Ursolic acid induces ER stress response to activate ASK1–JNK signaling and induce apoptosis in human bladder cancer T24 cells

doi:10.1016/j.cellsig.2012.09.012

Cellular Signalling

Volume 25, Issue 1, January 2013, Pages 206-213

https://doi.org/10.1016/j.cellsig.2012.09.012 Get rights and content

Abstract

Here we studied the cellular mechanisms of ursolic acid's anti-bladder cancer ability by focusing on endoplasmic reticulum stress (ER stress) signaling. We show that ursolic acid induces a significant ER stress response in cultured human bladder cancer T24 cells. ER stress inhibitor salubrinal, or PERK silencing, diminishes ursolic acid-induced anti-T24 cell effects. Salubrinal inhibits ursolic acid-induced CHOP expression, Bim ER accumulation and caspase-3 activation in T24 cells. Ursolic acid induces IRE1–TRAF2–ASK1 signaling complex formation to activate pro-apoptotic ASK1–JNK signaling. We suggest that ER stress contributes to ursolic acid's effects against bladder cancer cells.

Highlights

► Ursolic acid induces ER stress response in cultured bladder cancer T24 cells. ► Salubrinal inhibits ursolic acid-induced ER stress response. ► Salubrinal or PERK silencing inhibited ursolic acid-induced cytotoxic effects. ► Ursolic acid induces IRE1–TRAF2–ASK1 complex formation to activate ASK1-JNK.

Introduction

Recent studies including ours [1] have confirmed the anti-growth ability of ursolic acid against a number of human cancer cells [2], [3], [4], [5]. In addition, ursolic acid has showed a broad spectrum of activities to influence cancer progression and development, including interfering tumorigenesis, metastasis and angiogenesis, and inducing tumor cell apoptosis and differentiation [3], [4], [5]. Studies using in vivo animal models have confirmed the chemo-preventive ability of ursolic acid [[6], [7], [8], [9]]. Further, ursolic acid inhibits experimental metastasis of esophageal carcinoma [5], [10]. However, the potential mechanisms of its anti-tumor cells ability are not extensively studied. Here we focused on the potential role of endoplasmic reticulum stress (ER stress) in its anti-bladder cancer ability.

The normal function of endoplasmic reticulum (ER) is to regulate the synthesis, initial post-translational modification, proper folding, and maturation of newly synthesized proteins. It is also responsible for intracellular calcium homeostasis. Various stresses including multiple anti-cancer drugs disrupt proper function of ER and cause ER stress [11], [12]. Cells response to ER stress by activating the following major mechanisms known as unfolded protein response: (a) transcriptional up-regulation of ER chaperones and folding enzymes (for example C/EBP homologous protein (CHOP), GADD 45, GRP 78 and GRP 94); (b) translational attenuation to limit further accumulation of misfolded proteins; and (c) ER-associated degradation to eliminate misfolded proteins inside the ER [13]. It is now well-established that ER membrane receptors including double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) act as the sensors of stress, and eventually lead to unfolded protein response [13]. Although the unfolded protein response is primarily a pro-survival response, in the event of prolonged or severe ER stress that is not resolved, the unfolded protein response switches to initiation of apoptosis [13].

Recent studies have confirmed that anti-cancer drugs including cisplatin [14] and Tetrocarcin-A [15] as well as anti-cancer neutral compounds such as curcumin [16], [17], [18] all activate ER stress to promote cell apoptosis. Here we studied whether ER stress play a role in ursolic acid's anti-bladder cancer ability.

Section snippets

Reagents

Salubrinal, anti-tubulin, TNF receptor-associated factor 2 (TRAF2), AMP-activated protein kinase (AMPK), rabbit IgG-HRP and mouse IgG-HRP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were purchased from Cell Signaling Technology (Bevery, MA). Ursolic acid was purchased from Sigma (U6753, St. Louis, MO).

Cell culture

As reported early [1], the human bladder cancer cell T24 and BIU-87 lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine

Ursolic acid induces ER stress in cultured bladder cancer cells

The main focus of this study is to test the possible involvement of ER stress in ursolic acid's effect against bladder cancer cells. We first examined ER stress activation after ursolic acid (see structure in Fig. 1A) treatment in T24 cells using Western-blots detecting the expression of C/EBP homologous protein (CHOP, also known as GADD153), and phosphorylation of PERK (Thr 980) and eIF2α (Ser 51). Results in Fig. 1B and C demonstrated a significant ER stress activation after ursolic acid

Discussion

Certain components of ER stress-induced unfolded protein response are strongly linked to apoptosis. For instance, CHOP, which is induced by all three arms of unfolded protein response through x-box–binding protein–1 (XBP1), activating transcription factor 4 (ATF 4) and activating transcription factor 6 (ATF 6) transcription factors, is required for ER stress-induced apoptosis in many instances [19]. CHOP promotes mitochondrial apoptosis pathway through altering the balance between pro-survival

Contributors

Q. Z., P.L., F. J., C. Y., G. Z., T. Z., and X. A., wrote the paper, designed and performed the experiments. Q. Z., P.L., F. J., C. Y., did the stats and organized the data. Q. Z., F. J., C. Y., supervised the research design and the writing of the paper. All authors read and commented on the manuscript.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This research was supported in part by grants from the National Natural Science Foundation of China. Funding recourses have no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

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Ursolic acid induces ER stress response to activate ASK1–JNK signaling and induce apoptosis in human bladder cancer T24 cells

Abstract

Highlights

Introduction

Section snippets

Reagents

Cell culture

Ursolic acid induces ER stress in cultured bladder cancer cells

Discussion

Contributors

Conflict of interest

Acknowledgments

Biochemical and Biophysical Research Communications

Life Sciences

Cancer Letters

European Journal of Pharmacology

Leukemia Research

Journal of Biological Chemistry

Blood

Cell

Journal of Biological Chemistry

Cell

Journal of Biological Chemistry

Journal of Biological Chemistry

Journal of Enzyme Inhibition and Medicinal Chemistry

Neoplasma

Cancer Research