Study On The Effect And Mechanism Of Parthenolide In Inhibiting Liver Cancer Stem Cells By Downregulating SLC25A

Objective:Liver cancer is a major problem that needs to be solved urgently in global public health,ranking sixth in the incidence of malignant tumors and third in the cause of death from malignant tumors.The 5-year survival rate of liver cancer was only 18%.In China,less than 30%of liver cancer patients can be detected and diagnosed early,and the average survival time of advanced liver cancer patients is less than 1 year.Systemic therapy has played a pivotal role in the treatment of advanced liver cancer.We have witnessed the evolution from single-drug targeted therapy to a combination of immune checkpoint inhibitors and targeted therapy.Although systemic treatment of liver cancer has made some progress,only a small number of patients can obtain lasting clinical benefits.Moreover,the heterogeneity and distant metastasis of liver cancer lead to a high mortality rate of liver cancer patients,making liver cancer a highly fatal disease.Therefore,it is urgent to find effective drugs and methods to change the treatment of liver cancer.Cancer stem cells(CSCs)have unlimited self-renewal and differentiation potential,which may be the reason for clinical treatment failure.Studies on liver cancer stem cells(LCSCs)have shown that LCSCs play an important role in the growth,metastasis,drug resistance,recurrence,and prognosis of liver cancer.Therefore,it is of great significance to further search for therapeutic strategies for LCSCs.It has been shown that CSCs have different mitochondrial characteristics compared with its offspring cells,such as increased mitochondrial oxygen consumption rate,increased mitochondrial mass and increased membrane potential.Importantly,compared to the offspring cells,CSCs exhibit different metabolic characteristics.CSCs preferentially use mitochondrial oxidative phosphorylation(OXPHOS)to support selfrenewal and drug resistance rather than glycolysis.Some key genes responsible for enhancing CSCs mitochondrial OXPHOS have been identified.In recent years,in preclinical studies,therapies that directly inhibit CSCs mitochondria have emerged as potential anti-CSCs drugs.Therefore,the mitochondria of LCSCs can serve as an entry point for in-depth research on antiLCSCs.Traditional Chinese medicine is the treasure of Chinese medicine.It has attracted more and more researchers’ attention due to its unique diagnosis and treatment concept,multi-target,multi-link,non-resistant drug and immune regulation and many other clinical advantages.Importantly,the active ingredients of traditional Chinese medicine can be directly developed or used as the starting point of new drug optimization,and are important sources of new antitumor drugs and lead compounds.We have selected parthenolide(PTL)as the active ingredient of traditional Chinese medicine for research in the library of traditional Chinese medicine monomer compounds.PTL is a natural sesquiterpene lactone compound extracted from the traditional Chinese medicine of chrysanthemum.PTL has been reported to have significant antitumor effects on a variety of tumors,but hardly affects normal cells.In addition,existing studies have found that PTL may specifically kill leukemia stem cells,glioma stem cells,and nasopharyngeal carcinoma stem cells.However,the study of PTL in LCSCs has not been reported.Therefore,this study analyzes the anti-LCSCs effect of PTL through in vivo and in vitro experiments,and further clarifies its potential molecular mechanism.Methods:The effects of 30 kinds of active ingredients of anti-tumor traditional Chinese medicine on proliferation activity of LCSCs were detected by CCK-8 cell proliferation assay.The VOSviewer analysis software was conducted to preliminarily explore the target of PTL.The effects of PTL on the proliferation of LCSCs were detected by CCK-8 proliferation assay and clonal formation assay.DCFH-DA staining flow cytometry was used to detect the effect of PTL on reactive oxygen species of LCSCs.The effect of PTL on mitochondrial membrane potential of LCSCs was determined by JC-1 staining flow cytometry.Fluo-4 AM staining flow cytometry was used to detect the effect of PTL on mitochondrial calcium content of LCSCs.Seahorse assay was conducted to determine the effect of PTL on mitochondrial oxygen consumption rate of LCSCs.CD133-APC staining flow cytometry was used to detect the effect of PTL on CD 133-positive cells in LCSCs.Sphere formation assay was conducted to detect the effect of PTL on the self-renewal ability of LCSCs.qRT-PCR and Western blot were used to detect the effect of PTL on the expression of CD 133,Nanog and Oct4 of LCSCs.Annexin-V-FITC double staining flow cytometry was used to detect the effect of PTL on apoptosis of LCSCs.PI staining flow cytometry was conducted to detect the effect of PTL on the cell cycle of LCSCs.The effects of PTL on the expression of apoptosis-related proteins(Bax,Bcl2,cleaved caspase-3)and cell cycle related protein(Cyclin D1)of LCSCs were detected by Western blot.The effect of PTL on the growth of xenograft tumor of liver cancer was tested in nude mice.Transcriptome sequencing technology was used to detect the changes in transcription