Diallyl disulfide suppresses epithelial-mesenchymal transition, invasion and proliferation by downregulation of LIMK1 in gastric cancer

Abstract

Diallyl disulfide (DADS) has been shown to have multi-targeted antitumor activities. We have previously discovered that it has a repressive effect on LIM kinase-1 (LIMK1) expression in gastric cancer MGC803 cells. This suggests that DADS may inhibit epithelial-mesenchymal transition (EMT) by downregulating LIMK1, resulting in the inhibition of invasion and growth in gastric cancer. In this study, we reveal that LIMK1 expression is correlated with tumor differentiation, invasion depth, clinical stage, lymph node metastasis, and poor prognosis. DADS downregulated the Rac1-Pak1/Rock1-LIMK1 pathway in MGC803 cells, as shown by decreased p-LIMK1 and p-cofilin1 levels, and suppressed cell migration and invasion. Knockdown and overexpression experiments performed in vitro demonstrated that downregulating LIMK1 with DADS resulted in restrained EMT that was coupled with decreased matrix metalloproteinase-9 (MMP-9) and increased tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. In in vitro and in vivo experiments, the DADS-induced suppression of cell proliferation was enhanced and antagonized by the knockdown and overexpression of LIMK1, respectively. Similar results were observed for DADS-induced changes in the expression of vimentin, CD34, Ki-67, and E-cadherin in xenografted tumors. These results indicate that downregulation of LIMK1 by DADS could explain the inhibition of EMT, invasion and proliferation in gastric cancer cells.

Keywords: LIMK1; diallyl disulfide; gastric cancer cell epithelial-mesenchymal transition; invasion; proliferation.

Figure 1. LIMK1 expression is correlated with survival probability

A. LIMK1 expression in normal mucosa and tumor samples was detected by immunohistochemistry. A representative image is shown (×400 magnification). B. Increased LIMK1 expression was correlated with poor overall survival. The overall survival curves for patients with low or high LIMK1 expression are shown. These patients were right censored at the time of their last known date alive, and data until this point were used in the overall survival (OS) analysis.

Figure 2. The downregulation of LIMK1 and p-LIMK1 by DADS occurred concomitantly with the inhibition of MGC803 cell migration and invasion

Cells were treated with various concentrations of DADS for 24 h. A. Cell migration was analyzed using scratch wound assays. Migration rates are expressed as the ratio of the migration distance between treated and untreated cells. B. Invasion rates were determined by the ratio of the mean number of cells between treated and untreated cells. ^* P < 0.05 versus control. C. Western blot analysis was used to detect p-LIMK1, p-cofilin1, total LIMK1 and cofilin1 protein levels after the cells were treated with 30 mg/L DADS for the indicated times. β-actin was used as the internal control. The relative fold changes compared to controls were calculated. ^* P < 0.05 vs. control.

Figure 3. Analysis of the effects of DADS on Rac1, Rock1, Pak1 and destrin expression in MGC803 cells

Cells were treated with 30 mg/L DADS for the indicated times. A. RT-PCR was performed to detect the mRNA levels of Rac1, Rock1, Pak1 and destrin. β-actin was used as an internal control for normalization. B. Western blot analysis was performed to detect the protein levels of Rac1, Rock1, Pak1 and destrin. β-actin was used as an internal control. The relative fold-changes in mRNA or protein levels compared to the controls were calculated. *P < 0.05 versus control.

Figure 4. Knockdown of LIMK1 augmented the inhibitory effects of DADS on MGC803 cell migration and invasion

A. Cells were transfected with the LIMK1-microRNA (miR1, miR2, and miR3) or empty vectors and western blot analysis was performed to detect LIMK1 protein expression levels. B. p-LIMK1, p-cofilin1, total LIMK1 and cofilin1 protein levels were determined by western blot analysis after the transfected or untransfected cells were treated with 30 mg/DADS for 24 h or left untreated. β-actin was used as a loading control. The relative fold-changes compared to the empty vector or the MGC803 groups were calculated. *P < 0.05 vs. MGC803 or the empty vector group. Cell migration C. and invasion D. rates compared to the MGC803 or vector groups were determined. *P < 0.05 vs. MGC803 or the vector group, ^#P < 0.05 vs. the vector group, the vector + DADS group or the LIMK1-miR group.

