Inhibition of Motile and Invasive Properties of Ovarian Cancer Cells by ASODN Against Rho-associated Protein Kinase

Abstract

p160ROCK, a kinase effector of Rho GTPase mediating RhoA-induced assembly of focal adhesions and stress fibers, plays an important role in the invasive process of various tumor cells. The purpose of this study was to investigate the role of p160ROCK in the invasive behaviors of human ovarian cancer cells and their metastasis. Transfection with a dominant-active form of p160ROCK mutant (p160ROCKΔ 3) enhanced cell migration and invasion of ovarian cancer cells, while antisense oligodeoxynucleotide (ASODN) against p160ROCK inhibited the motile and invasive properties of the cells. Our data suggested that p160ROCK was involved in ovarian cancer cell invasion and metastasis by facilitating cancer cell migration, and that p160ROCK might be a potential new effective target for preventing metastasis of ovarian cancer.

Keywords:

• p160ROCK
• antisense oligodeoxynucleotide
• dominant active mutant
• invasion
• metastasis
• ovarian cancer

Abbreviation
ROCK	=	Rho-associated coiled-coil forming kinase
ASODN	=	antisense oligodeoxynucleotide
SODN	=	sense oligodeoxynucleotide
PBS	=	phosphate-buffered saline
BSA	=	bovine serum albumin
LIMK	=	LIM kinase
MLC	=	myosin light chain
ERM	=	ezrin-radixin-moesin.

INTRODUCTION

The incidence and mortality rate of ovarian cancer remains the highest among all gynecologic cancers [1], which is mainly due to the highly aggressive migration activity of ovarian cancer cells. Cancer cell metastasis is a multistep process comprising detachment from the primary tumor, entry into the vascular or lymphatic system, dispersal through the circulation, and extravasation and proliferation into the target organ [2]. Understanding the mechanisms of metastasis in ovarian cancer cells will promote new treatment strategies for preventing the progression of cancers.

Metastasis requires rearrangement of the actin cytoskeleton, including stress fiber and focal adhesion reassembly, which is regulated through the Rho/Rho–associated kinase pathway. Rho GTPase, a critical player during metastasis and tumorigenesis, is involved in various cellular activities, including cell migration, cell adhesion, cell proliferation, cell differentiation and apoptosis [3, 4, 5, 6, 7, 8]. The discovery of the high-level expression of some Rho GTPase family members in various human tumors indicated that the expression of the Rho molecule was related to the carcinogenic process. In the cases of breast cancer and testicular germ-cell tumors, the expression levels of RhoA are positively correlated with the progression of the disease [9, 10]. RhoC was shown to be overexpressed in pancreatic ductal adenocarcinoma and inflammatory breast cancer cells. Furthermore, a positive link was also shown between RhoC overexpression and inflammatory breast cancers [11, 12].

Rho GTPase regulates cellular activities through activating multiple signaling-targeted effectors and through altering gene expression patterns [8]. ROCK/ROK, Rho-associated coiled-coil forming protein kinase [13, 14], was the first Rho effector to be discovered, and was initially characterized regarding their role in mediating the formation RhoA-induced focal adhesions and stress fibers [15]. Two ROCK isoforms have been identified; ROCK-I (ROKβ and p160ROCK) and ROCK-II (known as ROKα and Rho-kinase) [16]. However, there is no evidence that p160ROCK and ROCK-II have different functions [17]. Recent studies showed that p160ROCK functions in the induction of focal adhesions and stress fibers in cultured rat MM1 hepatoma cells [18], suggesting that p160ROCK may play a role in cancer invasion. However, the details of ROCK regulatory mechanisms in ovarian cancer have not been clarified. In this study, we investigated the roles of p160ROCK for various cellular activities, such as motility, invasion, adhesion and proliferation in human ovarian cancer cells and demonstrated that changes in p160ROCK activity influence the metastasis of ovarian cancer cells.

