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. 2014 Jun 7:14:412.
doi: 10.1186/1471-2407-14-412.

The Rho-kinase inhibitor HA-1077 suppresses proliferation/migration and induces apoptosis of urothelial cancer cells

Hideyuki Abe, Takao Kamai  1 , Keitaro Hayashi, Naohiko Anzai, Hiromichi Shirataki, Tomoya Mizuno, Yoshiyuki Yamaguchi, Akinori Masuda, Hideo Yuki, Hironori Betsunoh, Masahiro Yashi, Yoshitatsu Fukabori, Ken-Ichiro Yoshida
Affiliations

The Rho-kinase inhibitor HA-1077 suppresses proliferation/migration and induces apoptosis of urothelial cancer cells

Hideyuki Abe et al. BMC Cancer. .

Abstract

Background: Activation of Rho, one of the small GTPases, and its major downstream target Rho-kinase (ROCK) promotes the development and metastasis of cancer. We previously showed that elevation of Rho and ROCK expression was associated with tumor invasion, metastasis, and an unfavorable prognosis in patients with urothelial cancer of the bladder or upper urinary tract.

Methods: We investigated the effects of a ROCK inhibitor on the growth, migration, and apoptosis of bladder cancer cells. We also examined phosphorylation of RhoA (RhoA activity) by measuring its GTP-bound active form and assessed the expression of ROCK to explore the underlying molecular mechanisms.

Results: Lysophosphatidic acid (LPA) and geranylgeraniol (GGOH) induced an increase of cell proliferation and migration in association with promotion of RhoA activity and upregulation of ROCK expression. The ROCK inhibitor fasudil (HA-1077) suppressed cell proliferation and migration, and also induced apoptosis in a dose-dependent manner. HA-1077 dramatically suppressed the expression of ROCK-I and ROCK-II, but did not affect RhoA activity.

Conclusions: These findings suggest that ROCK could be a potential molecular target for the treatment of urothelial cancer.

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Figures

Figure 1
Figure 1
The inhibitory effects of HA-1077 on cell proliferation. Cell proliferation was inhibited by HA-1077 in 5637 cell (A). In clonogenic assay (B), HA-1077 significantly decreased the number of colonies compared with control cultures in 5637 cells.
Figure 2
Figure 2
Immunohistochemical staining using anti-RhoA, anti-ROCK-I and anti-ROCK-II monoclonal antibodies in 5637 (x20 magnification). 70-80% of tumor cells showed moderate to strong cytoplasmic staining reaction for anti-RhoA antibody, and weak to moderate cytoplasmic staining for anti-ROCK-I and anti-ROCK-II antibodies. HA-1077 reduced the staining reactivity for anti-ROCK-I and anti-ROCK-II antibodies to very weak staining in only 20-30% of tumor cells, while it did not decrease the staining for anti-RhoA antibody with weak to moderate staining.
Figure 3
Figure 3
Representative bands identified by Western blotting in 5637 and UM-UC-3 cells. Expression of RhoA (24 kDa), ROCK-I (160 kDa), ROCK-II (164 kDa), and beta-actin (42 kDa). Hela cells were used as the positive control. In both cancer cells, LPA plus GGOH induced upregulation of RhoA activity, ROCK-I and ROCK-II. While HA-1077 significantly decreased ROCK-I and ROCK-II expression, this inhibitory effect of HA-1077 was not blocked by addition of LPA plus GGOH. HA-1077 did not influence RhoA activity.
Figure 4
Figure 4
Inhibiting effect of HA-1077 on proliferation and RhoA activity and ROCK expression in 5637 cell using the 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium (MTT) assay. The ratio of the cells treated with various doses (0.5–30 μM) of HA-1077 to control cells (HA-1077 0 μM) set as 1.0 were calculated. HA-1077 inhibited the growth of these cells in a dose-dependent manner (A). HA-1077 did not reduce the RhoA activity (B), but suppressed ROCK-I (C) and ROCK-II (D) in dose-dependent manner. The data show the 95% confidential interval.
Figure 5
Figure 5
Inhibiting effect of HA-1077 on proliferation and RhoA activity and ROCK expression in UM-UC-3 cell using the 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium (MTT) assay. The ratio of the cells treated with various doses (0.5–30 μM) of HA-1077 to control cells (HA-1077 0 μM) set as 1.0 were calculated. HA-1077 inhibited the growth of UM-UC-3 cell in a dose-dependent manner (A). HA-1077 did not decrease the RhoA activity (B), but inhibited ROCK-I (C) and ROCK-II (D) in dose-dependent manner. The data show the 95% confidential interval.
Figure 6
Figure 6
The effects of HA-1077 (0.5–30 μM) on bladder cancer cell viability. Fluorescence images generated by terminal deoxynucleotidyltransferase-mediated UTP end-labeling (TUNEL) analysis of the bladder cancer cells. Total cell population was visualized by staining with 4′,6-diamidine-2-phenylin- dole (DAPI). Graph at right shows apoptotic index, the percentage of apoptotic cells in 1000 cells. HA-1077 demonstrates apoptosis induction of 5637 (A) and UM-UC-3 cells (B). LPA plus GGOH reduced apoptotic cells in 5637 (C) and UM-UC-3 cells (D). The data show the 95% confidential interval.
Figure 7
Figure 7
The effects of HA-1077 (0–30 μM) on migration in 5637 cell. The cancer cells were incubated in supplemented with HA-1077 with/without LPA and GGOH. (A) The percentage of migrated cells decreased in dose-dependent manner. LPA and GGOH blocked HA-1077 induced suppression of migration. (B-D) RhoA activity was not statistically different between with and without HA-1077, but ROCK-I and ROCK-II expression was significantly reduced in the cells on the underside of each filter. The data show the 95% confidential interval.
Figure 8
Figure 8
The effects of HA-1077 (0–30 μM) on migration in UM-UC-3 cell. The cancer cells were incubated in supplemented with HA-1077 with/without LPA and GGOH. (A) The percentage of migrated cells decreased in dose-dependent manner. LPA and GGOH blocked HA-1077 induced suppression of migration. (B-D) RhoA activity was not statistically different between with and without HA-1077, but ROCK-I and ROCK-II expression was significantly reduced in the cells on the underside of each filter. The data show the 95% confidential interval.
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