Objective:To investigate the effects and possible mechanisms of the combination of DMDD(2-dodecyl-6-methoxycyclohexa-2-5-diene-1-4-dione),a traditional Chinese medicine monomer,and sorafenib on the malignant biologica...Objective:To investigate the effects and possible mechanisms of the combination of DMDD(2-dodecyl-6-methoxycyclohexa-2-5-diene-1-4-dione),a traditional Chinese medicine monomer,and sorafenib on the malignant biological behavior of human hepatocellular carcinoma Huh7 cells.Methods:The experiment was divided into four groups:Huh7 cells control group,DMDD group,sorafenib group and DMDD and sorafenib combination group.The CCK-8 assay was used to measure the viability of Huh7 cells,and the Kim's formula was used to determine the synergistic effect.The plate cloning experiment was conducted to test colony formation ability of Huh7 cells.The scratch and Transwell experiments were performed to evaluate the migration ability and the invasion ability of Huh7 cells.The cell cycle of Huh7 cells was detected by flow cytometry.RT-qPCR and Western blot were used to measure the mRNA transcription level and protein expression level of PHGDH in the serine synthesis pathway.Results:The plate cloning experiment,scratch experiment,and Transwell migration experiment showed that the combined application of DMDD and Sorafenib significantly enhanced the inhibitory effect on the proliferation,migration,and invasion ability of Huh7 cells compared to the control group,DMDD group,and Sorafenib group(P<0.05).According to the Kim's formula,the combination of DMDD(final concentrations of 2,4,8μmol/L)and Sorafenib(final concentrations of 1,2,4μmol/L)had a synergistic inhibitory effect on the proliferation of Huh7 cells(Q>1.15).6,10μmol/L DMDD combined with 3,5μmol/L Sorafenib showed additive effect.The cell cycle of Huh7 cells was detected by flow cytometry,and the results showed that after 48 hours of drug intervention,the proportion of G2/M phase cells in the control group,DMDD group,Sorafenib group,and combination group were(10.63±0.32)%,(35.77±1.22)%,(30.03±2.22)%,and(38.97±0.60)%,respectively.Compared with the control group,the proportion of G2/M phase cells in the three groups significantly increased(P<0.0001).Compared with the Sorafenib group,the proportion of G2/M phase cells in the combination group significantly increased(P<0.0001).RT-qPCR and Western blot results showed that the combined application of DMDD and Sorafenib significantly inhibited the mRNA transcription level and protein expression level of PHGDH(P<0.05).Conclusion:The combined application of DMDD and Sorafenib has a synergistic effect that can enhance the inhibitory effect on the proliferation,invasion,and migration ability of Huh7 cells.The mechanism of this effect is related to the synergistic inhibition of the gene transcription and protein expression of PHGDH in the serine synthesis pathway.展开更多
基金This study was supported by National Natural Foundation Project of China(81860504)。
文摘Objective:To investigate the effects and possible mechanisms of the combination of DMDD(2-dodecyl-6-methoxycyclohexa-2-5-diene-1-4-dione),a traditional Chinese medicine monomer,and sorafenib on the malignant biological behavior of human hepatocellular carcinoma Huh7 cells.Methods:The experiment was divided into four groups:Huh7 cells control group,DMDD group,sorafenib group and DMDD and sorafenib combination group.The CCK-8 assay was used to measure the viability of Huh7 cells,and the Kim's formula was used to determine the synergistic effect.The plate cloning experiment was conducted to test colony formation ability of Huh7 cells.The scratch and Transwell experiments were performed to evaluate the migration ability and the invasion ability of Huh7 cells.The cell cycle of Huh7 cells was detected by flow cytometry.RT-qPCR and Western blot were used to measure the mRNA transcription level and protein expression level of PHGDH in the serine synthesis pathway.Results:The plate cloning experiment,scratch experiment,and Transwell migration experiment showed that the combined application of DMDD and Sorafenib significantly enhanced the inhibitory effect on the proliferation,migration,and invasion ability of Huh7 cells compared to the control group,DMDD group,and Sorafenib group(P<0.05).According to the Kim's formula,the combination of DMDD(final concentrations of 2,4,8μmol/L)and Sorafenib(final concentrations of 1,2,4μmol/L)had a synergistic inhibitory effect on the proliferation of Huh7 cells(Q>1.15).6,10μmol/L DMDD combined with 3,5μmol/L Sorafenib showed additive effect.The cell cycle of Huh7 cells was detected by flow cytometry,and the results showed that after 48 hours of drug intervention,the proportion of G2/M phase cells in the control group,DMDD group,Sorafenib group,and combination group were(10.63±0.32)%,(35.77±1.22)%,(30.03±2.22)%,and(38.97±0.60)%,respectively.Compared with the control group,the proportion of G2/M phase cells in the three groups significantly increased(P<0.0001).Compared with the Sorafenib group,the proportion of G2/M phase cells in the combination group significantly increased(P<0.0001).RT-qPCR and Western blot results showed that the combined application of DMDD and Sorafenib significantly inhibited the mRNA transcription level and protein expression level of PHGDH(P<0.05).Conclusion:The combined application of DMDD and Sorafenib has a synergistic effect that can enhance the inhibitory effect on the proliferation,invasion,and migration ability of Huh7 cells.The mechanism of this effect is related to the synergistic inhibition of the gene transcription and protein expression of PHGDH in the serine synthesis pathway.
