| Background and aims:Abnormal metabolism is one of the tumor hallmarks.Usually,cancer cells increase their uptake of nutrients,mainly including glucose and glutamine,change the mode of metabolism to meet the energy demanding.Cell metabolic reprogramming and abnormal nuclear signal transduction are closely related to the key malignant behavior of tumor cells.HCC cells shows high glycolytic rate.The key enzymes including PKM2,HK2 and PFK are reported highly expressed in tumor cells.Not just as the previous studies stated that the bio-genesis and function of mitochondria was inhibited under hypoxia environment,the OXPHOS in mitochondria conducted actively in cancer cells as well.Targeting energy metabolism pathway is the focus of today’s cancer therapy.Natural products or compounds have long been recognized as valuable anticancer resources,since plant-derived products have few deleterious side effects than synthetic agents.Sanguinarine is a benzophenanthridine alkaloid isolated from the roots of Sanguinarine canadensis.In the following,SAG will be used as the abbreviation for sanguinarine.In vivo and in vitro preliminary studies on sanguinarine have documented a broad-spectrum pharmacological activities,including anti-tumor,antioxidant,and anti-bacterial actions.Sanguinarine has been reported to induce two modes of cell death in a range of human cancer cells,exhibits cytotoxic effects via modulating the activity of various signaling cascades.More importantly,sanguinarine shows non-toxic to healthy cells signifying its acceptance and potent as an anticancer agent.However,pathological mechanisms of Caspase-independent cell death and the metabolism changes caused by SAG are not defined.In this study,we aimed to investigate the anti-tumor and anti-metastatic action of sanguinarine on hepatocellular carcinoma,explore the underlying molecular mechanisms behind the phenomenon of cell death and energy changes.Methods:1.Sanguinarine induced two modes of tumor cell death:To explore the inhibitory effect of sanguinarine on hepatoma cell growth,CCK8-cell counting kit was used.Hepatoma cells were exposed to sanguinarine at 0μM,2μM,4μM,8μM,16μM for 6hours,morphologic alterations and ultrastructure changes were observed and photographed.Meanwhile,cell death was analyzed by cytometry and TUNEL assay.The ATP production,free Ca2+ level and LDH release were assessed.2.Differential genes of HCC cells exposed to low and high concentration of sanguinarine were screened.Enrichment analysis was performed based on differential analysis.Moreover,we measured the expression level of genes involved in lipid metabolism,glucose metabolism and energy pathways.PKM2 was screened out as the possible target of sanguinarine.3.To explore the effect of sanguinarine on mitochondria function in the process of cell death.Above cell models were used for exploration.The changes of ROS and MPTP,MMP,oxidative phosphorylation system and copy number of mitochondrial DNA in different groups were detected.The expression of genes related to mitochondrial biogenesis and dynamics were detected.The oxygen consumption rate were determined in this study.4.To explore the effect of sanguinarine on aerobic glycosis: the intracellular level of 2-DG and lactate in culture medium were used to evaluate the glycolytic activity.Extracellular acidification rate and the activity of PK were measured.Total proteins were extracted from cell samples and the expression levels of key enzyme were evaluated.5.Screening and exploring the molecular mechanism of SAG anti-tumor action:Above cell models were used for examination,the phosphorylation of ERK were detected using western blot.Cell immunofluorescent staining were used to exhibited the differential distribution of p ERK and PKM2 in cell regions and U0126 MEK/ERK inhibitor was used as control.Intracellular cytosolic and nuclear proteins were isolated,the expression level of ERK/PKM2/β-catenin were determined,including PKM2,p ERK,Y333,Y654,Y86 phosphorylation sites of β-catenin.EGF cytokine was used as the positive control.CHIP assay was performed to investigate the binding ability of β-catenin to the promoter of CCND1 under SAG treatment.Cell cycle distribution was analyzed by flow cytometry with PI staining,the check proteins related with cell cycle were detected by western analysis and RT-PCR.The PKM2over-expressing plasmids was constructed and transfected into HCC cells.Changes of ATP and 2-DG production in PKM2 over-expression cells were investigated.6.Subcutaneous xenograft tumor model and tail vein intravenous injection model were constructed.Mice were randomised into cancer group and SAG treatment group.During this period,the volume of subcutaneous tumor and weight of mice were recorded.At the end of modeling,blood