| Background and Objective:Hepatocellular carcinoma(HCC)is the most common pathological type of primary liver cancer and one of the most common malignancies in the world.It is a serious threat to human life and health.Sorafenib,an oral multi-kinase inhibitor,is the first molecular targeted drug approved by FDA in the United States for the treatment of advanced HCC,but it has low objective response rate,more adverse effects,drug resistance and other limitations.Therefore,it is particularly urgent to find effective drugs and methods to change the treatment dilemma of sorafenib.Hepatoma stem cells(HSCs)are a group of tumor cell populations with a very low proportion in liver tumors.With typical cancer stem cell characteristics,HSCs are the main driving factors of tumor progression,metastasis,recurrence and drug resistance,which greatly affect the therapeutic effect and may lead to treatment failure.Therefore,it is of great significance to further search for therapeutic strategies targeting HSCs.Cucurbitacin B(CuB)is a traditional Chinese medicine monomer extracted from cucurbitaceae plants,which shows significant anti-tumor effect in various malignant tumors.Based on this,the purpose of this project is to study CuB in combination with sorafenib whether has synergistic growth inhibitory effect on HCC,and explore whether CuB has inhibitory effect on the growth of CD133 + HepG2 hepatoma stem cell-like cells,as well as seek the possible mechanism of action from the level of molecular and cellular signal transduction aspects,to provide theoretical basis and experimental basis for improving the treatment status of liver cancer and expanding the clinical application of CuB.Materials and methods:First part:(1)CCK-8 assay was used to detect the effects of sorafenib,CuB and combination of them on the proliferation of HepG2 and Huh7 cells,and Calcu Syn software was used to calculate the combination index.(2)The morphological alterations and numbers of HepG2 and Huh7 cells were observed under inverted microscope.(3)The effects of drugs on the clonogenic ability of HepG2 and Huh7 cells were detected by colony formation assay.(4)Subcutaneous xenograft model of HepG2 and Huh7 cells in nude mice was established to study the effects of drugs on the growth of xenograft.(5)PI single staining assay and Annexin V-FITC/ PI double staining assay were used to detect the effects of drugs on cell cycle distribution and apoptotic rate of HepG2 and Huh7 cells.(6)Western blot assay was used to detect the effects of drugs on cleaved caspase-9,cleaved caspase-3,p-STAT3 and STAT3 protein expression levels in HepG2 and Huh7 cells.(7)CCK-8 assay and colony formation assay were used to detect the cell survival rate and colony formation rate of control group,combined group,EGF group and EGF+ combined group.Western blot assay was used to detect the protein expression levels of p-STAT3 and STAT3 in each group.Second part:(1)Growth characteristics of HepG2 cells under different culture conditions were observed under inverted microscope.(2)Immunofluorescence staining assay was used to detect the expression of CD90,CD133,CD326,CD24 and CD44 in parental HepG2 cells and the 12 th passaged HepG2 suspended cells by flow cytometry.(3)HepG2 suspended cells were sorted out by magnetic activated cell sorting(MACS),and the positive expression rate of CD133 was detected by flow cytometry.(4)The process of sphere forming and growth status of CD133+ HepG2 suspended cells cultured in different culture conditions were observed under inverted microscope.(5)CCK-8 assay was used to detect the effects of sorafenib on proliferation of HepG2 cells and CD133+HepG2 suspended cells,and the drug resistance index was calculated.(6)The protein expression levels of CD133,Nanog,Oct4 and Sox2 in HepG2 cells and CD133+HepG2 suspended cells were detected by Western blot assay.(7)The subcutaneous xenograft models of HepG2 cells and HepG2 suspended cells were established in nude mice,respectively.After 28 days of inoculation,the volume and weight of xenograft between the two groups were compared.(8)CCK-8 assay was used to detect the effect of CuB on the proliferation of CD133+HepG2 hepatoma stem cell-like cells,and the effect of CuB on suspended sphere-forming ability was observed under inverted microscope.(9)The effects of CuB on colony formation ability and cell cycle distribution of CD133+HepG2 hepatoma stem cell-like cells were detected by colony formation assay and PI staining assay.(10)The effects of CuB on the protein expression levels of CD133,CDK1,Cyclin B1,p-STAT3 and STAT3 in CD133+HepG2 hepatoma stem cell-like cells were detected by Western blot assay.(11)The protein expression levels of p-STAT3,STAT3,CD133 and Cyclin B1 in the control group,CuB group,IL-6 group and IL-6+ CuB group were detected by Western blot assay.(12)CCK-8 assay and subcutaneous xenograft assay were used to detect the effects of CuB,sorafenib and combination of them on the proliferation and the growth of xenograft of CD133+HepG2 hepatoma stem cell-like cells.Results:First part:(1)Sorafenib,CuB and combination of the two drugs could inhibit the proliferation of HepG2 and Huh7 cells,and the combination index of the two drugs is less than 1,which indicates that the two drugs have synergistic inhibitory effect on cell proliferation in vitro.(2)Sorafenib and CuB alone reduced the number of HepG2 and Huh7 cells,cells shrunk and became smaller,and combination of them had more obvious inhibitory effect.(3)The colony formation rate of HepG2 and Huh7 cells in the two-drug combination group was lower than that in the single drug group,suggesting that they had a long-term synergistic inhibitory effect on cell proliferation.(4)The volume and weight of subcutaneous xenograft in HepG2 and Huh7 cells in the two-drug combination group were lower than those in the single-drug group,which showed synergistic inhibitory effect on the growth of subcutaneous xenograft in vivo.