Department of Clinical Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China; Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China.
Department of Clinical Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
Biomed Pharmacother. 2019 Jan;109:2365-2374. doi: 10.1016/j.biopha.2018.11.124. Epub 2018 Nov 30.
The anti-tumor properties of Alpinia oxyphylla Miquel (A. oxyphylla) extracts and their petroleum ether (PE) fractions have long attracted scientific attention. These extracts' anti-tumor activity and mechanisms in vivo are still unclear. This study was designed to investigate the anti-tumor activity and the underlying mechanism of PE's effect on hepatocellular carcinoma (HCC) in vitro and in vivo.
The anti-tumor activity of PE was evaluated by MTT assay and xenograft study. Mechanistic studies of PE were analyzed by Hoechst 33342 staining, Annexin V-FITC/PI double-staining assay, immunohistochemical staining and western blot assay. The toxicity of the PE treatment was verified by the levels of liver and kidney function in nude mice and the H&E staining of their liver and kidney tissues.
PE significantly inhibited the growth of HepG2, BEL-7402, SMMC-7721 and Hep3B cells in a concentration- and time-dependent manner. Specifically, PE inhibited the growth of Hep3B cells by inducing apoptosis. PE treatment at the doses of 0.25, 0.5 and 1 g/kg for 21 days caused a respective 35.7 percent, 49.3 percent and 58.8 percent inhibition of the tumor volume, and a 14.8 percent, 40.2 percent and 55.6 percent decrease in the tumor weight, respectively, as compared with the vehicle group in tumor-loaded mice in vivo. PE promoted the release of cytochrome c from mitochondria to cytosol in a concentration-dependent manner. The expression levels of BAX (p < 0.01), cleaved caspase-9 (p < 0.01) and cleaved caspase-3 (p < 0.05) were increased significantly in the PE-treated group at the dose of 1 g/kg; the expression level of BAX (p < 0.05) was increased significantly in the PE-treated group at the dose of 0.5 g/kg, and the expression level of Bcl-2 (p < 0.01) was decreased significantly in the PE-treated group in a concentration-dependent manner. Apoptosis was induced by PE through up-regulating the expression of PTEN, down-regulating the expression of PI3K and inhibiting the phosphorylation of Akt. The liver and kidney function of the plasma and the morphology of the liver and kidney were normal in each group.
These findings suggested that PE exhibited anti-cancer efficacy on Hep3B cell in vitro and in vivo. The induction of apoptosis might be one mechanism that underlies PE's ability to combat cancer by inhibiting the PI3K/Akt pathway. PE has no obvious toxicity in vivo when it exerts anti-tumor effects and has the potential to develop into an alternative anti-cancer drug for HCC treatment.
阳春砂仁（A. oxyphylla）提取物及其石油醚（PE）部分的抗肿瘤特性一直引起科学界的关注。这些提取物在体内的抗肿瘤活性和机制尚不清楚。本研究旨在探讨 PE 在体外和体内对肝癌（HCC）的抗肿瘤活性及其潜在机制。
采用 MTT 法和异种移植研究评价 PE 的抗肿瘤活性。通过 Hoechst 33342 染色、Annexin V-FITC/PI 双染色法、免疫组织化学染色和 Western blot 分析研究 PE 的作用机制。通过检测裸鼠肝肾功能水平和肝肾功能组织的 H&E 染色，验证 PE 处理的毒性。
PE 呈浓度和时间依赖性地显著抑制 HepG2、BEL-7402、SMMC-7721 和 Hep3B 细胞的生长。具体而言，PE 通过诱导细胞凋亡抑制 Hep3B 细胞的生长。PE 以 0.25、0.5 和 1 g/kg 的剂量处理 21 天，与荷瘤小鼠体内的对照组相比，分别使肿瘤体积抑制 35.7%、49.3%和 58.8%，肿瘤重量降低 14.8%、40.2%和 55.6%。PE 呈浓度依赖性地促进细胞色素 c 从线粒体向细胞质释放。在 1 g/kg 的 PE 处理组中，BAX（p<0.01）、cleaved caspase-9（p<0.01）和 cleaved caspase-3（p<0.05）的表达水平显著增加；在 0.5 g/kg 的 PE 处理组中，BAX（p<0.05）的表达水平显著增加，并且在浓度依赖性方式中，Bcl-2（p<0.01）的表达水平显著降低。PE 通过上调 PTEN 的表达、下调 PI3K 的表达和抑制 Akt 的磷酸化诱导细胞凋亡。各组血浆肝肾功能正常，肝肾功能形态正常。
这些发现表明，PE 在体外和体内对 Hep3B 细胞均具有抗癌活性。诱导细胞凋亡可能是 PE 通过抑制 PI3K/Akt 通路发挥抗癌作用的一种机制。PE 在发挥抗肿瘤作用时，体内无明显毒性，具有开发为 HCC 治疗的替代抗癌药物的潜力。
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