Kim Hyo-Jin, Hwang Ki-Eun, Park Do-Sim, Oh Seon-Hee, Jun Hong Young, Yoon Kwon-Ha, Jeong Eun-Taik, Kim Hak-Ryul, Kim Young-Suk
Department of Internal Medicine, Institute of Wonkwang Medical Science, Wonkwang University, School of Medicine, Iksan, Jeonbuk, South Korea.
Department of Laboratory Medicine, Wonkwang University, School of Medicine, Iksan, Jeonbuk, South Korea.
J Transl Med. 2017 May 31;15(1):123. doi: 10.1186/s12967-017-1223-7.
Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action.
Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo.
Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin.
Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.
紫草素是从紫草中纯化得到的一种天然萘醌色素，可诱导多种癌症类型发生坏死性凋亡，但紫草素在肺癌中的抗癌活性机制尚未完全明确。本研究旨在阐明紫草素是否会导致非小细胞肺癌（NSCLC）细胞发生坏死性凋亡，并探究其作用机制。
采用多重检测和半胱天冬酶8检测分析紫草素对A549细胞的作用。运用膜联蛋白V/碘化丙啶（PI）染色细胞计数法和MTT检测分析细胞死亡模式。采用蛋白质免疫印迹法确定紫草素诱导坏死性凋亡和自噬的作用。利用A549细胞的异种移植和原位模型评估紫草素在体内的抗肿瘤作用。
紫草素诱导的大部分细胞死亡可被特异性坏死性凋亡抑制剂 Necrostatin-1挽救，但不能被通用的半胱天冬酶抑制剂Z-VAD-FMK挽救。接受紫草素治疗的动物肿瘤生长明显低于对照组。紫草素还增加了肿瘤组织中RIP1蛋白的表达。自噬抑制剂，包括甲基腺嘌呤（3-MA）、ATG5小干扰RNA（siRNA）和巴弗洛霉素A，增强了紫草素诱导的坏死性凋亡，而RIP1 siRNA对紫草素的凋亡潜力没有影响。
我们的数据表明，紫草素处理可诱导NSCLC细胞发生坏死性凋亡和自噬。此外，抑制紫草素诱导的自噬可增强坏死性凋亡，这表明紫草素可能是一种针对NSCLC的新型治疗策略。