Figure 5. Overexpression of LIMK1 attenuated the inhibitory effects of DADS on MGC803 cell migration and invasion

A. Cells were transfected with LIMK1-expressing or empty vectors, and western blot analysis was performed to determine the LIMK1 protein expression level. B. p-LIMK1, p-cofilin1, total LIMK1 and cofilin1 protein levels were determined by western blot analysis after transfected or untransfected cells were treated with 30 mg/DADS for 24 h or left untreated. β-actin was used as a loading control. The relative fold-changes compared to the empty vector group or the MGC803 group were calculated. *P < 0.05 vs. the MGC803 group or the empty vector group. Cell migration C. and invasion D. rates compared with the MGC803 group or the empty vector groups were determined. *P < 0.05 vs. the MGC803 group or the empty vector group, ^#P < 0.05 vs. the vector + DADS group or the LIMK1 group.

Figure 6. DADS inhibits EMT by downregulating LIMK1 in MGC803 cells

A. Western blot analysis was used to detect E-cadherin and vimentin protein levels after cells were treated with 30 mg/DADS for 12, 24, and 48 h. Cells transfected with the empty, B. LIMK1-miR-expressing or C. LIMK1-expressing vectors were treated with or without 30 mg/DADS for 24 h. E-cadherin, vimentin, MMP-9 and TIMP-3 protein levels were determined by western blot analysis. β-actin was used as a loading control. The relative fold-changes compared to the empty vector groups were calculated. *P < 0.05 vs. the empty vector group. D. The untransfected, LIMK1-miR-expressing and LIMK1-overexpressing MGC803 cells were treated with 30 mg/DADS for 24 h or left untreated. Representative images were captured by phase-contrast microscopy.

Figure 7. Effects of LIMK1 knockdown and overexpression on the DADS-induced inhibition of growth in MGC803 cells in vitro and in vivo

A. Cell proliferation assays were performed in vitro after the untransfected, LIMK1-miR-expressing and LIMK1-overexpressing MGC803 cells were treated with 30 mg/DADS or left untreated for the indicated times. B. Cells were treated with 30 mg/L DADS for 24 h or left untreated. The percentages of cells in the G0/G1, S and G2/M phases of the cell cycle were determined by flow cytometry. C. Untransfected, LIMK1-miR-expressing and LIMK1-overexpressing MGC803 cells were injected into the subcutis of nude mice. The mice were treated with normal saline or DADS via intraperitoneal injection every 2 days. The effect of DADS, LIMK1 knockdown and LIMK1 overexpression on tumor volume was examined every 6 days. Average tumor volumes are represented (n=5 per group) starting from the twelfth day and continuing until sacrifice at 48 days. D. The xenografts were collected at 48 days. Tumor sizes are shown for each group of MGC803 model mice. E. The mean ± SD tumor weight for each group was calculated at the termination of the experiment. *P < 0.05 vs. the MGC803 group, vector group, or the LIMK1 + DADS group. ^#P < 0.05 vs. the MGC803 group, DADS group, miR(−) group, or LIMK1-miR group.

Figure 8. Effects of LIMK1 knockdown and LIMK1 overexpression on DADS-induced vimentin, CD34, Ki-67 and E-cadherin expression in vivo

Untransfected, LIMK1-miR-expressing and LIMK1-overexpressing MGC803 cells were injected into the subcutis of nude mice. The mice were treated with normal saline or DADS via intraperitoneal injection every 2 days. The xenografts were collected at 48 days. Immunohistochemistry was performed to detect the expression of vimentin, CD34, Ki-67 and E-cadherin in the tumor tissue specimens obtained from the xenografts. A representative tissue section is shown for each group (×400 magnification).