Inhibition Effects Of Dihydroartemisinin On Human Gastric Cancer Cell MKN-45 And The Mechanism Of Action

Objective:To investigate the inhibitory effects of dihydroartemisinin(DHA)on human gastric cancer cell MKN-45 and elucidate the possible mechanism.Methods:1.CCK8 assay was used to detect the cell growth and proliferation:(1)Detect and compare the anti-proliferation effects of DHA on human gastric cancer cell line MKN-45,human chronic myeloid leukemia cell line K562,human colon cancer cell line HT-29.(2)Detect the role of ferrous ions(Fe2+)and reactive oxygen species(ROS)in the anti-proliferation effects of DHA towards the most sensitive cell line MKN-45,including:?Modulation effects of ferrous sulfate(FeSO4)and ammonium ferric citrate(FAC)on the anti-tumour effects of DHA;?Modulation effects of iron chelator deferoxamine(DFO)on the anti-tumour effects of DHA;?Modulation effects of ROS scavenger N-acetyl-L-cysteine(NAC)on the anti-tumour effects of DHA.2.Flow cytometry(FCM)was used to detect the ROS level and cell cycle distribution in MKN-45 cells:?Detect the reactive oxygen species(ROS)level in MKN-45 cells.?Detect the cell cycle distribution of MKN-45 cells.?Detect the apoptosis induced by DHA in MKN-45 cells.?Detect the mitochondrial membrane potential(??m)changes in MKN-45 cells.3.RQ-PCR or Western-blot technology were used to detect the effects of DHA on cell cycle-associated factors:(1)The effects of DHA on the expression of CyclinDl,CDK2,CDK4,CDK6,p21,p27,p53,Rb,E2F1 genes in MKN-45 cells were analyzed using RT-qPCR.(2)The effect of DHA on the expression of CyclinD1,p-Cdk4,CDC25A,p21,p53,p-Rb protein in MKN-45 cells was detected by Western-blot technology.4.Detection of cell death:?Using fluorescence microscopy to detect the morphological changes.Hoechst33342/PI staining was used to observe the morphological changes of DHA-induced MKN-45 cell death by fluorescence microscope.?CCK-8 assay or western-blot technology were used to verify ferroptosis:CCK-8 assay was used to detect the effect of erastin,an iron death activator,and ferrostatin-1,an iron death inhibitor,on the proliferation of MKN-45 cells;Western blot technology was used to detect the expression of glutathione peroxidase 4(GPx4),a ferroptosis inhibitory protein.Results:1.DHA showed stronger inhibitory effects on human gastric cancer(GC)cell line MKN-45.The inhibitory effects of DHA towards three tumor cells showed a dose-dependent manner,and the IC50 for HT-29,K562 and MKN-45 was 384.91±48.03,54.35±26.38,19.66±3.76?M at 48h,respectively.2.The presence of Fe2+significantly affected the inhibitory activity of DHA on MKN-45 cells.When combined with FeSO4 or FAC,the cytotoxicity of DHA were enhanced.The cytotoxicity of DHA was ameliorated when combined with DFO or NAC,showing with increased cell numbers and enhanced cell activity.3.DHA significantly induced ROS accumulation in MKN-45 cells in a dose-dependent manner.The accumulation level of ROS is dependent on the presence of Fe2+,showing that combination with FAC significantly enhanced the DHA-produced ROS level.When chelating the Fe2+(combination with DFO)and removing ROS(combination with NAC),they both ameliorated the DHA-produced ROS,4.DHA treatment induced MKN-45 cell cycle arrest with a significant increase proportion of G0/G1 phase cells.When combinated with DFO or NAC,the proportion of G0/G1 phase cells was significantly reduced.5.DHA treatment up-regulated the expression of cyclinD1,CDK4,CDK6,p21,p27,p53,Rb genes in MKN-45 cells,but the expression of CDK2,E2F genes decreased.DHA up-regulated the expression of CDC25A,p21,p53 and p-Cdk4 proteins in MKN-45 cells and inhibited the expression of p-Rb and cyclinD1 protein in a dose-dependent manner.6.DHA induced MKN-45 cells apoptosis.Morphological observations showed that the number of MKN-45 cells was reduced,the chromatin density was densely stained,and the nucleus pyknosis and crescent core were observed.The mitochondrial membrane potential(??m)of MKN-45 did not change after DHA treatment,neither did DFO,NAC,FeSO4 alter ??m when compared with DHA treatment alone.The iron death activator erastin and inhibitor ferrostatin-1 had no significant effect on the growth and proliferation of MKN-45 cells.The expression of GPx4 protein was up-regulated after DHA treatment in MKN-45 cells.Conclusion:1.DHA exerts significant anti-tumor effect on human gastric cancer cells line MKN-45.The anti-tumor effect of DHA was enhanced by the presence of Fe2+ and accumulation of ROS level in the cells.2.The cytotoxicity of DHA towards GC is implicated in the cell cycle arrest and apoptosis induced by DHA.3.DHA treatment arrests MKN-45 cells at the G0/G1 phase by suppressing the mRNA expression of CDK2 and E2F,and by increasing that of P21,p27,P53,Cyclin D1,CDK4 and CDK6.DHA increases p53,p21,p-Cdk4 protein expression and decreases the expression of p-Rb,cyclinDl,CDC25 A protein.4.DHA do not induced MKN-45 cells ferroptosis.