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. Xi Huang Wan 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 Xi Huang Wan in hepatocellular carcinoma remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of hepatocellular carcinoma. However, no study has focused on the Xi Huang Wan-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that Xi Huang Wan might play a role in inhibiting hepatocellular carcinoma through the PI3K/Akt/mTOR signalling pathway.
Aim: To confirm the effect of Xi Huang Wan on hepatocellular carcinoma and the possible mechanisms involved.
Methods: The chemical constituents and active components of Xi Huang Wan were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of Xi Huang Wan on hepatocellular carcinoma tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of Xi Huang Wan (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 Xi Huang Wan 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 Xi Huang Wan on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of Xi Huang Wan on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.
Results: The following 12 compounds were identified in Xi Huang Wan 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 0.625 mg/mL Xi Huang Wan extract decreased hepatocellular carcinoma cell viability after 12 h, and the effects were dose- and time-dependent.
The results of the cell scratch assay showed that the migration of hepatocellular carcinoma cells was significantly inhibited in a time-dependent manner by the administration of Xi Huang Wan extract (0.625 mg/mL). Moreover, Xi Huang Wan significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, Xi Huang Wan downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of Xi Huang Wan on hepatocellular carcinoma cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.
Conclusion: Xi Huang Wan inhibited hepatocellular carcinoma 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 Xi Huang Wan on hepatocellular carcinoma cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that Xi Huang Wan may be a potential complementary therapy for hepatocellular carcinoma.