MATERIALS AND METHODS

Cell Culture and Reagents

The human ovarian cancer cell line, Caov-3, was obtained from American Type Culture Collection (Rockville, MD) and cultured in DMEM supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 100 μ g/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. Rabbit polyclonal anti-p160ROCK antibody and mouse anti-Myc (9E10) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Expression Vector, ASODN and Transfection

Myc-tagged dominant-active mutant of p160ROCK was constructed in the pCAG mammalian expression vector, or pCAG-myc-p160ROCKΔ3, was described previously [15]. Phosphorothioated 23-mer oligodeoxynucleotides were synthesized through β -cyanothylphoramidite chemistry, which can minimize nucleotide degradation. The ASODN (base sequence 5′-TCAAAACTGTCCCCAGTCGACAT-3′) was designed to direct against the translation start site (AUG codon) of the human p160ROCK gene. As a control of ASODN, we used its corresponding SODN-sense oligodeoxynucleotide (5′- ATGTCGACTGGGGACAGTTTTGA-3′). Transfection was performed by using the LipofectAMINE2000 system from Invitrogen and the protocol used in this study followed the manufacturer's instructions.

Western Blot Analysis

Cells were lysed in cold solubilization buffer containing 50 mM HEPES (pH 7.5), 1% Triton X-100, 150 mM NaCl, 2 mM Na₃VO₄, 100 mM NaF, 100 units/ml aprotinin, 20 μ M leupeptin, and 0.2 mg/ml PMSF. Cell extract was clarified (14,000g, 15 min, 4°C) and aliquots of lysate were diluted in 4 x SDS-PAGE sample buffer (62 mM Tris-HCl, 8% SDS, 40% glycerol, 20% 2-mercaptoethanol, and 0.16% bromophenol blue), boiled for 5 min and resolved in 8% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto nitrocellulose membranes that were blocked using 5% (w/v) nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 and exposed to the antibodies overnight at 4°C in the same buffer. After incubation with the secondary antibody conjugated to horseradish peroxidase, blots were revealed by using the enhanced chemiluminescence method.

Reverse Transcription-PCR

Total RNA was extracted from cells. The cDNA was synthesized using a reverse transcription system (Promega corp., Madison, WI). Reverse transcription products were amplified by PCR using specific primers for human p160ROCK (forward 5′-AAGAAGGCAGAGGAAGAATA-3′; reverse 5′-TAACAGCAGCATATCTCTTG-3′). Amplification was performed following 32 cycles with denaturing at 94°C for 30 sec, annealing at 52°C for 1 min, and extension at 72°C for 1 min. The PCR products were subjected to electrophoresis on a 1% agarose gel containing ethidium bromide and visualized under UV light. β –actin primers were used as an internal control.

Immunofluorescence

To visualize the actin cytoskeleton, treated cells grown on covered glasses were washed with PBS and fixed with 3.7% formaldehyde in PBS for 10 min. Cells were washed twice with PBS followed by permeabilization with 0.1% TRITON®X-100 for 5 min. After washing with PBS, cells were then preincubated with PBS containing 1% BSA for 30 min and stained with a preparation of rhodamine-phalloidin (Molecular Probes, Eugene, USA). Fluorescent images were recorded using Olympus microscope (Model BX40F4, Tokyo, Japan).

Invasion and Migration Assays

The in vitro invasive ability of ovarian cancer cells was measured in a modified Boyden chamber. Polycarbonate filters in 8 μ m pore size (Costar), were first coated with an extract of basement membrane component (Matrigel, 5 μ g/filter; Collaborative Research Co.), dried out, and reconstituted with DMEM. The coated filters were placed in blind-well Boyden chambers. The cells to be tested were seeded in the upper compartment of the Boyden chamber at a concentration of 2 × 10⁵ cells/chamber. NIH3T3 fibroblast-conditioned medium was used as a chemoattractant in the lower compartment of the chamber. After incubation for 10h at 37°C, the noninvasive cells were removed with a cotton swab. The cells that had migrated through the membrane and stuck to the lower surface of the membrane were fixed with methanol and stained with hematoxylin. Cells were counted at × 400 in 10 randomly selected fields, and the mean number of cells per field was recorded. The protocol for migration assays was similar to the invasion assay, except that filters were not precoated with matrigel. As for cell directional migration, or chemotaxis, the lower compartment of the chamber was filled with NIH3T3 fibroblast-conditioned medium. As for cell random migration, or chemokinesis, the lower compartment of the chamber was filled with the same medium as the upper compartment of the chamber.

