锐尖杜英三萜皂苷 III 诱导人黑色素瘤 A375 细胞自噬并通过 Akt/mTOR/p70S6K 通路促进其凋亡。

Sasanquasaponin ΙΙΙ from Schima crenata Korth induces autophagy through Akt/mTOR/p70S6K pathway and promotes apoptosis in human melanoma A375 cells.

机构信息

Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Institute of Traditional Chinese Medicine and Natural Product, College of Pharmacy, Jinan University, Guangzhou, Guangdong 510632, China.

College of Pharmacy, Guangzhou Medical University, Guangzhou, Guangdong 510632, China.

出版信息

Phytomedicine. 2019 May;58:152769. doi: 10.1016/j.phymed.2018.11.029. Epub 2018 Nov 20.

DOI:10.1016/j.phymed.2018.11.029

PMID:31005714

Abstract

BACKGROUND

Melanoma is a high fatality skin cancer which lacks effective drugs. Sasanquasaponin, an important sort of constituents in theaceae, has been demonstrated to have potent anti-tumor effect in breast cancer and hepatocellular carcinoma. As a sasanquasaponin, we speculate that Sasanquasaponin III (SQS III) isolated from Schima crenata Korth may also have anti-tumor activity.

PURPOSE

This study aims to investigate whether SQS III has anti-melanoma activity and examine the underlying mechanisms of SQS III against melanoma.

METHODS/STUDY DESIGNS: The anti-proliferative effect of SQS III was assessed by cells viability assay. Annexin V-FITC/PI double staining assay was utilized for detection of apoptosis. Mitochondrial membrane potential and reactive oxygen species (ROS) production were detected using JC-1 and DCFH-DA assay, respectively. Autophagy was monitored using transmission electron microscopy (TEM) and GFP-LC3 transfection fluorescence analysis. Autophagosome-lysosome fusion and lysosomal degradation were determined using a GFP-LC3 & LAMP1 co-localization assay and DQ-BSA staining. Proteins related to apoptosis and autophagy were analyzed by Western blotting.

RESULTS

Our results demonstrated that the SQS III exhibited potent anti-cancer activity in A375 cells by inducing both apoptosis and autophagy. In melanoma cells treated with SQS III, caspases were activated and PARP was cleaved, proving the occurrence of apoptosis. Mechanistic studies indicated that the pro-apoptosis activity of SQS III was mediated by death receptor pathway and mitochondrial dysfunction which was induced by ROS accumulation and reversed by the ROS inhibitor N-acetyl-cysteine (NAC). In addition to triggering apoptosis, SQS III may also cause autophagy in melanoma cells. Our results demonstrated that SQS III induced up-regulated expression of GFP-LC3, autophagosome-lysosomal fusion and lysosomal degradation. Additionally, the ROS accumulation was also involved in the activation of autophagy. Meanwhile, it was also found that after SQS III treatment, the expression of LC3-II was up-regulated and the AKT/mTOR signaling pathway was inhibited. The autophagy inhibitor 3-MA converted cytotoxicity and apoptosis of SQS III in A375 cells, which indicated that autophagy promoted the SQS III-induced apoptosis.

CONCLUSION

SQS III showed potent anti-cancer activity by inducing apoptosis and autophagy, which provides insights into its possible use as a therapy for melanoma.

摘要

背景

黑色素瘤是一种高致死率的皮肤癌，目前缺乏有效的治疗药物。三萜皂苷是山茶科植物的重要组成部分之一，已被证实具有很强的抗肿瘤作用，如在乳腺癌和肝癌中。作为一种三萜皂苷，我们推测从杨桐中分离得到的三叶海棠苷（SQS III）可能也具有抗肿瘤活性。

目的

本研究旨在探讨 SQS III 是否具有抗黑色素瘤活性，并研究 SQS III 对黑色素瘤的作用机制。

方法/研究设计：通过细胞活力测定评估 SQS III 的抗增殖作用。采用 Annexin V-FITC/PI 双染色法检测细胞凋亡。使用 JC-1 和 DCFH-DA 测定法分别检测线粒体膜电位和活性氧（ROS）的产生。使用透射电子显微镜（TEM）和 GFP-LC3 转染荧光分析监测自噬。通过 GFP-LC3 和 LAMP1 共定位测定和 DQ-BSA 染色测定自噬体-溶酶体融合和溶酶体降解。通过 Western blot 分析检测与凋亡和自噬相关的蛋白。

结果

我们的结果表明，SQS III 通过诱导细胞凋亡和自噬在 A375 细胞中表现出很强的抗癌活性。在 SQS III 处理的黑色素瘤细胞中，半胱天冬酶被激活，PARP 被切割，证明发生了细胞凋亡。机制研究表明，SQS III 的促凋亡活性是由死亡受体途径介导的，线粒体功能障碍是由 ROS 积累引起的，并可被 ROS 抑制剂 N-乙酰半胱氨酸（NAC）逆转。SQS III 不仅可以触发细胞凋亡，还可能在黑色素瘤细胞中引起自噬。我们的结果表明，SQS III 诱导 GFP-LC3 的表达上调、自噬体-溶酶体融合和溶酶体降解。此外，ROS 积累也参与了自噬的激活。同时，研究还发现，SQS III 处理后 LC3-II 的表达上调，AKT/mTOR 信号通路被抑制。自噬抑制剂 3-MA 可使 SQS III 在 A375 细胞中的细胞毒性和细胞凋亡发生转化，表明自噬促进了 SQS III 诱导的细胞凋亡。