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

Yu-Jen Wu,Guo-Fong Dai,Chih-I Liu,Tzu-Rong Su,Jui-Hsin Su

doi:10.3390/md17050287

Abstract

Flaccidoxide-13-acetate, an active compound isolated from cultured-type soft coral Sinularia gibberosa, 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, p-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-eIF2α-ATF6-CHOP pathway. Moreover, we examined the PI3K/AKT signal pathway, and found that the expressions of phosphorylated PI3K (p-PI3K) and AKT (p-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.

Highlights

In the US, bladder cancer is the second most frequent genitourinary malignant tumor, and is a fatal disease [1]
An 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and a colony formation assay were used to study the cytotoxic effects of flaccidoxide-13-acetate on T24 and RT4 formation assay were used to study the cytotoxic effects of flaccidoxide-13-acetate on T24 and RT4 bladder cancer cell lines
The results showed that the expressions of activating transcription factor 6 (ATF6)-f and GRP78 proteins increased in both cell lines treated with flaccidoxide-13-acetate;of and increased in as both linesand treated with flaccidoxide-13-acetate; inATF6-f addition, theGRP78 levels ofproteins p-eukaryotic initiation factor 2α (eIF2α) and p-PKR-like ER-associated kinase (PERK), wellcell as ATF4

Summary

Introduction

In the US, bladder cancer is the second most frequent genitourinary malignant tumor, and is a fatal disease [1]. Most human bladder cancers belong to the category of transitional cell carcinoma, which can be further divided into invasive and non-invasive bladder cancer. T2 and higher stages of muscle invasive bladder cancer have a poor prognosis and a high risk of metastasis [2,3,4]. Non-muscle-invasive bladder cancer is usually treated with transurethral resection of the bladder tumor, followed by close monitoring or Bacillus Calmette–Guérin (BCG) instillation [5]. The recurrence rate in patients with high-grade non-muscle-invasive bladder cancer after transurethral resection exceeds 75%, with a low survival rate [6]. Since the recurrence rate of bladder cancer is still high, and many medications currently used for bladder cancer have strong side effects [3], the development of new drugs for bladder cancer treatment remains an important issue

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