| BackgroundHepatocellular carcinoma(HCC),also known as liver cancer,is a common malignant tumor in China.Chinese medicine can be highly effective in preventing and treating this disease.Jinmaojiedu Formula(JDF)was developed by our unit as a treatment option.There has been evidence from previous multicenter clinical trials that JDF reduces the postoperative recurrence rate of small liver cancer,but the mechanism of action remains elusive.There is a high rate of liver cancer associated with liver fibrosis and cirrhosis,and liver fibrosis is primarily caused by the activation of hepatic stellate cells(HSCs),which are also the main stromal cells of the postoperative remnant liver.Liver cancer stem cells(LCSCs)are crucial in postoperative liver cancer recurrence.Activated HSCs increase the stemness of LCSCs and promote the postoperative recurrence of liver cancer.This study explores whether JDF and its main component oleanolic acid can prevent liver cancer recurrence by inhibiting HSCs-mediated stemness of HCC cells.Research Objective1.To observe whether JDF reduces the stemness of HCC cells and inhibits tumor growth when administered to HSCs;2.To observe how JDF affects tumorigenesis and stemness expression in HCC cells based on liver fibrosis.Methods1.Conditioned medium(CM)preparationThe conditioned medium was prepared using human HSCs LX2.There were four groups:LX2 cell conditioned medium(LX2-CM group);10 ng/m L TGFβ-1-activated LX2-CM(TGFβ-1 group);10 ng/m L TGFβ-1-activated and 10μM oleanolic acid-treated LX2-CM(QDGS group);and 10 ng/m L TGFβ-1-activated and 1 mg/m L JDF-treated LX2-CM(JDF group).After drug treatment for 24 hours,DMEM containing 1%FBS was used for starvation for 24 hours.The supernatant was collected,centrifuged,and stored at 4°C in a refrigerator.2.Effect of JDF and oleanolic acid-regulated HSCs on EMT and stemness of HCC cellsHuman hepatoma cells namely Huh7 cells were cultivated with collected LX2-CM group,TGFβ-1 group,oleanolic acid group,LX2-CM in JDF group,and standard complete medium(C-DMEM)(control group).After 24 hours,Western blot was performed to detect epithelial-mesenchymal transition(EMT)related protein expression in hepatoma cells,and RT-PCR was conducted to detect stemness-related gene expression in hepatoma cells.3.Effect of JDF and QDGS-regulated HSCs on the sphere-forming ability of HCC cellsIn the presence of 2%B27,20 ng/m L EGF,20 ng/m L b FGF,and 1%N2 in the collected LX2-CM,TGFβ-1,oleanolic acid,and JDF groups,Huh7 cells with a density of 1,000/m L were added,and then the sphere-forming size and the number of suspended spheres exceeding70μm were observed after seven days.4.Effect of JDF and QDGS-regulated HSCs on Wnt/β-catenin signaling pathway in HCC cellsHuh7 cells were cultivated with collected LX2-CM group,TGFβ-1 group,oleanolic acid group,JDF group,and standard C-DMEM(control group).Protein expression related to Wnt/β-catenin signaling pathway in HCC cells was detected by Western blot after 24 hours.5.Effect of JDF and QDGS-regulated HSCs on tumorigenesis and stemness of HCC cells in vivoThe LX2 cells were treated with oleanolic acid and JDF for 24 hours.After that,untreated and drug-treated LX2 cells were mixed in equal proportions with human HCC cells LM3 for the LM3+LX2 group,the LM3+LX2(QDGS treatment)group,and the LM3+LX2(JDF treatment)group,with the LM3 cells alone for the LM3 group.Subcutaneous tumor was implanted in nude mice with cells from each group,and the tumor long axis and short axis were measured every three days.After 14 days,subcutaneous tumors were taken out,tumor weight was measured,and immunohistochemistry was used to detect tumor EMT and stemness index of HCC.6.Effect of JDF on tumorigenesis and stemness expression of HCC cells in hepatic fibrosis miceFor the preparation of 10 liver fibrosis mice models,5 mg/kg DMN was injected intraperitoneally,while saline was injected intraperitoneally for the preparation of five blank control mice.When the subcutaneous tumors grew to a diameter of approximately 1 cm,all mice were implanted with hepatic in situ tumors.Three days after in situ tumor implantation,blank control mice(control group)and five randomly selected hepatic fibrosis model mice(DMN group)received saline gavage,and the remaining five hepatic fibrosis