levels of LCSCs treated with PTL,and to explore the molecular mechanism of PTL on LCSCs.The expression of SLC25A1 in HCC was analyzed by UALCAN,TNM Plot and HCCDB databases.The clinical significance and prognostic value of SLC25A1 in liver cancer were analyzed by TIMER2.0 and SangerBox.Based on TCGA dataset,the potential molecular mechanism of SLC25A1 was analyzed by GSEA.TIMER2.0 was used to analyze the correlation between SLC25A1 and genes related to mitochondrial respiratory chain complex.Verification of SLC25A1 and detection of mRNA and protein levels of the downstream gene IDH2 of SLC25A1 through qRTPCR and Western blot.SLC25A1 knockout and SLC25A1 inhibitor CTPI-2 were used to detect whether SLC25A1 mediates the anti-LCSCs effect of PTL.The expression levels of SLC25A1,Cleaved caspase-3 and Oct4 in the transplanted tumor tissue were detected by Western blot.The expressions of Ki67,SLC25A1 and Oct4 in the transplanted tumor tissue were detected by IHC.Results:(1)The result of the Sphere formation assay showed that the number of spheres formed by T3A-A3 cells was higher than that of MHCC97H and Huh7 cells(P<0.05).Western blot analysis showed that compared with MHCC97H and Huh7 cells,the expressions of CD133,Nanog and Oct4 proteins in T3A-A3 cells were higher(P<0.05),indicating that T3A-A3 cells had the characteristics of LCSCs.(2)The results of CCK-8 cell proliferation assay showed that the proliferation inhibition rates of PTL at 10 μM and 20 μM concentrations on LCSCs were 37.06%and 12.48%,respectively,lower than those of other 29 active ingredients of traditional Chinese medicine.The screening results showed that PTL could specifically kill LCSCs.(3)Through keyword cluster analysis,VOS viewer found that CSCs and mitochondria were the research hotspots of antitumor of PTL.(4)The results of CCK-8 proliferation assay showed that the IC50 of LCSCs treated with PTL was 9.651μM,significantly lower than that of MHCC97H and Huh7 cells(17.49 μM and 17.02 μM)(P<0.01),and the inhibitory effect of PTL on LCSCs proliferation was concentration-dependent and time-dependent.The results of mouse liver cancer xenograft experiments showed that compared to the control group,PTL significantly inhibited the growth of transplanted liver cancer tumors in mice(P<0.05).(5)The results of flow cytometry and Seahorse assay showed that compared with control group,PTL significantly increased mitochondrial calcium content and reactive oxygen species,and decreased mitochondrial membrane potential and oxygen consumption rate of LCSCs(P<0.01),(6)The results of Sphere formation assay and Western blot detection showed that compared with the control group,PTL inhibited the size and number of spheres formed by LCSCs(P<0.01),and inhibited the mRNA and protein expressions of CD 133,Nanog and Oct4 in LCSCs(P<0.01).(7)The results of flow cytometry and Western blot detection of cell apoptosis and cycle showed that compared with the control group,PTL significantly induced apoptosis of LCSCs cells(P<0.01),increased pro-apoptotic protein Cleaved caspase-3 and Bax expression,and decreased anti-apoptotic protein Bcl2 expression(P<0.01).Compared with control group,PTL significantly increased the percentage of G1 phase cells in LCSCs(P<0.01)and decreased the expression of Cyclin D1 protein in LCSCs(P<0.01).(8)The KEGG enrichment analysis results showed that the genes with significant differences after the treatment of PTL were mainly concentrated in the mitochondria-related signaling pathways including OXPHOS.(9)SLC25A1 was screened by RNA-Seq as a potential target for the antiLCSCs effect of PTL.qRT-PCR and Western blot confirmed that PTL inhibited SLC25A1 expression(P<0.05).The analysis results of the public database showed that the expression level of SLC25A1 in hepatocellular carcinoma was higher than that in normal tissues(P<0.001),and SLC25A1 expression was correlated with liver cancer grade and OS in patients with liver cancer(P<0.05).The GSEA analysis results showed that the high expression of SLC25A1 was correlated with mitochondrial OXPHOS signaling pathway(P<0.01).(10)The results of SLC25A1 knockdown and SLC25A1 inhibitor CTPI-2 experiments showed that PTL inhibited mitochondrial function,stemness characteristics and proliferation of LCSCs by SLC25A1.(11)Western blot results showed that compared with control group,PTL significantly inhibited the expression of SLC25A1 and Oct4(P<0.05),and increased the expression of Cleaved caspase3(P<0.01).The results of IHC showed that compared with the control group,the positive areas of cell proliferation markers Ki67,SLC25A1 and Oct4 in transplanted tumor tissue after the treatment of PTL were significantly reduced(P<0.05).Conclusions:The active ingredient PTL which can kill LCSCs was successfully screened.Functional studies showed that PTL significantly inhibited the proliferation,mitochondrial function,and self-renewal ability of LCSCs.Molecular mechanism studies showed that PTL could significantly inhibit mitochondrial OXPHOS signaling pathway,and preliminary identification showed that SLC25A1 was a potential target of PTL against LCSCs.PTL may be a potential active ingredient of traditional Chinese medicine targeting LCSCs for the treatment of liver cancer.