Wound-closure Assay

For the wound-closure assay, cells were seeded into 60-mm diameter dishes at 2× 10⁶/dish density and allowed to grow to 90% confluence in DMEM supplemented with 10% fetal bovine serum. Cell monolayers were wounded using a plastic pipette tip and then washed three times with PBS to remove cell debris. For each dish, three sites of regular wounds were selected and marked. After 12-h culture, cell movement into the wound area was examined at different time points by using a Nikon light microscope. The distance between the leading edges of the wounds was compared.

Cell Adhesion Assay

Matrigel-coated 96-well plates were blocked with 2% fatty acid-free BSA overnight. The treated cells and control cells (5 × 10⁴ cells in100 μ l/well) were suspended in the recommended medium, added to each well, and incubated for 30 min at 37°C. Then the plates were washed three times with PBS gently and the number of remaining adherent cells was measured by MTT assay as described previously, with minor modifications [12]. Briefly, MTT (0.5 mg/ml) (from Sigma Chemical Co.) was added to each well. After incubation for 4 h at 37°C, the supernatants were aspirated, and 100 μ l of DMSO were added. The absorbance at 570 nm was measured using a microplate reader. All experiments and measurements were repeated three times.

Cell Proliferation Assay

In the cell proliferation assay, transfected cells and control cells (1 × 10⁴cells/well) were prepared in 96-well plates in serum-containing medium. After incubation at 37°C in 5% CO₂ for 72 h, the living cells were counted by using the MTT assay as described above.

Statistical Analysis

Student's t test was used to compare data between two groups. Values were expressed as mean ± SD of at least triplicate samples. P < 0.05 was considered statistically significant.

RESULTS

Expression of p160ROCK in caov-3 Cells

The expression level of p160ROCK was estimated by Western blot analysis of cell lysates derived from a human ovarian cancer cell line, called caov-3. The antibody specific for p160ROCK was used to detect endogenous 160KD p160ROCK which is shown in Figure 1A.

Figure 1 A, Expression of endogenous p160ROCK and p160ROCKΔ3 mutant in Caov-3 cells. Western blot analysis of p160ROCK in Caov-3 cells. p160ROCK protein was stained with anti-p160ROCK antibody in the left column. The right column shows lysates from p160ROCKΔ3 mutant cells immunoblotted with an anti-myc antibody. p160ROCK Δ3 was tagged with a MYC epitope at its N-terminus. B, C, D and E show the inhibition of ASODN to p160ROCK expression. p160ROCK immunoblotting and RT-PCR demonstrated the decrease of p160ROCK expression after treatment with the ASODN at different does (10 and 20 μ M) or 20 μ M within various period time (for 24 h, 48 h and 72 h).

