared with sh-AMPKα1 group,the expression of AMPKα1 and LDHA protein in sh-AMPKα1+DADS group was not significantl y different.Compared with mock group,the expression of AMPKα1 and LDHA protein in sh-AMPKα1 group and sh-AMPKα1+DADS group wer e significantly decreased.Conclusion:1.DADS suppresses energy metabolism in MGC-803 human gastric cancer cells,and the concentration of 20 mg/L DADS inhibition energy metabolism is best.2.DADS suppressed aerobic glycolysis in MGC-803 human gastric cancer cells by down-regulation of AMPKα1.It These results indicated that AMPKα1 may serve as a potential gene therapy target. |
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