Graduate Institute of Acupuncture Science, China Medical University, Taichung 40402, Taiwan; Division of Surgery, Department of Medical Research, China Medical University Hospital, Taichung 40447, Taiwan.
Department of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, No. 123, Dapi Road, Niaosong District, Kaohsiung 83300, Taiwan.
Phytomedicine. 2021 Nov;92:153720. doi: 10.1016/j.phymed.2021.153720. Epub 2021 Aug 17.
Bladder cancer (BC) is a very common type of malignant cancer in men and new therapeutic strategies are urgently needed to reduce mortality. Several studies have demonstrated that Rhopaloic acid A (RA), a compound isolated from marine sponges, fights cancer but its potential anti-tumor effect on BC is still unknown.
The present study was aimed to explore the potential anti-tumor effects of RA against human BC cells and the underlying molecular mechanism.
Cell cytotoxicity was determined using the MTT and colony formation assays. Cell cycle distribution, apoptosis induction and generation of mitochondrial reactive oxygen species (ROS) were analyzed by flow cytometry. Mitochondrial membrane potential, acridine orange staining and intracellular ROS levels were observed using fluorescence microscopy. Levels of various signaling proteins were assessed using Western blotting. Furthermore, a zebrafish BC xenotransplantation model was used to confirm the anti-tumor effect of RA in vivo.
Treatment with RA significantly suppressed the proliferation of BC cells that resulted from G2/M cycle arrest. Additionally, RA induced mitochondrial-mediated apoptosis and autophagy in BC cells. The death of BC cells induced by RA was rescued by treatment with inhibitors of apoptosis (Z-VAD-FMA) or autophagy (3-MA). RA activated the MAPK pathway and increased the production of cellular and mitochondrial ROS. Treatment with the ROS scavenger N-acetyl cysteine, effectively reversed the induction of apoptosis, autophagy, JNK activation and DNA damage elicited by RA. Finally, RA significantly inhibited tumor growth in a zebrafish BC xenotransplantation model.
Taken together, our findings indicate that RA induces apoptosis and autophagy and activates the MAPK pathway through ROS-mediated signaling in human BC cells. This RA-induced pathway offers insights into the molecular mechanism of its antitumor effect and shows that RA is a promising candidate for the treatment of BC.
膀胱癌（BC）是男性中非常常见的恶性肿瘤，迫切需要新的治疗策略来降低死亡率。几项研究表明，从海洋海绵中分离出的化合物罗巴酸 A（RA）具有抗癌作用，但它对 BC 的潜在抗肿瘤作用尚不清楚。
本研究旨在探讨 RA 对人 BC 细胞的潜在抗肿瘤作用及其潜在的分子机制。
采用 MTT 和集落形成实验测定细胞毒性。通过流式细胞术分析细胞周期分布、凋亡诱导和线粒体活性氧（ROS）的产生。荧光显微镜观察线粒体膜电位、吖啶橙染色和细胞内 ROS 水平。采用 Western blot 检测各种信号蛋白的水平。此外，还使用斑马鱼 BC 异种移植模型在体内证实 RA 的抗肿瘤作用。
RA 处理显著抑制了 BC 细胞的增殖，导致 G2/M 期细胞周期阻滞。此外，RA 诱导 BC 细胞中线粒体介导的凋亡和自噬。凋亡抑制剂（Z-VAD-FMA）或自噬抑制剂（3-MA）处理可挽救 RA 诱导的 BC 细胞死亡。RA 激活 MAPK 通路并增加细胞和线粒体 ROS 的产生。ROS 清除剂 N-乙酰半胱氨酸处理可有效逆转 RA 诱导的凋亡、自噬、JNK 激活和 DNA 损伤。最后，RA 显著抑制了斑马鱼 BC 异种移植模型中的肿瘤生长。
综上所述，我们的研究结果表明，RA 通过 ROS 介导的信号通路诱导人 BC 细胞凋亡和自噬，并激活 MAPK 通路。RA 诱导的这种通路为其抗肿瘤作用的分子机制提供了新的见解，并表明 RA 是治疗 BC 的有前途的候选药物。
