Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Ginsenoside Rk3, an important and rare saponin in heat-treated ginseng, is generated from Rg1 and has a smaller molecular weight. However, the anti-HCC efficacy and mechanisms of ginsenoside Rk3 have not yet been characterized. Here, we investigated the mechanism by which ginsenoside Rk3, a tetracyclic triterpenoid rare ginsenoside, inhibits the growth of HCC. We first explored the possible potential targets of Rk3 through network pharmacology. Both in vitro (HepG2 and HCC-LM3 cells) and in vivo (primary liver cancer mice and HCC-LM3 subcutaneous tumor-bearing mice) studies revealed that Rk3 significantly inhibits the proliferation of HCC. Meanwhile, Rk3 blocked the cell cycle in HCC at the G1 phase and induced autophagy and apoptosis in HCC. Further proteomics and siRNA experiments showed that Rk3 regulates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway to inhibit HCC growth, which was validated by molecular docking and surface plasmon resonance. In conclusion, we report the discovery that ginsenoside Rk3 binds to PI3K/AKT and promotes autophagy and apoptosis in HCC. Our data strongly support the translation of ginsenoside Rk3 into novel PI3K/AKT-targeting therapeutics for HCC treatment with low toxic side effects.

肝细胞癌（HCC）是全球第三大癌症死亡原因。人参皂苷Rk3是热处理人参中一种重要且稀有的皂苷，由Rg1生成，具有较小的分子量。然而，人参皂苷 Rk3 的抗 HCC 功效和机制尚未得到表征。在这里，我们研究了人参皂苷 Rk3（一种四环三萜类稀有人参皂苷）抑制 HCC 生长的机制。我们首先通过网络药理学探索了Rk3可能的潜在靶点。体外（HepG2和HCC-LM3细胞）和体内（原发性肝癌小鼠和HCC-LM3皮下荷瘤小鼠）研究均表明Rk3显着抑制HCC的增殖。同时，Rk3将HCC细胞周期阻断在G1期，并诱导HCC自噬和凋亡。进一步的蛋白质组学和 siRNA 实验表明，Rk3 调节磷脂酰肌醇 3 激酶 (PI3K)/蛋白激酶 B (AKT) 通路来抑制 HCC 生长，并通过分子对接和表面等离子共振得到验证。总之，我们报告了人参皂苷 Rk3 与 PI3K/AKT 结合并促进 HCC 中自噬和细胞凋亡的发现。我们的数据强烈支持将人参皂苷 Rk3 转化为新型 PI3K/AKT 靶向疗法，用于低毒副作用的 HCC 治疗。