Vincristine enhances amoeboid-like motility via GEF-H1/RhoA/ROCK/Myosin light chain signaling in MKN45 cells

Abstract

Background: Anti-cancer drugs are widely used in cancer treatment frequently combined with surgical therapy and/or radiation therapy. Although surgery and radiation have been suggested to facilitate invasion and metastasis of tumor cells in some cases, there is so far little information about the effect of anti-cancer drugs on cellular invasive ability and metastasis. In this study, using four different anti-cancer drugs (vincristine, paclitaxel, cisplatin and etoposide), we examined whether these drugs influence the invasive ability of tumor cells.

Methods: Human gastric adenocarcinoma MKN45 cells were used to evaluate the effect of anti-cancer drugs. After drug treatment, cellular invasive ability was assessed using the Matrigel invasion chamber. Cytoskeletal changes after treatment were examined microscopically with F-actin staining. In addition, we monitored cellular motility in 3D matrigel environment by time-lapse microscopic analysis. The drug-induced activation of RhoA and ROCK was evaluated by pull-down assay and Western blotting using an antibody against phosphorylated myosin light chain (MLC), respectively. Where necessary, a ROCK inhibitor Y27632 and siRNA for guanine nucleotide exchange factor-H1 (GEF-H1) were applied.

Results: Among all drugs tested, only vincristine stimulated the invasive ability of MKN45 cells. Microscopic analysis revealed that vincristine induced the formation of non-apoptotic membrane blebs and amoeboid-like motility. Vincristine significantly enhanced RhoA activity and MLC phosphorylation, suggesting the involvement of RhoA/ROCK pathway in the vincristine-induced cytoskeletal reorganization and cellular invasion. Furthermore, we found that Y27632 as well as the siRNA for GEF-H1, a RhoA-specific activator, attenuated MLC phosphorylation, the formation of membrane blebs and the invasive ability after vincristine treatment.

Conclusions: These results indicate that vincristine activates GEF-H1/RhoA/ROCK/MLC signaling, thereby promoting amoeboid-like motility and the invasive ability of MKN45 cells.

Figure 1

Invasive ability enhanced by vincristine.A. The number of invading cells measured by the invasion assay. Cells (1 × 10⁶) were seeded into the upper chamber in the presence or absence of anti-cancer drugs. After 24 h incubation, the invading cells were fixed, and stained by toluidine blue. Total numbers of the stained cells were counted using a microscope. Con, non-treated control; Vin, vincristine; Pac, paclitaxel; Cis, cisplatin (15 μM); Eto, etoposide (20 μM). The graph shows mean ± S.E. of three independent experiments. *, P < 0.05; **, P < 0.01 versus Con. B. Cells (1 × 10⁴) were seeded into wells of a 96-well plate in the presence or absence of anti-cancer drugs. After 24 h incubation and the following 1 h incubation with WST-1 solution, the absorbance at 450 nm was recorded using a microplate reader. Cell viability is expressed as percentages relative to the viability obtained for non-treated control. Con, non-treated control; Vin, vincristine; Pac, paclitaxel; Cis, cisplatin (15 μM); Eto, etoposide (20 μM). The graph shows mean ± S.E. of three independent experiments. N.S., not-significant.

Figure 2

Amoeboid-like motility observed after vincristine treatment. A. Cellular morphology after 24 h drug treatment. Cells (2.5 × 10⁵) were seeded into wells of 6-well plates and incubated for 24 h on gelatin-coated coverslips with or without vincristine or paclitaxel. F-actin was labeled with Alexa Fluor 488 phalloidin, and nuclei were labeled with DAPI. F-actin (green) and nuclei (blue) were analyzed using confocal microscopy. Arrows indicate cell protrusions. Arrowheads indicate membrane blebs. White bars, 10 μm. B. Detection of blebbing cells and apoptosis. Cells on gelatin-coated coverslips were treated with vehicle or drugs, fixed and stained with TRITC phalloidin, cleaved caspase-3 antibody and DAPI. F-actin (red), cleaved caspase-3 (green) and DAPI (blue) were analyzed using confocal microscopy. Arrows indicate the cells with membrane blebs. White bars, 10 μm. C. DIC images from supplemental videos of the control cell (Additional file 1: Video S1) and the 15 μM vincristine-treated cell (Additional file 2: Video S2). Cells (6.25 × 10³) were confined to a 50% Matrigel environment with or without 15 μM vincristine on a 35 mm glass-bottom dish by a coverslip. Frames show the cells at the indicated times. Arrows indicate cell protrusions. Arrowheads indicate membrane blebs.

