| Objective:Liver cancer is the sixth most common cancer in the world in 2020 and is one of the major threat factors that seriously affect human health worldwide.Primary liver cancer is the most predominant type of liver cancer,and its mortality rate is the third highest in the ranking of cancer mortality rates.Traditional treatment methods are facing the challenges of short survival,poor prognosis and drug resistance of advanced liver cancer patients,which cannot treat liver cancer from the root cause and improve the efficacy,so it is urgent to find effective treatment methods to eradicate primary liver cancer.Cancer stem cells(CSC)are a small group of cancer cells with stem cell characteristics that exist in tumor tissues,which are the root cause of tumor development,as well as an important cause of a series of immune escape and drug resistance responses.Liver cancer stem cells(LCSCs),obtained through screening and isolation,are likely to be the underlying factors leading to the development,metastasis and recurrence of liver cancer and resistance to treatment.Therefore,it is wise and inevitable to deeply explore and to take LCSCs as an important research direction to overcome clinical liver cancer treatment.The metabolism properties of CSC have special characteristics to meet their survival needs.A large amount of research evidence suggests that CSC tend to favor oxidative phosphorylation(OXPHOS)rather than glycolysis.Mitochondria are an important organelles and major sites of cellular OXPHOS,which are involved in regulating cellular energy conversion,apoptosis,redox homeostasis,resctive oxygen species(ROS)production,calcium homeostasis and mitochondrial membrane potential(MMP)regulation are closely related to the maintenance of CSC.Therefore,finding the vulnerability of LCSCs from the mitochondrial perspective and developing novel targeted drugs for LCSCs are expected to be an effective way for clinical treatment of liver cancer.In China,the use of traditional Chinese medicine has a long history,and the therapeutic advantages of natural products of traditional Chinese medicine in various clinical diseases are becoming more and more significant,especially in the application of tumor therapy with high safety,multi-target and specificity.Dioscin is the main active ingredient of Dioscorea opposita(also known as yam),which is known as the "food of the immortals" in Chinese medicine,and has various biological effects such as immunomodulation,anti-inflammatory,anti-tumor,lipid-lowering,and hepatoprotective,and has high medicinal value.Studies have shown that dioscin can produce antitumor effects by participating in several biological processes such as cell proliferation,metabolism,and cell cycle.Studies have confirmed that dioscin has therapeutic effects on CSC in osteosarcoma and breast cancer,but there are no studies on whether dioscin can specifically target LCSCs.Therefore,this study mainly investigated the inhibitory effects of dioscin on LCSCs and the potential molecular mechanisms through in vitro and in vivo experiments.Methods:1 The results of Sphere formation assay showed that T3 A-A3 could form more Sphere spheres with strong self-renewal ability,and dioscin significantly inhibited the self-renewal of T3A-A3 cells;immunofluorescence staining showed that LCSCs T3A-A3 highly expressed surface marker CD 133(P<0.05);Western blot assay showed higher expression levels of stemness markers CD133,CD44 and stemness-related genes Nanog,SOX2 and Oct4 in LCSCs T3A-A3(P<0.05),confirming that LCSCs T3A-A3 cells have the characteristics of CSC.2 The effects of dioscin on the viability of LCSCs T3A-A3 and liver cancer cell line MHCC97H and their proliferation ability were assessed using CCK-8 and cell colony formation assays.3 The effect of dioscin on the cell cycle of LCSCs T3A-A3 and liver cancer cell line MHCC97H was examined by PI staining flow cytometry.4 The effect of dioscin on the apoptosis level of LCSCs T3A-A3 and liver cancer cell line MHCC97H cells was detected by Annexin V-FITC double-stained flow cytometry.5 Western blot was performed to detect the expression of apoptosis-related proteins Bax,Bcl-2 and Cleaved caspase-3 to assess the effect of dioscin on apoptosis in LCSCs T3A-A3 cells.6 ROS assay was performed to assess the difference in ROS levels between LCSCs T3A-A3 and liver cancer line MHCC97H and the effect of dioscin on ROS levels in LCSCs T3 A-A3,combined with mitochondrial membrane potential assay to investigate the effect of dioscin on the mitochondrial function of LCSCs T3A-A3.7 A subcutaneous transplantation tumor assay in nude mice was used to compare the tumorigenic ability of LCSCs T3 A-A3 and liver cancer cell line MHCC97H and to detect the effect of dioscin on subcutaneous transplantation tumors of liver cancer.8 The results of the reactive oxygen species assay showed that the reactive oxygen species level in LCSCs T3A-A3 cells without any treatment was lower than that in MHCC97H cells;the reactive oxygen species level in LCSCs T3A-A3 cells in the dioscin-treated group was increased(P<0.05).9 Western blot to detect the effect of dioscin on the expression of stemness markers CD 133,CD44,stemness-related genes Nanog,SOX2 and Oct4 and apoptosis-related protein Cleaved caspase-3 in transplanted tumor tissues of nude mice in vivo.10 Using transcriptome sequencing technology,we examined the changes of transcript levels before and after dioscin treatment of LCSCs T3A-A3,explored the molecular mechanism of dioscin action on LCSCs T3A-A3,and searched for which biological functions and signal transduction