Shuyu pills inhibit immune escape and enhance chemosensitization in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is characterized by dysregulation of the immune microenvironment and the development of chemoresistance.Specifically,expression of the programmed cell death protein 1(PD-1)/programmed cell death 1 ligand 1(PD-L1)axis,an immune checkpoint,may lead to tumour immune escape,resulting in disease progression.The latest research shows that tumour immune escape may be caused by the upregulation of PD-L1 mediated by hypoxia-inducible factor-1 alpha(HIF-1α),and simultaneous inhibition of HIF-1αand PD-L1 has the potential to enhance the host’s antitumour immunity.Moreover,inhibition of the PD-1/PD-L1 axis may mitigate tumour chemoresistance.Shuyu pills(SYPs)contain immunity-enhancing and antitumour components,making them a potential HCC treatment.AIM To investigate the efficacy of SYPs for HCC treatment via simultaneous HIF-1α and PD-L1 inhibition and the mechanism involved.METHODS A subcutaneous xenograft tumour model was first established in BALB/c nude mice by the subcutaneous injection of 1×107 SMMC-7721 cells.Male mice(male,5 weeks old;n=24)were then randomly divided into the following four groups(n=6):Control(0.9%normal saline),SYP(200 mg/kg),SYP+cisplatin(DDP)(200 mg/kg+5 mg/kg DDP weekly via intraperitoneal injection),and DDP(5 mg/kg cisplatin weekly via intraperitoneal injection).The dose of saline or SYPs for the indicated mouse groups was 0.2 mL/d via intragastric administration.The tumour volumes and body weights of the mice were measured every 2 d.The mice were euthanized by cervical dislocation after 14 d of continuous treatment,and the xenograft tissues were excised and weighed.Western blot assays were used to measure the protein expression of HIF-1α,PD1,PD-L1,CD4+T cells,and CD8+T cells in HCC tumours from mice.Quantitative reverse transcription polymerase chain reaction was used for real-time quantitative detection of PD-1,PD-L1,and HIF-1α mRNA expression.An immunofluorescence assay was conducted to examine the expression of CD4+T cells and CD8+T cells.RESULTS Compared to mice in the control group,those in the SYP and SYP+DDP groups exhibited reduced tumour volumes and tumour weights.Moreover,the protein and mRNA expression levels of the oncogene HIF1α and that of the negative immunomodulatory factors PD-1 and PD-L1 were decreased in both the SYP and SYP+DDP groups,with the decrease effects being more prominent in the SYP+DDP group than in the SYP group(HIF-1α protein:Control vs SYP,P=0.0129;control vs SYP+DDP,P=0.0004;control vs DDP,P=0.0152,SYP+DDP vs DDP,P=0.0448;HIF-1αmRNA:control vs SYP,P=0.0009;control vs SYP+DDP,P<0.0001;control vs DDP,P=0.0003,SYP vs SYP+DDP,P=0.0192.PD-1 protein:Control vs SYP,P=0.0099;control vs SYP+DDP,P<0.0001,SPY vs SYP+DDP,P=0.0009;SYP+DDP vs DDP,P<0.0001;PD-1 mRNA:control vs SYP,P=0.0002;control vs SYP+DDP,P<0.0001;control vs DDP,P=0.0003,SPY vs SYP+DDP,P=0.0003;SYP+DDP vs DDP,P=0.0002.PD-L1 protein:control vs SYP,P<0.0001;control vs SYP+DDP,P<0.0001;control vs DDP,P<0.0001,SPY vs SYP+DDP,P=0.0040;SYP+DDP vs DDP,P=0.0010;PD-L1 mRNA:Control vs SYP,P<0.0001;control vs SYP+DDP,P<0.0001;control vs DDP,P<0.0001,SPY vs SYP+DDP,P<0.0001;SYP+DDP vs DDP,P=0.0014).Additionally,the quantitative and protein expression levels of CD4+T cells and CD8+T cells were simultaneously upregulated in the SYP+DDP group,whereas only the expression of CD4+T cells was upregulated in the SYP group.(CD4+T cell quantitative:Control vs SYP+DDP,P<0.0001,SYP vs SYP+DDP,P=0.0005;SYP+DDP vs DDP,P=0.0002.CD4+T cell protein:Control vs SYP,P=0.0033;Control vs SYP+DDP,P<0.0001;Control vs DDP,P=0.0021,SYP vs SYP+DDP,P=0.0004;SYP+DDP vs DDP,P=0.0006.Quantitative CD8+T cells:Control vs SYP+DDP,P=0.0013;SYP vs SYP+DDP,P=0.0347;SYP+DDP vs DDP,P=0.0043.CD8+T cell protein:Control vs SYP+DDP,P<0.0001;SYP vs SYP+DDP,P<0.0001;SYP+DDP vs DDP,P<0.0001).Finally,expression of HIF-1αwas positively correlated with that of PD-1/PD-L1 and negatively correlated with the expression of CD4+T cells and CD8+T cells.CONCLUSION SYPs inhibit immune escape and enhance chemosensitization in HCC via simultaneous inhibition of HIF-1α and PD-L1,thus inhibiting the growth of subcutaneous xenograft HCC tumours.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase- B/mechanistic target of rapamycin pathway

BACKGROUND The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin(PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills(XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma(HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHPassociated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIM To confirm the effect of XHP on HCC and the possible mechanisms involved.METHODS The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS). Cellbased experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP(0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay.Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction(RT-qPCR), respectively.Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway.Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTS The following 12 compounds were identified in XHP using high-resolution mass spectrometry:Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose-and timedependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract(0.625mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins(e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSION XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3.Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.