Yang Yan, Sun Xiangdong, Yang Yuehua, Yang Xi, Zhu Hongcheng, Dai Shengbin, Chen Xiaochen, Zhang Hao, Guo Qing, Song Yaqi, Wang Feng, Cheng Hongyan, Sun Xinchen
Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China.
Department of Radiotherapy, The 81st Hospital of PLA, Nanjing, Jiangsu Province, China.
Tumour Biol. 2016 Feb;37(2):1853-62. doi: 10.1007/s13277-015-3974-1. Epub 2015 Aug 30.
Radiotherapy is a widespread treatment in human solid tumors. However, therapy resistance and poor prognosis are still problems. Gambogic acid (GA), extracted from the dried yellow resin of gamboges, has an anticancer effect against various types of cancer cells. To explore the radiosensitivity of GA on esophageal cancer cell line TE13, cell viability was tested by Cell Counting Kit-8 (CCK-8) assay, colony formation assay was used to assess the effects of GA on the radiosensitivity of TE13, and flow cytometry was performed to meter the percentage of apoptosis. The protein levels of microtubule-associated protein 1 light chain 3 (LC3), caspase3, caspase8, casepase9, pAkt, and p-mammalian target of rapamycin (p-mTOR) were tested using Western blot. The distribution of LC3 was detected by immunofluorescence. Additionally, we also examined reactive oxygen species (ROS) expression by laser scanning confocal microscope (LSCM). The cells were transfected with adenovial vector to monitor the autophagy through the expression of green fluorescent protein (GFP-red fluroscent protein (RFP)-LC3. The rates of apoptotic cells in combined-treated TE13 increased significantly compared with the control groups in accordance with the results of Western blot. The clonogenic survival assay showed that GA enhances radiosensitivity with a sensitizing enhancement ratio (SER) of 1.217 and 1.436 at different concentrations. The LC3-II protein level increased in the combined group indicating the increase of autophagy incidence, and the results of GFP-RFP-LC3 experiment showed that GA may block the process of autophagic flux in TE13 cells. Moreover, we successfully demonstrated that ROS is involved in the induction of autophagy. ROS-mediated autophagy depends on the inhibition of the Akt/mTOR pathway. Taken together, GA induced radiosensitivity involves autophagy and apoptosis which are regulated by ROS hypergeneration and Akt/mTOR inhibition.
放射疗法是人类实体瘤中一种广泛应用的治疗方法。然而，治疗抗性和预后不良仍然是问题。从藤黄干燥黄色树脂中提取的藤黄酸（GA）对多种类型的癌细胞具有抗癌作用。为了探究GA对食管癌细胞系TE13的放射敏感性，通过细胞计数试剂盒-8（CCK-8）法检测细胞活力，采用集落形成试验评估GA对TE13放射敏感性的影响，并进行流式细胞术检测凋亡百分比。使用蛋白质免疫印迹法检测微管相关蛋白1轻链3（LC3）、半胱天冬酶3、半胱天冬酶8、半胱天冬酶9、磷酸化蛋白激酶B（pAkt）和磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）的蛋白水平。通过免疫荧光检测LC3的分布。此外，我们还通过激光扫描共聚焦显微镜（LSCM）检测活性氧（ROS）表达。用腺病毒载体转染细胞，通过绿色荧光蛋白（GFP）-红色荧光蛋白（RFP）-LC3的表达监测自噬。与对照组相比，联合处理的TE13中凋亡细胞率显著增加，这与蛋白质免疫印迹法的结果一致。克隆形成存活试验表明，GA在不同浓度下以1.217和1.436的增敏增强率（SER）增强放射敏感性。联合组中LC3-II蛋白水平升高，表明自噬发生率增加，GFP-RFP-LC3实验结果表明GA可能阻断TE13细胞中的自噬流过程。此外，我们成功证明ROS参与自噬的诱导。ROS介导的自噬依赖于Akt/mTOR途径的抑制。综上所述，GA诱导的放射敏感性涉及自噬和凋亡，它们由ROS过度生成和Akt/mTOR抑制调节。