Figure 3

RhoA activated by vincristine. A and B. Cells were starved in serum-free RPMI1640 for 24 h, and then exposed to vincristine. After vincristine treatment, the cells were harvested to evaluate RhoA activity by rhotekin-based pull-down assay. RhoA in pull-down samples (active RhoA) and in total lysates (total RhoA) were detected by Western blotting using an anti-RhoA antibody. A, time-course; B, concentration-response at 15 min. The blots in (A) and (B) are representative of three independent experiments. The blots were quantified by densitometry, and the results were expressed as ratio relative to the values obtained in non-treated control cells (0 min or 0 μM). The graphs in (A) and (B) show means ± S.E. of three independent experiments. *, P < 0.05 versus control.

Figure 4

MLC phosphorylation promoted by vincristine. A and B. Cells were starved in serum-free RPMI1640 for 24 h, and then treated with or without vincristine. After vincristine treatment, the cells were harvested to evaluate MLC phosphorylation by Western blotting. A, time-course; B, concentration-response at 15 min. The blots in (A) and (B) are representative of three independent experiments. The blots were quantified by densitometry, and the results were expressed as a ratio relative to the values obtained in non-treated control cells (0 min or 0 μM). The graphs in (A) and (B) show means ± S.E. of three independent experiments. *, P < 0.05 versus control.

Figure 5

Involvement of ROCK in vincristine-enhanced invasive ability. A. Cells were starved in serum-free RPMI1640 for 24 h, and then treated with or without 15 μM vincristine and/or 50 μM Y27632 for 15 min. The cells were then harvested to evaluate MLC phosphorylation by Western blotting using anti-MLC and anti-pMLC antibodies. The blots in (A) are representative of four independent experiments. The blots were quantified by densitometry, and the results were expressed as ratio relative to the values obtained in non-treated control cells. The graph in (A) shows mean ± S.E. of four independent experiments. *, P < 0.05; **, P < 0.01. B. Cells on gelatin-coated coverslips were treated with drugs, fixed and stained with Alexa Fluor 488 phalloidin and DAPI. F-actin (green) and nuclei (blue) were analyzed using confocal microscopy. Arrows indicate the cells with membrane blebs. White bars, 10 μm. The graph shows means ± S.E. of three independent experiments. *, P < 0.05. C. Cells (1 × 10⁶) were seeded into the upper chamber with or without 15 μM vincristine and/or 50 μM Y27632. After 24 h incubation, the invading cells were fixed and stained with toluidine blue. Total numbers of the stained cells were counted using a microscope. The graphs show means ± S.E. of three independent experiments. *, P < 0.05.

Figure 6

Involvement of GEF-H1 in vincristine-enhanced invasive ability. A. At 72 or 92 h after transfection of GEF-H1-specific siRNA or negative control siRNA, cells were harvested to analyze GEF-H1 expression by Western blotting using anti-GEF-H1 and anti-actin antibodies. Actin was used as a loading control. B. At 72 h after transfection, starved cells (24 h) were treated with or without 15 μM vincristine for 15 min. The cells were then lysed and analyzed by Western blotting. The blots were quantified by densitometry, and the results were expressed as a ratio relative to the values of pMLC/MLC obtained in non-treated cells. The graph shows mean ± S.E. of three independent experiments. **, P < 0.01. C. Control siRNA- or GEF-H1 siRNA-transfected cells on gelatin-coated coverslips were treated with vehicle or 15 μM vincristine. The cells were fixed and stained with Alexa Fluor 488 phalloidin and DAPI. F-actin (green) and nuclei (blue) were analyzed using confocal microscopy. Arrows indicate the cells with membrane blebs. White bars, 10 μm. The graph shows means ± S.E. of three independent experiments. **, P < 0.01. D. The number of invading cells measured by invasion assay. At 72 h after transfection, cells (1 × 10⁶) were seeded into the upper chamber with or without 15 μM vincristine. After 24 h incubation, the invading cells were fixed, and stained with toluidine blue. Total numbers of the stained cells were counted using a microscope. The graph shows means ± S.E. of three independent experiments. *, P < 0.05.

Figure 7

Summary of this study.