Investigating The Anti-hepatocellular Carcinoma Mechanism Of Pseudolaric Acid B And Its Synergistic Effect With Sorafenib

Research Background: Hepatocellular carcinoma(HCC,referred to as liver cancer)is a significant public health problem worldwide.Its high incidence and high lethality have severely threatened the health of human and animal.For patients with early-stage HCC whose liver function remains good,surgical resection,liver transplantation,and tumor ablation are the main treatment options for HCC.However,the incidence of HCC is insidious,and most patients have lost the best opportunity for surgery by the time they seek for treatment.Although traditional chemotherapy drugs increase patients’ survival benefits,their resistance problems have been criticized.In recent years,Immune Checkpoint Inhibitors(ICIs)therapy targeting PD-1/PD-L1 and CTLA-4 targets have shown good anti-HCC activity in clinical trials.However,with the constant development of ICI drugs and the continuous improvement of drug availability,the immune-related adverse events(ir AEs)have gradually been discovered and focused.Therefore,it is urgent to enrich existing treatments and develop new treatment strategies.Natural products have always been a treasure trove for drug development.Finding new anti-HCC drugs from natural drugs is essential for treating HCC.Pseudolaric Acid B(PAB),as a new type of diterpene acid isolated from the traditional Chinese medicine "Pseudolaric Acid B," has various biological activities such as antibacterial,anti-angiogenic,and antiviral.As shown by previous in vitro experiments,PAB can induce the death of prostate,cervical,and breast cancer cells.Still,the anticancer target,specific molecular mechanism,and application potential of PAB on HCC are not yet fully understood.Research purposes:(1)To systematically elucidate the molecular mechanism of PAB inhibiting the growth of HCC cells and reveal the specific signaling molecular pathways of PABinduced apoptosis of HCC cells through in vitro cell biology experiments and C57BL/6J mouse HCC syngeneic mouse model experiments;(2)This study investigate its synergistic effect when combined with the clinical anti-hepatoma drug sorafenib,and clarify the anti-HCC effect and safety of combined drugs;To lay the foundation for preclinical research on PAB and provide the theoretical and experimental basis for its further development and clinical application.Research methods:This study used cell proliferation toxicity assay(CCK8),cell proliferation assay(Ed U),and plate colony formation assay(CFU)to detect cell proliferation,and flow cytometry to observe mitochondrial membrane potential and cell apoptosis.Western blot assay was adopted to detect cell apoptosis and mitochondrial respiratory chain and the expression of mitochondrial fission-related proteins.Laser confocal microscopy was used to observe mitochondrial fission,membrane potential changes,and colocalization of target proteins and mitochondria.Transmission electron microscopy was applied to observe mitochondrial morphology and structure.ATP content in HCC cells was detected through chemiluminescence.Differences in enrichment of genes and pathways were detected through RNA-seq.Immunohistochemical detection of cell proliferation and apoptosis-related protein expression in tumor tissue was conducted.Tissue H&E staining was used to see the degree of damage to major organs of mice in each group.Research content and results:(1)Inhibition of HCC cell proliferation and induction of apoptosis by PABIn vitro,PAB can inhibit the proliferation of HCC cells and reduce the colony formation ability of HCC cells in a concentration-dependent manner,but it has little effect on the viability of normal human tissue cells(LO2 and HK2).In addition,coincubation of inhibitors of different forms of Death with PAB suggests that PAB can induce apoptosis in HCC cells.As confirmed by flow cytometry results,PAB can induce apoptosis in Hepa1-6 HCC cells in a concentration-dependent manner.According to Western blot testing,PAB can increase the expression of apoptotic proteins CleavedCaspase3,Cleaved-PARP,and Bax and reduce the expression of anti-apoptotic protein Bcl-2.(2): PAB’s promotion of Drp1(Ser 616)phosphorylation and increase mitochondrial translocation to mediate excessive mitochondrial division and activate the mitochondrial apoptosis pathwayAccording to transmission electron microscopy,after PAB treatment,some mitochondria of HCC cells were swollen and rounded,and mitochondrial cristae were broken or even disappeared.As shown by laser confocal microscopy observation,PAB treatment caused excessive mitochondria fission in HCC cells,and the number of fragmented mitochondria increased and broke into "spots." As revealed by Western blot analysis,PAB could promote the phosphorylation of Drp1(Ser 616)and increase mitochondrial translocation,but it had no significant effect on the phosphorylation level of Drp1(Ser 637).Subsequently,the Drp1 mitochondrial translocation inhibitor Mdivi-1 and PAB were co-incubated.It was found that co-incubation reduced the phosphorylation level of Drp1(Ser 616).Moreover,mitochondrial translocation effectively inhibited excessive mitochondrial fission,restored the balance of division and fusion,and effectively improved viability and apoptosis of HCC cells.Stable knockdown of Drp1 expression in the Hepa1-6 HCC cell line directly blocked excessive mitochondrial fission caused by PAB,restored the balance of fission and fusion as well as the morphology and function of mitochondria,and dramatically weakened the activation of the mitochondrial apoptosis pathway.