and tissue samples were collected for follow-up analysis.ALT and AST were detected in the serum of mice.The numbers of metastatic nodules on the surface of organs were counted macroscopically,and tissue protein was extracted for detection of gene expression.Wound healing assay was performed to determine the migratory ability of hepatoma cells exposed to SAG,the biomarkers of EMT and MMPs expression level were detected.Results:1.Sanguinarine treatment inhibits hepatoma cell growth,time-dependent cytotoxicity of drug was found in two cell lines.After incubated with sanguinarine for6 hours,hepatoma cells mainly showed two different morphological changes.Transmission electron microscopy imaged the swollen mitochondria with disorganized cristae.In the flow cytometry assay,one group of cell clustered in the Annexin-V-/PI + quadrant was found under high SAG treatment,these sub-population cells could not be reduced by using Z-VAD-FMK or Necrostatin-1.TUNEL positive labeled regions were found in both low and high dose group.Caspase activation was detected in SAG induced cell apoptosis,while there was no statistically increased Caspases level in oncotic cells.High dose SAG significantly increased the expression of capn2 and porimin.Besides,the amount of ATP production,Ca2+ content and LDH releases were statistically changed under high dose of SAG stimulation.2.Transcriptome sequence analyses: Illumina sequencing was performed to sequence the transcriptomes of tumor tissues in the blank control and SAG treated groups.The differential genes were visualized using volcano plot and analyzed via R platform.GO and KEGG pathway enrichment analysis showed that DEGs were enriched in lipid metabolism,glucose metabolism,energy pathway and cell cytoskeleton modulation signal.Based on the results of RT-PCR and the data from bioinformatic analysis,PKM2 was screened out as the target of SAG treatment.3.“Mitochondria events” is the critical element in cell death.Apoptotic could be initiated by high oxidative stress,and mitochondria is the main organelle which produces ROS.We found that the ROS level was up-regulated with the increasing of external SAG stimulation.Besides,anti-oxidant agent NAC could not rescue the loss of ATP caused by high SAG application.Next,findings from mito-tracker staining and OCR showed that mitochondria completeness and respiration function was destroyed after high dose of SAG treatment.Moreover,MMP depolarization and MPTP opening were found in oncosis cells.4.Sanguinarine reduced the intracellular content of 2-DG,which represents the uptake rate of glucose in tumor cells.The content of lactic acid and extracellular acidation rate in liver cancer cells were determined,high SAG treated group showed more overwhelming inhibitory effects on cell aerobic glycolysis.5.SAG decreased the level of p ERK in total cell protein and mitochondria protein.PKM2 expression level was decreased in both cytosolic and nuclei regions in high dose group.EGF activates ERK phosphorylation,promotes p ERK-mediate intra-nuclear translocation of PKM2 and phophorylation level of β-catenin Y333 site.Sanguinarine reduced the binding ability of β-catenin on CCND1 promoter,and almost treated tumor cells were arrested in G0/G1 phase.Results showed Cyclin D1 and c-myc expression were inversely related with the sanguinarine concentration.Besides,metabolite function of PKM2 was upregulated in PKM2 plasmids transfected tumor cells,accompanied with more ATP production and higher 2-DG uptake.Furthermore,PKM2-oe transfection could reverse the changes of capn2,porimin,ERK/PKM2/β-catenin,EMT biomarkers and MMPs caused by SAG treatment.6.SAG treatment significantly inhibits tumor growth in xenograft model,and decreased amounts of nodules on liver and lung were found in tail vein injection metastasis mice.Fewer nuclear atypia in liver or lung tissues was observed in SAG-treated group.H&E staining showed numerous swollen pale-staining cells with loss of nuclear staining are present in hepatocytes and transplanted tumors.SAG treat increased the expression of capn2,porimin,E-cadherin,while decreased the level of p ERK,PKM2,β-catenin,N-cadherin,vimentin,MMP2 and MMP9 in vivo and in vitro.Conclusions:In summary,results from our study showed that SAG treatment on liver cancer cells suppressed cell viability in bimodal cell death patterns,apoptosis and oncosis.SAG treatment inactivated ERK,which modulated the entry of PKM2/β-catenin into cell nucleus.SAG-induced cell death involves the changes of mitochondria function,energy and metabolic alternations via regulating the metabolic and non-metabolic function of PKM2,ultimately inhibited the growth and invasion of tumor cells via ERK/PKM2/β-catenin axis. |
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