Expression of p160ROCK Δ3 and Suppression of p160ROCK Protein by p160ROCK ASODN

To study the effect of changing p160ROCK activity on the phenotype of ovarian cancer cells, the dominant-active mutant of p160ROCK, p160ROCKΔ3, was transfected into Caov-3 cells. The expression of p160 ROCKΔ 3 in Caov-3 cells was detected at a significant level by Western analysis (Figure 1A). To down-regulate p160ROCK expression in the cancer cells, we transfected caov-3 cells with p160ROCK ASODN, which was designed against the translation start site (AUG codon). The expression levels of p160ROCK incubated with 20μ M ASODN for 24, 48 and 72 hours separately were analyzed by Western blot assay and RT-PCR as shown in Figure 1B and 1C. After 48 hours incubation with ASODN, p160ROCK was significantly decreased. The inhibition by ASODN was strengthened when the concentration of ASODN was increased from 10μ M to 20μ M, as shown in Figure 1D and 1E. Taken together, the ASODN against p160ROCK was able to substantially down-regulate the expression of p160ROCK in a dose-dependent manner, and the maximum suppression was seen at 48 hrs. No significant difference was seen in the expression level of p160ROCK comparing the transfectants with p160ROCK SODN and the negative control with LipofectAMIN2000 only. Therefore, we used an incubation period of 48 hr for the subsequent experiments.

Changes in Morphology and Cytoskeleton of the p160ROCKΔ 3 Transfected Cells

Cell migration is strictly regulated by reorganization of the actin cytoskeleton and focal adhesions. Previous studies of metastasis revealed that strongly and weakly invasive cells differ in terms of morphology, and that such differences might be attributable, at least in part, to the difference in motile activities and the reorganization of the actin cytoskeleton, which generates intracellular tension. Therefore, invasive cells tend to be more elongated than noninvasive cells [19]. To study how the changes in expression and activity of p160ROCK influence the reorganization of cytoskeleton, the transfected Caov-3 cells were stained for F-actin with rhodamine-phalloidin. The cell shapes of control Caov-3 cells were flat and well spread, as shown in Figure 2A, while cells transfected with p160 ROCKΔ3 showed pointed pseudopodia or polygonal cell shapes with pointed edges, in which brightly-stained, longitudinal actin bundles were formed (Figure 2B). The cells transfected with p160ROCK ASODN had shrunken cell shapes without visible stress fibers (Figure 2C).

Figure 2 Changes in actin stress fibers and morphology in Caov-3 cells by expression of p160ROCKΔ3 mutant or ASODN against p160ROCK. A, control (transfectants with p160ROCK SODN, pCAG-myc vector and LipofectAMIN2000 show similar phenotypes). B, Caov-3 cells transfected with p160ROCKΔ3 construct. Arrows indicate brightly stained, longitudinal actin bundles formed in the cells. C, Caov-3 cells transfected with ASODN against p160ROCK (20 μ M). Cells were cultured for 48 h after transfection and subsequently stained for actin with rhodamine-phalloidin. Cells were examined under fluorescence microscope with ×400 magnification. Scale bar = 20 μ m.

Effect of p160ROCKΔ 3 Mutant and p160ROCK ASODN on Cell in Vitro Invasion and Motility

Rho molecules and their effector p160ROCK are well known for their abilities to control cell polarity, membrane protrusion, and focal adhesion during cell movement by rearranging the actin cytoskeleton [20, 21, 22, 23]. Since some clinical observations suggested that Rho/ROCK pathways are associated with invasion and metastasis of cancer [24, 25, 26, 27], we sought to investigate whether the change in p160ROCK activity and its expression could affect the invasive ability and motility of ovarian cancer cells. The invasive ability of mutant and control cells was measured by the in vitro invasion assay. The number of the mutants transfected with p160ROCKΔ3 at the lower side of the filter after 10 hr was much greater than the number of control cells, 139 % of the total (P < 0.05). When compared with the control, p160ROCK ASODN transfected at a concentration of 20 μM inhibited the invasion of Caov-3 cells downward to the extent of 50%. In addition, the inhibitory effect was dose-dependent. As shown in Figure 3A, the invasive ability of the ovarian cancer cells was significantly changed by altering p160ROCK activity or inhibiting its expression. We next investigated whether the changed invasion of the cancer cells by p160ROCKΔ3 mutant and p160ROCK ASODN was related to their mobile activity. We therefore designed two transwell experiments cultured under two different conditions: First, cells were put into the upper compartment of the Boyden chamber, with NIH3T3 fibroblast-conditioned medium in the lower chamber to test directed migration or chemotaxis; second, cells were put into the upper compartment of the Boyden chamber, with the same medium in both lower and upper chambers for measuring random motility and chemokinesis. The results are shown in Figure 3B and 3C. In each experimental condition, a number of p160ROCKΔ 3-transfected cells successfully migrated through to the undersurface of the filter, and p160ROCK ASODN transfected cancer cells exhibited a dose-dependent inhibitory transwell migration effect. In the wound-closure assay, the p160ROCKΔ 3 mutant cells and the cells with ASODN against p160ROCK showed the same effect on cell migration (Figure 4).

