Table_4_Uncovering the Mechanisms of Cryptotanshinone as a Therapeutic Agent Against Hepatocellular Carcinoma.xlsx

Hepatocellular carcinoma (HCC) is a fatal and dominant form of liver cancer that currently has no effective treatment or positive prognosis. In this study, we explored the antitumor effects of cryptotanshinone (CPT) against HCC and the molecular mechanisms underlying these effects using a systems pharmacology and experimental validation approach. First, we identified a total of 296 CPT targets, 239 of which were also HCC-related targets. We elucidated the mechanisms by which CPT affects HCC through multiple network analysis, including CPT-target network analysis, protein-protein interaction network analysis, target-function network analysis, and pathway enrichment analysis. In addition, we found that CPT induced apoptosis in Huh7 and MHCC97-H ells due to increased levels of cleaved PARP, Bax, and cleaved caspase-3 and decreased Bcl-2 expression. CPT also induced autophagy in HCC cells by increasing LC3-II conversion and the expression of Beclin1 and ATG5, while decreasing the expression of p62/SQSTM1. Autophagy inhibitors (3-methyladenine and chloroquine) enhanced CPT-induced proliferation and apoptosis, suggesting that CPT-induced autophagy may protect HCC cells against cell death. Furthermore, CPT was found to inhibit the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, activation of PI3K by insulin-like growth factor-I inhibited CPT-induced apoptosis and autophagy, suggesting that the PI3K/AKT/mTOR signaling pathway is involved in both CPT-induced apoptosis and autophagy. Finally, CPT was found to inhibit the growth of Huh7 xenograft tumors. In conclusion, we first demonstrated the antitumor effects of CPT in Huh7 and MHCC97-H cells, both in vitro and in vivo. We elucidated the potential antitumor mechanism of CPT, which involved inducing apoptosis and autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway. Our findings may provide valuable insights into the clinical application of CPT, serving as a potential candidate therapeutic agent for HCC treatment.

肝细胞癌（Hepatocellular carcinoma, HCC）是一种致死性且最为高发的肝癌亚型，目前尚无有效的治疗手段及理想预后方案。本研究采用系统药理学结合实验验证的研究策略，探究了隐丹参酮（cryptotanshinone, CPT）抗肝细胞癌的作用及其潜在分子机制。首先，本研究共筛选得到296个CPT的作用靶点，其中239个同时为肝细胞癌相关靶点。通过包括CPT-靶点网络分析、蛋白质相互作用网络分析、靶点-功能网络分析及通路富集分析在内的多组网络分析手段，本研究阐明了CPT调控肝细胞癌的潜在机制。此外，研究发现CPT可通过上调剪切型多聚ADP核糖聚合酶（cleaved PARP）、Bcl-2相关X蛋白（Bax）及剪切型半胱氨酸天冬氨酸蛋白酶3（cleaved caspase-3）的表达，同时下调B细胞淋巴瘤-2（Bcl-2）的表达，诱导Huh7与MHCC97-H细胞发生细胞凋亡。CPT还可通过提升LC3-II转化水平、上调Beclin1蛋白与自噬相关基因5（ATG5）的表达，并下调p62/SQSTM1的表达，诱导肝癌细胞发生细胞自噬。自噬抑制剂（3-甲基腺嘌呤与氯喹）可增强CPT诱导的增殖抑制与凋亡效应，提示CPT诱导的自噬可能对肝癌细胞起到了死亡保护作用。进一步研究证实，CPT可抑制磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase, PI3K）/蛋白激酶B（protein kinase B, Akt）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）信号通路。有趣的是，胰岛素样生长因子-I介导的PI3K激活可抑制CPT诱导的细胞凋亡与自噬，表明PI3K/Akt/mTOR信号通路同时参与了CPT诱导的细胞凋亡与自噬过程。最终实验证实，CPT可抑制Huh7细胞异种移植瘤的生长。综上，本研究首次在体内外实验中证实了CPT对Huh7与MHCC97-H细胞的抗肿瘤活性，阐明了CPT潜在的抗肿瘤机制：通过抑制PI3K/Akt/mTOR信号通路，诱导细胞凋亡与自噬。本研究结果可为CPT的临床转化应用提供重要理论参考，有望成为肝细胞癌治疗的潜在候选药物。