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-1α and 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 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 × 10 SMMC-7721 cells. Male mice (male, 5 weeks old; = 24) were then randomly divided into the following four groups ( = 6): Control (0.9% normal saline), SYP (200 mg/kg), SYP + cisplatin (DDP) (200 mg/kg + 5 mg/kg DDP weekly intraperitoneal injection), and DDP (5 mg/kg cisplatin weekly intraperitoneal injection). The dose of saline or SYPs for the indicated mouse groups was 0.2 mL/d 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, PD-1, 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 HIF-1α 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 SYP, = 0.0129; control SYP + DDP, = 0.0004; control DDP, = 0.0152, SYP + DDP DDP, = 0.0448; HIF-1α mRNA: control SYP, = 0.0009; control SYP + DDP, < 0.0001; control DDP, = 0.0003, SYP SYP + DDP, = 0.0192. PD-1 protein: Control SYP, = 0.0099; control SYP + DDP, < 0.0001, SPY SYP + DDP, = 0.0009; SYP + DDP DDP, < 0.0001; PD-1 mRNA: control SYP, = 0.0002; control SYP + DDP, < 0.0001; control DDP, = 0.0003, SPY SYP + DDP, = 0.0003; SYP + DDP DDP, = 0.0002. PD-L1 protein: control SYP, < 0.0001; control SYP + DDP, < 0.0001; control DDP, < 0.0001, SPY SYP + DDP, = 0.0040; SYP + DDP DDP, = 0.0010; PD-L1 mRNA: Control SYP, < 0.0001; control SYP + DDP, < 0.0001; control DDP, < 0.0001, SPY SYP + DDP, < 0.0001; SYP + DDP DDP, = 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 SYP + DDP, < 0.0001, SYP SYP + DDP, = 0.0005; SYP + DDP DDP, = 0.0002. CD4+ T cell protein: Control SYP, = 0.0033; Control SYP + DDP, < 0.0001; Control DDP, = 0.0021, SYP SYP + DDP, = 0.0004; SYP + DDP DDP, = 0.0006. Quantitative CD8+ T cells: Control SYP + DDP, = 0.0013; SYP SYP + DDP, = 0.0347; SYP + DDP DDP, = 0.0043. CD8+ T cell protein: Control SYP + DDP, < 0.0001; SYP SYP + DDP, < 0.0001; SYP + DDP DDP, < 0.0001). Finally, expression of HIF-1α was 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 simultaneous inhibition of HIF-1α and PD-L1, thus inhibiting the growth of subcutaneous xenograft HCC tumours.