model mice received JDF gavage(DMN+JDF group)for 14 days.Then,the mice were sacrificed to obtain liver weight and tumor weight.The tumor weight ratio and tumor volume were calculated,and the liver tissueα-SMA,tumor tissue EMT and liver cancer stemness indexes were detected by immunohistochemistry.Research Results1.The results of Western blot showed that EMT-related proteins E-cadherin and Vimentin phenotypes were up-regulated in the LX2-CM and TGFβ-1 groups,but down-regulated in the oleanolic acid and JDF groups.2.The results of RT-PCR demonstrated elevated expression of stemness-related genes Nanog,SOX2,and CD133 in Huh7 cells in the LX2-CM and TGFβ-1 groups.In the QDGS group and JDF group Nanog,CD133 expression was lower than that in the TGFβ-1 group(P<0.05).There was a decrease in the expression of stemness-related gene SOX2 in Huh7 cells in the QDGS group and JDF group.However,the differences were not statistically significant(P>0.05).3.The results of tumor stem cell sphere-forming assay suggested that the volume of Huh7 cell suspension spheres was smaller in the LX2-CM group,and the number of suspension spheres larger than 70μm was 23.67±2.49.In contrast,the volume of Huh7 cell suspension spheres became larger in the TGFβ-1 group,and the number of suspension spheres larger than 70μm(56±5.35)increased compared with the LX2-CM group(P<0.05).The QDGS group had smaller Huh7 cell suspension spheres,and the number of greater than 70μm suspension spheres(28.67±2.05)reduced compared with the TGFβ-1 group(P<0.05).The JDF group had the smallest Huh7 cell sphere-forming spheres and the least number of greater than 70μm suspension spheres(19.33±1.24)(P<0.05).4.The results of Western blot revealed thatβ-catenin and p-GSK3β(ser9)protein expression of Huh7 cells was significantly elevated in the LX2-CM group as well as in the TGFβ-1 group,withβ-catenin expression more significantly elevated in the TGFβ-1 group.However,β-catenin and p-GSK3β(ser9)expressions of Huh7 cells decreased in the QDGS group and JDF group.5.The results of subcutaneous tumor experiments in nude mice indicated that the tumor weight of LM3+LX2 group(0.178±0.029 g)was greater than that of LM3 group(0.118±0.019 g)(P<0.05).The tumor weight of LM3+LX2(QDGS treatment)group(0.094±0.017g)was smaller than that of LM3+LX2 group(P<0.05).The tumor weight of LM3+LX2(JDF treatment)group(0.089±0.041 g)was smaller than that of LM3+LX2 group(P<0.05).The immunohistochemical results displayed that tumor cell proliferation-related marker Ki67,EMT marker Vimentin,and liver cancer stemness-related markers Ep CAM and Nanog were significantly higher in the LM3+LX2 group compared with the LM3 group.Ki67,Vimentin,Ep CAM,and Nanog expressions decreased in the LM3+LX2(QDGS treatment)and LM3+LX2(JDF treatment)groups compared with the LM3+LX2 group.6.The experiments on the effect of JDF on tumorigenesis and stemness expression of HCC cells in liver fibrosis mice in vivo found that the tumor volume was larger in the DMN modeling group(1.66±0.42 cm~3)compared with the blank control group(0.45±0.20 cm~3)(P<0.05),and it was smaller in the DMN+JDF group compared with the DMN modeling group(1.36±0.51 cm~3)(P>0.05),with the difference not statistically significant.The immunohistochemical results exhibited thatα-SMA expression in liver tissues increased in the DMN modeling group and decreased in the DMN+JDF group.The EMT markers E-cadherin and Vimentin as well as the stemness-related markers Ep CAM and Nanog were significantly up-regulated in the tumor tissues of the DMN group,while E-cadherin,Vimentin,Ep CAM and Nanog were not significantly up-regulated in the tumor tissues of DMN+JDF group.Summary1.JDF and its main component QDGS inhibited EMT and decreased stemness in HCC cells by regulating HSCs which was associated with Wnt/β-catenin signaling pathway;2.JDF and its main component QDGS inhibited the growth of subcutaneous tumors in nude mice and reduced tumor stemness and JDF had the ability to inhibit tumor stemness and tumor growth on the basis of liver fibrosis;3.On the whole,JDF had a greater effect than its main component QDGS.ConclusionJDF can suppress stemness characteristics of HCC cells by inhibiting the activation of HSCs. |
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