Flaccidoxide-13-Acetate-Induced Apoptosis in Human Bladder Cancer Cells is through Activation of p38/JNK, Mitochondrial Dysfunction, and Endoplasmic Reticulum Stress Regulated Pathway

• Main Authors: Yu-Jen Wu, Tzu-Rong Su, Guo-Fong Dai, Jui-Hsin Su, Chih-I Liu
• Format: Article
• Language: English
• Published: MDPI AG 2019-05-01
• Series: Marine Drugs
• Subjects: flaccidoxide-13-acetate; cultured soft coral <i>Sinularia gibberosa</i>; human bladder cancer; apoptosis; mitochondrial dysfunction; PERK-eIF2<i>α</i>-ATF6-CHOP pathway; p38/JNK
• Online Access: https://www.mdpi.com/1660-3397/17/5/287

Flaccidoxide-13-acetate, an active compound isolated from cultured-type soft coral <i>Sinularia gibberosa</i>, has been shown to have inhibitory effects against invasion and cell migration of RT4 and T24 human bladder cancer cells. In our study, we used an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation assay, and flow cytometry to determine the mechanisms of the anti-tumor effect of flaccidoxide-13-acetate. The MTT and colony formation assays showed that the cytotoxic effect of flaccidoxide-13-acetate on T24 and RT4 cells was dose-dependent, and the number of colonies formed in the culture was reduced with increasing flaccidoxide-13-acetate concentration. Flow cytometry analysis revealed that flaccidoxide-13-acetate induced late apoptotic events in both cell lines. Additionally, we found that flaccidoxide-13-acetate treatment upregulated the expressions of cleaved caspase 3, cleaved caspase 9, Bax, and Bad, and down-regulated the expressions of Bcl-2, <i>p</i>-Bad, Bcl-x1, and Mcl-1. The results indicated that apoptotic events were mediated by mitochondrial dysfunction via the caspase-dependent pathway. Flaccidoxide-13-acetate also provoked endoplasmic reticulum (ER) stress and led to activation of the PERK-eIF2<i>&#945;</i>-ATF6-CHOP pathway. Moreover, we examined the PI3K/AKT signal pathway, and found that the expressions of phosphorylated PI3K (<i>p</i>-PI3K) and AKT (<i>p</i>-AKT) were decreased with flaccidoxide-13-acetate concentrations. On the other hand, our results showed that the phosphorylated JNK and p38 were obviously activated. The results support the idea that flaccidoxide-13-acetate-induced apoptosis is mediated by mitochondrial dysfunction, ER stress, and activation of both the p38 and JNK pathways, and also relies on inhibition of PI3K/AKT signaling. These findings imply that flaccidoxide-13-acetate has potential in the development of chemotherapeutic agents for human bladder cancer.