Experimental study on the synergistic inhibition of malignant biological behavior of hepatocellular carcinoma cells by the combination of DMDD and sorafenib

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.

DMDD与索拉非尼联合应用协同抑制肝癌细胞恶性生物学行为的实验研究

目的:探讨中药杨桃根提取物DMDD单体(2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione)与索拉非尼联合应用对人肝癌Huh7细胞恶性生物学行为的影响及可能的机制。方法:实验分4组:Huh7细胞对照组、DMDD组、索拉非尼组及DMDD与索拉非尼联合应用组(简称联合应用组)。CCK-8法检测Huh7细胞活力,以金氏公式判定协同效应;平板克隆实验检测Huh7细胞的集落形成能力;划痕实验、Transwell迁移实验和侵袭实验检测Huh7细胞的迁移能力和侵袭能力;流式细胞术检测Huh7细胞周期;RT-qPCR和Western blot检测丝氨酸合成通路中的PHGDH mRNA转录水平和蛋白表达水平。结果:平板克隆实验、划痕实验和Transwell迁移实验结果显示,与Huh7细胞对照组、DMDD组、索拉非尼组比较,DMDD和索拉非尼联合应用组对Huh7细胞增殖、迁移和侵袭能力的抑制作用显著增强(均P<0.05),经金氏公式计算,2、4、8μmol/L DMDD分别联合1、2、4μmol/L索拉非尼具有协同作用(Q>1.15),6、10μmol/L DMDD分别联合3、5μmol/L索拉非尼具有相加作用(0.85<Q<1.15);流式细胞周期检测显示,各组药物干预48 h后,对照组、DMDD组、索拉非尼组及联合应用组G2/M期细胞比例分别为(10.63±0.32)%、(35.77±1.22)%、(30.03±2.22)%、(38.97±0.60)%。与对照组比较,3个组的G2/M期细胞比例均显著上升(均P<0.0001),与索拉非尼组比较,联合应用组G2/M期细胞比例显著上升(P<0.0001)。RT-qPCR和Western blot结果显示,DMDD与索拉非尼联合应用可显著抑制PHGDH的mRNA转录水平和蛋白表达水平(P<0.05)。结论:DMDD与索拉非尼联合应用具有协同增效作用,可协同抑制Huh7细胞的增殖、侵袭和迁移能力,其机制与协同抑制丝氨酸合成通路中的PHGDH的基因转录和蛋白表达有关。

Qishen Yiqi Dripping Pills for Cardiovascular Diseases: Effects and Mechanisms

Qishen Yiqi Dripping Pills(QSYQ) is a compound of Chinese medicine, which has been used to treat coronary heart disease and cardiac dysfunction. Its natural components include astragaloside Ⅳ, flavonoids, danshensu, protocatechualdehyde, salvianolic acid B, salvianolic acid A, ginsenosides Rg1, ginsenosides Rb1, and essential oils, etc. It exerts effects of nourishing qi and promoting blood circulation to relieve pain. In this review, the bioactive components of QSYQ and its effects for treating cardiovascular diseases and possible mechanism were summarized, providing references for further study and clinical application of QSYQ.

细胞周期蛋白依赖性激酶4对肝癌细胞生物学行为的影响

通过癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库、基因表达综合数据库(Gene Expression Omnibus,GEO)和体外细胞实验,研究了细胞周期蛋白依赖性激酶4(cyclin-dependent kinase 4,CDK4)在肝细胞癌(hepatocellular carcinoma,HCC)中的临床预后价值及其对HCC细胞系恶性生物学行为的影响。从TCGA数据库中下载371例HCC肿瘤样本、50例非肿瘤样本,GEO数据库中下载225例肿瘤样本和220例非肿瘤样本的CDK4 mRNA表达量及临床生存数据,采用Kaplan-Meier法分析CDK4表达量与HCC临床预后的关系。采用siRNA技术抑制HCC细胞中CDK4的表达,通过qPCR和Western blotting检测CDK4的表达抑制效果;体外细胞实验检测沉默CDK4对HCC细胞系增殖、侵袭、转移和细胞凋亡、细胞周期等恶性生物学行为的影响。TCGA和GEO(GSE14520)数据分析结果显示,在HCC组织中CDK4表达量显著高于非肿瘤组织(均P<0.01),并且高表达提示不良预后(均P<0.05)。细胞功能实验结果显示,沉默Hep3B和Huh-7细胞系中CDK4后,与对照组si-Control相比,si-CDK4实验组肿瘤细胞的增殖、侵袭和转移潜能均显著增加(均P<0.05),并且能抑制细胞凋亡的发生和促进细胞周期进展(均P<0.05)。综上所述,CDK4高表达提示不良预后,具有一定的临床预后预测价值,然而抑制CDK4表达后反而促进HCC细胞系恶性生物学潜能,因此CDK4可能无法作为单独阻断靶点以抑制肿瘤细胞的增殖,是否能作为潜在的联合治疗靶点应用于HCC的治疗还需进一步的探索。