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

PLoS One. 2017 Feb 10;12(2):e0171860. doi: 10.1371/journal.pone.0171860. eCollection 2017.

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.

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Cell Cycle / drug effects
  • Cell Line, Tumor
  • Cell Movement / drug effects
  • Cell Proliferation / drug effects
  • Checkpoint Kinase 2 / metabolism
  • Garlic / chemistry*
  • HSP70 Heat-Shock Proteins / metabolism*
  • Humans
  • MAP Kinase Signaling System
  • Matrix Metalloproteinase 9 / metabolism*
  • Plant Extracts / pharmacology*
  • Urinary Bladder Neoplasms / metabolism*

Substances

  • Antineoplastic Agents
  • HSP70 Heat-Shock Proteins
  • HSPA6 protein, human
  • Plant Extracts
  • Checkpoint Kinase 2
  • CHEK2 protein, human
  • MMP9 protein, human
  • Matrix Metalloproteinase 9

Grant support

This study was supported by a National Research Foundation of Korea (NRF) grant by the government of Korea (MSIP) (no. 2014007036), http://www.nrf.re.kr/.