(3)The effect of PAB activating AMPK-JNK signaling pathway in Drp1 regulating the growth and apoptosis of HCC cellsAs shown by transcriptome sequencing analysis,the expression of 399 genes was significantly upregulated after PAB treatment,while the expression of 1330 genes significantly decreased.According to GSEA enrichment analysis,these upregulated genes were mainly enriched in the MAPK signaling pathway,especially the JNK cascade-related genes.As verified by Western blot,the expression of proteins encoded by JNK cascade-related genes upregulated after PAB treatment.Apart from that,JNK inhibitor SP600125 can significantly block the phosphorylation of Drp1(Ser616)induced by PAB as well as the excessive division of mitochondria,restore the morphology and function of mitochondria,and then block the activation of mitochondrial apoptosis pathway by PAB.However,the co-incubation of Drp1 mitochondrial translocation inhibitor Mdivi-1 and PAB did not significantly affect the phosphorylation level of JNK.Moreover,JNK regulated apoptosis by acting on the upstream of Drp1.Similarly,the AMPK inhibitor Compound C was used to demonstrate that AMPK also acted upstream of Drp1.As revealed by Western blot experiments,the AMPK inhibitor Compound C can reduce the phosphorylation level of JNK.Still,the JNK inhibitor SP600125 failed to significantly affect the phosphorylation level of AMPK.In summary,PAB induces phosphorylation of Drp1(Ser616)by activating the AMPK/JNK pathway,leading to excessive mitochondrial fission and ultimately triggering the mitochondrial apoptotic pathway.(4)The anti-tumor effect of PAB on the subcutaneous HCC syngeneic model of mouse HCC in vivoIn the mouse HCC Hepa1-6 HCC cells subcutaneous HCC syngeneic mouse model based on C57BL/6J mice,PAB can significantly inhibit the tumor growth of the subcutaneous syngeneic tumor model of mouse HCC,reduce the weight of tumor tissue,and present good dose dependence.According to immunohistochemical detection,PAB can reduce the expression of tumor cell proliferation protein Ki67 and increase the expression of apoptosis protein Cleaved-Caspase3.Consistent with the in vitro results,PAB can also activate the AMPK/JNK/Drp1 signaling pathway in vivo.According to body weight tracking and monitoring,this study found that PAB had no significant effect on the body weight of HCC syngeneic mouse model.There were no abnormalities in H&E staining of the mice’s lung,liver,spleen,and kidney and detecting liver and kidney function indicators.(5)The synergistic effect of PAB combined with sorafenib on anti-hepatocellular carcinomaBased on cell proliferation toxicity experiments,cell colony formation experiments,and Calcein-AM/PI co-staining experiments,this study found that PAB’s in vitro killing activity combined with sorafenib on Hepa1-6 HCC cells was more substantial than that of a single drug.As demonstrated by laser confocal and Western blot experiments,PAB combined with sorafenib induced apoptosis in HCC cells by activating the mitochondrial apoptosis pathway and presented a synergistic effect.PAB combined with sorafenib enhanced the inhibitory effect of a single drug on tumor growth in the subcutaneous transplantation tumor model of Hepa1-6 HCC cells and significantly reduced the weight of tumor tissue.According to immunohistochemistry testing,the PAB combined with sorafenib could inhibit tumor tissue cell proliferation.The expression of protein Ki67 enhanced the expression of apoptotic protein CleavedCaspase3.Based on body weight tracking and monitoring,this study found that PAB combined with sorafenib had no significant effect on the body weight of HCC transplanted tumor model mice.At the same time,no abnormalities were observed in the H&E staining of the mice’s heart,liver,spleen,and kidneys and in detection of liver and kidney function indicators.Analysis conclusion:(1)PAB significantly reduces the viability of HCC cells in vitro without apparent inhibitory effects on normal human tissue cells(LO2 and HK2).No significant damage is observed in major tissues and organs,such as the spleen and kidney,and mice’s body weight and liver and kidney function indicators remain normal.(2)PAB induces the phosphorylation of Drp1(Ser 616)and increase mitochondrial translocation by activating the AMPK/JNK pathway,breaks the balance of mitochondrial fission and fusion,promotes excessive mitochondrial division,induces mitochondrial structural abnormalities and dysfunction,and then activates the mitochondrial apoptosis pathway,which leads to apoptosis of HCC cells.(3)PAB combined with sorafenib can activate the mitochondrial apoptosis pathway to enhance the killing activity of Hepa1-6 HCC cells in vitro.(4)In comparison to the drug treatment alone,PAB combined with sorafenib has a synergistic anti-HCC effect in vivo.The primary organ tissues of mice such as the liver,spleen,and kidneys are not damaged.There are no apparent abnormalities in the weight and liver and kidney function indicators of mice.