Background: Epirubicin (EADM) is a common chemotherapeutic agent in hepatocellular carcinoma (HCC). The accumulation of hypoxia-inducible factor-1α (HIF-1α) is an important cause of drug resistance to EADM in HCC. Tanshinone I (Tan I) is an agent with promising anti-cancer effects alone or with other drugs. Some tanshinones mediate HIF-1α regulation via PI3K/AKT. However, the role of Tan I combined with EADM to reduce the resistance of HCC to EADM has not been investigated. Therefore, this study aimed to investigate the combined use of Tan I and EADM in HCC and the underlying mechanism of PI3K/AKT/HIF-1α.
Methods: HCC cells were treated with Tan I, EADM, or the combined treatment for 48 hrs. Cell transfection was used to construct HIF-1α overexpression HCC stable cells. Cell viability, colony formation, and flow cytometric assays were used to detect the viability, proliferation, and apoptosis in HCC cells. Synergism between Tan I and EADM were tested by calculating the Bliss synergy score, positive excess over bliss additivism (EOBA), and the combination index (CI). Western blotting analyses were used to detect the levels of β-actin, HIF-1α, PI3K p110α, p-Akt Thr308, Cleaved Caspase-3, and Cleaved Caspase-9. Toxicity parameters were used to evaluate the safety of the combination in mice. The xenograft model of mice was built by HCC stable cell lines, which was administrated with Tan I, EADM, or a combination of them for 8 weeks. Immunohistochemistry staining (IHC) was used to assess tumor apoptosis in mouse models.
Results: Hypoxia could upregulate HIF-1α to induce drug resistance in HCC cancer cells. The combination of Tan I and EADM was synergistic. Although Tan I or EADM alone could inhibit HCC cancer cells, the combination of them could further enhance the cytotoxicity and growth inhibition by targeting the PI3K/AKT/HIF-1α signaling pathway. Furthermore, Tan I and EADM synergistically reversed HIF-1α-mediated drug resistance to inhibit HCC. The results of toxicity parameters showed that the combination was safe in mice. Meanwhile, animal models showed that Tan I not only improved the anti-tumor effect of EADM, but also reduced the drug reactions of EADM-induced weight loss.
Conclusion: Our results suggested that Tan I could effectively improve the anti-tumor effect of EADM, and synergize EADM to reverse HIF-1α mediated resistance via targeting PI3K/AKT/HIF-1α signaling pathway.



中文翻译：

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丹参酮 I 和表柔比星的联合治疗显示通过靶向 PI3K/AKT/HIF-1α 增强对肝细胞癌的抑制作用

背景：表柔比星 (EADM) 是肝细胞癌 (HCC) 中常见的化疗药物。缺氧诱导因子-1α（HIF-1α）的积累是肝细胞癌对 EADM 耐药的重要原因。丹参酮 I (Tan I) 是一种单独或与其他药物联合使用具有良好抗癌作用的药物。一些丹参酮通过 PI3K/AKT 介导 HIF-1α 调节。然而，Tan I 联合 EADM 降低 HCC 对 EADM 耐药性的作用尚未得到研究。因此，本研究旨在探讨 Tan I 和 EADM 在 HCC 中的联合应用以及 PI3K/AKT/HIF-1α 的潜在机制。
方法：HCC 细胞用 Tan I、EADM 或联合治疗处理 48 小时。细胞转染用于构建HIF-1α过表达HCC稳定细胞。细胞活力、集落形成和流式细胞术检测用于检测 HCC 细胞的活力、增殖和凋亡。Tan I 和 EADM 之间的协同作用通过计算 Bliss 协同分数、幸福加成性 (EOBA) 和组合指数 (CI) 的正过量来测试。蛋白质印迹分析用于检测 β-肌动蛋白、HIF-1α、PI3K p110α、p-Akt Thr308、Cleaved Caspase-3 和 Cleaved Caspase-9 的水平。毒性参数用于评估组合在小鼠中的安全性。小鼠异种移植模型由 HCC 稳定细胞系构建，并给予 Tan I、EADM 或它们的组合 8 周。
结果：缺氧可上调 HIF-1α 诱导 HCC 癌细胞产生耐药性。Tan I 和 EADM 的组合具有协同作用。虽然单独使用 Tan I 或 EADM 可以抑制 HCC 癌细胞，但它们的组合可以通过靶向 PI3K/AKT/HIF-1α 信号通路进一步增强细胞毒性和生长抑制。此外，Tan I 和 EADM 协同逆转 HIF-1α 介导的耐药性以抑制 HCC。毒性参数结果表明该组合在小鼠中是安全的。同时，动物模型显示Tan I不仅提高了EADM的抗肿瘤作用，而且降低了EADM引起的体重减轻的药物反应。
结论：我们的结果表明，Tan I 可以有效提高 EADM 的抗肿瘤作用，并通过靶向 PI3K/AKT/HIF-1α 信号通路协同 EADM 逆转 HIF-1α 介导的耐药性。