HSPA6 augments garlic extract-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; Implication for cell cycle dysregulation, signaling pathway alteration, and transcription factor-associated MMP-9 regulation.

Seung-Shick Shin,Byungdoo Hwang,Sung-Soo Park,Dae-Hwa Noh,Wun-Jae Kim,Won Tae Kim,Sung-Kwon Moon,Sung Lyea Park,Jun-Hui Song

doi:10.1371/journal.pone.0171860

Abstract

Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies.

Highlights

Bladder cancer is the most common of all human genitourinary tumors
These results indicate that garlic extract (GE) inhibited proliferation of EJ cells through inducing cell cycle arrest at the G2/M-phase
In order to verify the result of the microarray data, we examined whether the expression of Heat shock protein A6 (HSPA6) protein was upregulated by GE treatment in different periods (12 h and 24 h)

Summary

Introduction

Bladder cancer is the most common of all human genitourinary tumors. The worldwide incidence of bladder cancer has been sharply increasing over the past 10 years [1,2,3]. Accumulation of cyclin B1 increases the activity of CDK1, whose activity is negatively regulated by phosphorylation of its T14/Y15 residues [4]. This inhibitory phosphorylation at T14/Y15 is removed by Cdc25C phosphatases [4]. Activated ATM stimulates the activity of CHK1 and CHK2 by phosphorylation [5]. Cumulated studies have suggested that mitogen-activated protein kinase (MAPK) and AKT signaling cascades are frequent main events involved in multiple biologic processes, such as cell proliferation, differentiation, migration, invasion, and inflammation [6]. Recent studies have shown that the phosphorylation of MAPK and AKT is implicated in the growth inhibition of tumor cells and leads to the induction of cell death [7, 8]

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Results

Conclusion