Abstract: Objective To investigate the effects of Pifithrin-μ on the proliferation of TSC2 mutant hepatocellular carcinoma cells. Methods The effect of Pifithrin-μ on cell proliferation was evaluated using CCK8 assay in multiple lines of Tsc2 mutant mouse embryonic fibroblasts (MEFs) and human hepatocellular carcinoma cells. Lentiviral infection was used to construct hepatocellular carcinoma cells with knockdown or overexpression of TSC2. The protein expression of TSC2 and the downstream effectors of the mTOR pathway were detected by Western blot. Cellular sensitivity to Pifithrin-μ was detected by CCK8. The nude mouse subcutaneous transplantation tumor model was established for evaluation of drug efficacy in vivo. RNA sequencing was used for differential gene and pathway enrichment analysis, which was verified by Western blot and CCK8. Results Pifithrin-μ selectively inhibited the proliferation of Tsc2 mutated MEFs and hepatocellular carcinoma cells in a dose-dependent manner(P＜0.05). Hepatocellular carcinoma cells with TSC2 knockdown showed increased sensitivity to Pifithrin-μ, whereas those with TSC2 overexpression were resistant (P＜0.05). Pifithrin-μ significantly inhibited the growth of Tsc2 mutant transplantation tumors in nude mice (P＜0.05). Pifithrin-μ downregulated the expression of Smad7 protein in Tsc2 mutant cells, resulting in elevated levels of p-Smad2/3 and over-activation of the TGF-β/Smad signaling pathway (P＜0.05). The combination of each of the six cell death inhibitors with Pifithrin-μ revealed that reactive oxygen species inhibitor and TGF-β/Smad pathway inhibitor were able to reverse Pifithrin-μ-induced cell death in TSC2 mutant cells (P＜0.05). Conclusions Pifithrin-μ selectively inhibits the proliferation of TSC2 mutant hepatocellular carcinoma cells. This effect is associated with the downregulation of Smad7 and the disruption of the TGF-β/Smad signaling pathway balance.

Key words: TSC2, hepatocellular carcinoma, Pifithrin-μ, TGF-β/Smad signaling pathway, cell proliferation