(5)Sorafenib and CuB arrested the HepG2 and Huh7 cell cycle at G1 and G2/M phases,respectively,and the cell cycle was arrested at G2/M phase by the combination treatment,suggesting that the cell cycle was not arrested in a synergistic manner by the two drugs.(6)The apoptotic rate of HepG2 and Huh7 cells in the two-drug combination group was higher than that in the single drug group,suggesting that the two drugs induced cell apoptosis in a synergistic manner.(7)The expression levels of apoptosisrelated proteins cleaved caspase-9 and cleaved caspase-3 in HepG2 and Huh7 cells in the two-drug combination group were higher than those in the single drug group,indicating that the two drugs synergistically induced caspase-dependent cell apoptosis.(8)Sorafenib did not significantly change the expression level of p-STAT3,while CuB and combination treatment inhibited the expression of p-STAT3,and combination treatment had a stronger inhibitory effect than single drug,meanwhile,the total protein of STAT3 did not change significantly in each group,suggesting that the phosphorylation of STAT3 may be involved in the molecular mechanism of the antiHCC effect of CuB and sorafenib.(9)After providing STAT3 signaling pathway agonist EGF(Epidermal Growth Factor),we found that EGF could promote the proliferation of HepG2 and Huh7 cells,colony formation and p-STAT3 up-regulation,and partially rescue the proliferation inhibition,colony formation inhibition and p-STAT3 downregulation caused by combination treatment,further demonstrating that STAT3 phosphorylation may be the molecular mechanism of the anti-HCC effect of CuB and sorafenib.Second part:(1)Parent HepG2 cells exhibited anchorage-dependent growth in medium containing 10% FBS,however,HepG2 cells formed floating cell spheres in serum-free culture,suggesting the growth characteristics of cancer stem cells.(2)Compared with parental HepG2 cells,there was no statistical difference in the positive expression rates of stem cell surface markers CD90,CD44 and CD326 in the 12 th generation HepG2 suspended cells,while there was statistical difference in the positive expression rates of CD133 and CD24,suggesting that suspended cells expressed some stem cell surface markers.(3)The positive expression rates of CD133 in HepG2 suspended cells of the20 th generation before and after MACS were 79.13% and 97.29%,respectively,showing that MACS further improved the CD133 purity of cells.(4)A single CD133+HepG2 suspended cell could gradually form a floating three-dimensional spherical colony within 7 days,suggesting the ability of self-renewal.(5)CD133+ HepG2 suspended cells gradually differentiated into adherent cells in 10% FBS culture medium,suggesting the potential of self-differentiation.(6)The IC50 of sorafenib in CD133+HepG2 suspended cells was higher than that of parental HepG2 cells,and the drug resistance index was 3.72,suggesting that CD133+ HepG2 suspended cells were resistant to sorafenib.(7)CD133+ HepG2 suspended cells had higher expression levels of stemness-related proteins CD133,Nanog,Oct4 and Sox2 than parental HepG2 cells,suggesting characteristics of stemness.(8)The volume and weight of subcutaneous xenograft in CD133+ HepG2 suspended cell group were both greater than that of parental HepG2 cell group,suggesting stronger tumorigenic potential.The above experiments indicated that the prepared CD133+ HepG2 suspended cells were hepatoma stem cell-like cells(9)CuB could inhibit the proliferation,suspension sphereforming ability and colony formation of CD133+ HepG2 hepatoma stem cell-like cells in a concentration-dependent manner,suggesting that CuB had a growth inhibitory effect on hepatoma stem cell-like cells.(10)CuB arrested CD133+ HepG2 hepatoma stem cell-like cell cycle at G2/M phase in a concentration-dependent manner,suggesting that the growth inhibitory effect of CuB was related to the cell cycle arrest.(11)CuB inhibited the expression levels of stemness-related protein CD133,G2/M phase-associated cell cycle molecules CDK1 and Cyclin B1 in a concentrationdependent manner,suggesting that inhibition of stemness and arrest of cell cycle may be the specific mechanism of the growth inhibitory effect of CuB.(12)CuB inhibited the expression of p-STAT3 protein,but had no significant effect on the expression of STAT3 protein,suggesting that STAT3 phosphorylation may play an important regulatory role in the inhibitory growth of hepatoma stem cell-like cells by CuB.(13)After providing STAT3 signaling pathway agonist IL-6,we found that IL-6 could upregulate protein expression levels of p-STAT3,Cyclin B1 and of CD133 in CD133 +HepG2 hepatoma stem cell-like cells,and partially rescue p-STAT3,Cyclin B1 and CD133 down-regulation caused by CuB,further demonstrating that CuB exhibited growth inhibitory effect on hepatoma stem cell-like cells through the regulation of pSTAT3,Cyclin B1 and CD133.(14)CuB combined with sorafenib could synergically inhibit the proliferation and the growth of subcutaneous xenograft of CD133+ HepG2 hepatoma stem cell-like cells in vivo and in vitro,suggesting that CuB could improve the phenomenon of sorafenib resistance.Conclusion:(1)CuB combined with sorafenib could exert synergistic anti-HCC effects in vitro and in vivo.(2)Caspase-dependent cell apoptosis may be the mechanism of apoptosis induced by the combination of the two drugs,and may also be the pathway of synergistic inhibitory growth of HCC cell by the two drugs.(3)Phosphorylation of STAT3 was involved in the molecular mechanism of the synergistic anti-HCC effects of CuB combined with sorafenib.(4)CD133+ HepG2 suspended cells obtained by serum-free suspension culture combined with MACS were hepatoma stem cell-like cells with characteristics of cancer stem cell.(5)CuB could inhibit the growth of CD133+ HepG2 hepatoma stem cell-like cells by down-regulating STAT3 phosphorylation and thereby inhibiting the expression of Cyclin B1,CDK1 and CD133.(6)CuB could improve the resistance of CD133+ HepG2 hepatoma stem cell-like cells to sorafenib in vivo and in vitro. |
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