pathways the differential genes were mainly associated with by GO analysis and KEGG analysis.11 Western blot and real-time fluorescence quantitative PCR to detect the effect of dioscin on MBNL1 and PKM2.12 An siRNA knockdown of MBNL1 was used to test whether MBNL1 mediates the inhibition effect of dioscin against T3A-A3 in LCSCs.13 The ROS assay was used to detect the level of ROS in LCSCs T3A-A3 after siRNA knockdown of MBNL1 and PKM2,and to test whether dioscin affects the mitochondrial function of LCSCs T3A-A3 through MBNL1.14 The mitochondrial membrane potential levels of LCSCs T3A-A3 after siRNA knockdown of MBNL1 and PKM2 were detected using mitochondrial membrane potential assay to test whether dioscin could affect the mitochondrial function of LCSCs T3A-A3 through MBNL1.Results:1 The results of Sphere formation assay showed that LCSCs T3A-A3 could form more Sphere spheres with high self-renewal ability,and dioscin significantly inhibited the self-renewal of T3A-A3 cells;immunofluorescence staining showed that LCSCs T3A-A3 highly expressed surface marker CD 133(P<0.05);Western blot showed higher expression levels of stemness markers CD133,CD44 and stemness-related genes Nanog,SOX2 and Oct4 in LCSCs T3A-A3 compared with MHCC97H and BEL7402 cells(P<0.05),confirming that LCSCs T3A-A3 cells have the characteristics of CSC.2 The results of CCK-8 assay showed that dioscin inhibited the viability and proliferation ability of T3A-A3,MHCC97H and BEL7402 cells with IC50 of 2.62±0.133 μM,7.58± 0.69μM and 4.70±0.16 μM at 48 h.Dioscin significantly inhibited the viability of T3A-A3 cells of LCSCs in a time-and concentration-dependent(P<0.05).3 The results of cell colony formation assay showed that LCSCs T3A-A3 had strong proliferation ability and formed more number of colonies;dioscin showed concentration-dependent inhibition of T3A-A3 proliferation ability.4 Western blotting experiments showed that dioscin significantly reduced the expression levels of stemness markers CD 133,CD44 and stemness-related genes Nanog,SOX2 and Oct4 in LCSCs T3A-A3 in a concentration-dependent manner(P<0.05).5 Cell cycle assays showed that T3A-A3 cells were more often in the G1 phase;dioscin induced a concentration-dependent block in the G1 phase of the T3A-A3 cell cycle in LCSCs compared with the control group(P<0.05).6 Flow cytometric analysis of apoptosis showed that dioscin significantly induced apoptosis in LCSCs T3A-A3 cells in a concentration-dependent manner(P<0.05).7 Western blotting assays showed that dioscin induced a concentration-dependent increase in the expression levels of pro-apoptotic protein Bax and Cleaved caspase3 and a decrease in the expression level of anti-apoptotic protein Bcl-2 in T3A-A3 cells of LCSCs(P<0.05).8 Without any treatment,the results of ROS experiment showed that the ROS level in LCSCs T3A-A3 cells was lower than that in MHCC97H cells;compared with the control group,the ROS level in LCSCs T3A-A3 cells was increased in the dioscin-treated group(P<0.05).9 The subcutaneous transplantation tumor experiment in nude mice showed that LCSCs T3A-A3 cells were more tumorigenic(P<0.05);dioscin significantly inhibited the growth of LCSCs T3A-A3 cells in vivo in a concentration-dependent manner(P<0.05);dioscin combined with sorafenib increased the tumor suppression rate(P<0.05).10 The results of immunohistochemical experiments showed that dioscin significantly reduced the expression levels of stemness markers CD 133 and CD44 and upregulated the expression of apoptotic protein Cleaved caspase3 in transplanted tumor tissues(P<0.05).11 Western blotting experiments showed that dioscin was able to decrease the expression levels of stemness markers CD133,CD44 and stemness-related genes Nanog,SOX2 and Oct4 in transplanted tumor tissues in a concentration-dependent manner,and upregulated the expression of apoptotic protein Cleaved caspase3(P<0.05).12 Transcriptome sequencing showed that dioscin could elevate MBNL1 expression in LCSCs T3A-A3 cells.13 Western blotting assay and real-time fluorescence quantitative PCR showed that dioscin was able to increase the expression level of MBNL1 and decrease the expression level of PKM2 in LCSCs T3A-A3 cells in a concentration-dependent manner(P<0.05).14 The results of the reactive oxygen species assay showed that dioscin could affect ROS levels in T3A-A3 cells through MBNL1 regulation of PKM2 and increase the accumulation of ROS in LCSCs T3A-A3 cells(P<0.05).15 Mitochondrial membrane potential experiments showed that dioscin induced MMP reduction in LCSCs T3A-A3 cells through MBNL1 regulation of PKM2(P<0.05).16 Taken together,the above experimental results indicate that dioscin can inhibit the CSC characteristics of LCSCs T3A-A3 cells via MBNL1 and affect their mitochondrial functions.Conclusions and significance:LCSCs T3A-A3 has biological properties such as self-renewal and high tumorigenicity.In vitro,dioscin could significantly inhibit the viability,proliferation and self-renewal ability of LCSCs T3A-A3,induce the elevated intracellular ROS levels in LCSCs T3A-A3 through MBNL1 and PKM2,reduce MMP and affect the mitochondrial function of LCSCs T3A-A3;in vivo,dioscin could inhibit the transplantation tumor formed by LCSCs T3A-A3.Dioscin inhibited the stemness characteristics and proliferative capacity of LCSCs T3A-A3 from in vitro and in vivo,respectively.In addition,MBNL1 and PKM2 may be therapeutic targets for primary liver cancer,and dioscin may be a candidate herbal natural product for the treatment of primary liver cancer. |
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