Figure 3 Effect of expression of p160ROCKΔ3 mutant and ASODN against p160ROCK on the in vitro invasion and motility of Caov-3 cells. A, quantitation of the invasive activity of Caov-3 cells expressing p160ROCKΔ3 mutant or transfected with the vector alone, p160ROCK ASODN, SODN and their controls. The control indicates cells transfected with the LipofectAMIN2000 only. Columns indicate the mean of three studies performed in triplicate, bars indicate SD. *, P < 0.05. B, chemotaxis assay of ovarian cancer cells expressing p160ROCKΔ3 mutant and cells transfected with vectors alone, ROCK-I ASODN, SODN. Data are expressed as a percentage of controls. C, effect of p160ROCKΔ3 mutant or ROCK-I ASODN on the random motility of the cells.

Figure 4 Wound assay analysis of p160ROCKΔ3 mutant and anti-p160ROCK oligos on the migrating activity of Caov-3 cells. A, control example representing transfectants with p160ROCK SODN, pCAG-myc vector and LipofectAMIN2000. B, cells transfected with p160ROCKΔ3. C, cells transfected with ASODN against p160ROCK (20 μ M). Wounds were created with a sterile tip in 90% confluent monolayers of cells (see Materials and Methods). The monolayers were cultured for 24 h in DMEM. Movement of the cells into the scarred region resulted in a decrease in the surface area of scar. Scale bar = 300 μ m.

Effect of Changing p160ROCK Activity and Expression on Cell Adhesion

Dynamic rearrangement of cell adhesions with the substratum allows cells to carry out various functions, including cell polarity establishment, morphological change and cell migration. In order to investigate whether p160ROCK is involved in regulating cell adhesion, we examined the potential effect of p160ROCKΔ 3 mutant and p160ROCK ASODN transfection on adhesion to Matrigel in Caov-3 cells. In this experiment, the transfected cells were plated onto a Matrigel-coated 96-well plate and incubated for 30 minutes to allow cell adhesion. Subsequently, the cells were washed and the number of adherent cells was quantified by MTT assay. No obvious changes in adhesive ability were observed in Caov-3 cells transfected with either p160ROCKΔ 3 mutant or p160ROCK ASODN (Figure 5A). However, significant changes on cell migration were observed in ovarian cancer cells transfected by either p160ROCKΔ 3 mutant or p160ROCK ASODN, suggesting that changes in p160ROCK activity or expression impaired cell migration. Since cell migration involves both formation and release of adhesion complexes [28], exploring the mechanism of p160ROCK regulating focal adhesion will be further needed.

Figure 5 Adhesion and proliferation analysis of p160ROCKΔ3 mutant or ASODN against p160ROCK in Caov-3 cells. A, cell culture with p160ROCKΔ3 DNA or p160ROCK ASODN for 48 hr. Cells were first detached from dishes, suspended in the recommended medium, and then were added to matrigel-coated 96-well plates (5 × 10⁴ cells in100 μ l/well), and cultured to attach for 30 min at 37°C. Then the plates were washed three times with PBS gently and the number of remaining adherent cells was measured by MTT assay. B, Caov-3 cells were cultured for three days after being treated with p160ROCKΔ3 or p160ROCK ASODN. The cell proliferation rate was measured by MTT assay. Results are show as the value of absorbance at 570 nm. The experiments were repeated three times, and mean was calculated.

