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

<div><p>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.</p></div

The 10 most up-regulated genes found with MF analysis following GE treatment.

<p>The 10 most up-regulated genes found with MF analysis following GE treatment.</p

Up-regulated genes following GE treatment (800 μg/ml).

<p>Up-regulated genes following GE treatment (800 μg/ml).</p

Effect of GE on G₂/M-phase associated cell-cycle regulators and signaling pathways in EJ cells.

<p>(A) Changes in cell cycle regulators by treatment of different concentrations of GE. Immunoblots were performed using specific antibodies indicated. The bar graphs were presented as a fold ratio to the control. (B, C) Effect of GE on MAPK (ERK1/2, JNK1/2, and p38) and AKT signaling. Phosphorylation levels of each molecule were assessed by immunoblots. Bar graphs were presented as fold changes compared to the control. (D) EJ cells were pre-incubated with U0126 (0.5 μM), SB203580 (10 μM), SP600125 (10 μM), and LY 294002 (10 μM) for 40 min prior to treatment with GE (800 μg/ml). The ratio of phosphorylated to non-phosphorylated form was measured and presented as fold changes compared to the control. Results in bar graphs are represented as mean ± SE from three different triplicate experiments. *P<0.05 compared with the control.</p

The 10 most down-regulated genes found with BP analysis following GE treatment.

<p>The 10 most down-regulated genes found with BP analysis following GE treatment.</p

The 10 most up-regulated genes found with BP analysis following GE treatment.

<p>The 10 most up-regulated genes found with BP analysis following GE treatment.</p

GE inhibited the migration and invasion of EJ cells through diminished MMP-9 activity by suppressing binding activity of transcription factor AP-1, Sp-1, and NF-κB.

<p>(A) Changes of migratory potential were assessed by scratch wound-healing assays. The cells were pre-treated with mitocycin C (5 μg/ml) for 2 h. The surface area of migrating cells was photographed by a phase contrast microscope. The recovery rate was measured as fold changes compared with the control. (B) Invasive capacity was measured using Matrigel^®-coated transwell plates in GE-treated EJ cells. The cells were incubated with mitocycin C (5 μg/ml) for 2 h before the invasion assays. Cellular images were taken by a phase contrast microscope. The amount of invading cells was presented as a fold change relative to the control. (C) Gelatinase activity of MMP-2 and -9 were assessed by different concentrations of GE using zymography. Bar graph was presented as a fold change contrasted with the control. (D) Binding activity of transcription factor AP-1, Sp-1, and NF-κB, was measured by EMSA in GE-treated EJ cells. Bar graph was presented as a fold change compared with the control. Results in bar graphs are represented as mean ± SE from three different triplicate experiments. *P<0.05 compared with the control.</p

HSPA6 gene enhanced regulatory proteins participated in GE-mediated G₂/M-phase cell cycle and early signaling pathways in EJ cells.

<p>EJ cells were transfected with an EV (A, C) or HSPA6 (B, D) followed by incubation in the culture medium with or without GE. Cell cycle regulators and signaling molecules were assessed by immunoblots using specific antibodies indicated. Expression levels of proteins were normalized by corresponding total forms or GAPDH. *P < 0.05, compared with the control and **P < 0.05, compared with GE treatment.</p

The 10 most down-regulated genes found with MF analysis following GE treatment.

<p>The 10 most down-regulated genes found with MF analysis following GE treatment.</p

HSPA6 intensified GE-mediated inhibitory effect of MMP-9 activity via suppression of binding activity of AP-1, Sp-1, and NF-κB in EJ cells.

<p>EJ cells were transfected with an EV (A, C) or HSPA6 (B, D) followed by incubation either in the presence or absence of GE. (A, B) Proteolytic activity of MMP-9 was assessed by gelatin zymography. (C, D) EMSA was performed to detect the binding activity of AP-1, Sp-1, and NF-κB using radiolabeled oligonucleotide probes. Unlabeled AP-1, Sp-1, and NF-κB oligonucleotides were used as competitors. Relative fold changes were indicated to compare against the control versus GE treatment. In each bar graph, results are presented as the mean ± SE from three different triplicate experiments.*P < 0.05, compared with the control and **P < 0.05, compared with GE treatment.</p