Effect of Cell Proliferation by p160ROCKΔ3 Mutant and p160ROCK ASODN

p160ROCK has been shown to contribute to reorganization of the actin cytoskeleton and this process is required for cell migration and cytokinesis. Therefore, it is possible that up-regulation of p160ROCK activity or reduction of p160ROCK protein level might either promote or inhibit proliferation. However, MTT proliferation assays showed no significant difference in the growth of the ovarian cancer cells transfected with either p160ROCKΔ3 or p160ROCK ASODN over three days when compared with their controls (Figure 5B), indicating that the change of p160ROCK activity or its expression level did not affect tumor cell proliferation. It is possible that migration of tumor cells might be more sensitive than their cytokinesis to changes in p160ROCK activity or its expression. Indeed the p160ROCK ASODN did not totally eliminate expression of p160ROCK, and maybe the activity of exogenous p160ROCK was not sufficient to increase the proliferation of cancer cells. Moreover, regulation of various pathways might exist to safeguard the essential processes of proliferation.

DISCUSSION

The high incidence of overexpression of Rho GTPases and their effectors in human tumors suggests that these proteins are important in the carcinogeneic process, and are therefore potential candidates for therapeutic intervention. In recent years, progress in characterizing downstream effectors of Rho GTPase has been made to increase our understanding of the general cellular effects that permit aberrant proliferation and motility of tumor cells. However, directly targeting the expression and activity of Rho GTPase has proven to be a difficult and unsuccessful task. The amount of information gathered so far about signaling downstream of Rho GTPase might offer some hope for successful approaches against several human cancers.

One of these downstream effectors is p160ROCK. p160ROCK binds selectively to Rho-GTP and is activated by this binding. The activated p160ROCK phosphorylates various substrates, such as MLC phosphatase (MLCP), LIM kinase (LIMK) and ERM. In addition, several proteins that are involved in regulating actin-filament assembly and contractility are phosphorylated by p160ROCK [29, 30, 31, 32, 33, 34]. Therefore, p160ROCK is an important regulator of actin cytoskeleton. In this study, up-regulating p160ROCK activity or down-regulating its expression was shown to produce a striking change in actin stress fibers, accompanied by obvious changes in cell morphology (Figure 2). After transfection with p160ROCKΔ 3, the cells showed brightly staining longitudinal actin bundles and became extended and flattened. All of these changes should facilitate cell motility. The cells treated with p160ROCK ASODN appeared more stationary with a shrunken morphology, and the actin stress fibers disappeared. Because serum can induce the activation of endogenous Rho in a variety of cells [35], these results strongly suggest that acting downstream of Rho, p160ROCK is involved in mediating the motile activity of human ovarian cancer cells.

Horiuchi et al. [25] showed that in ovarian cancer cells, the expression of both RhoA and RhoC, which are considered to regulate p160ROCK, was significantly higher in carcinomas cells than in benign tumors and significantly higher in metastatic than in primary tumor cells. These findings suggest that up-regulation of Rho GTPase plays an important role in the progression of ovarian cancer. In the present study, Matrigel invasion assay using the ovarian cancer cell line Caov-3 showed that up-regulation of p160ROCK activity was associated with the enhanced invasion of the cancer cells, while down-regulation of the expression level of p160ROCK caused a significant decrease of the invasion of these cells (Figure 3A). Therefore, both the level of p160ROCK activation and its expression were crucial for invasion of the cells, suggesting a significant association of the Rho/ROCK pathway with invasion and metastasis of ovarian cancer cells.

During metastasis, cancer cells are thought to migrate from the primary tumor mass into the surrounding tissue and also the circulatory system. Therefore, cell motility represents a critical parameter in the pathobiology of cancers. In the case of human ovarian cancer, it is likely that cancer cells depart from the surface of tumors and then adhere and invade tissues and organs in the peritoneal cavity. In this way, a poor outcome is to a large extent, because of the peritoneal dissemination caused by the aggressive migration activity of ovarian cancer cells. As p160ROCKΔ3 and p160ROCK ASODN had dramatic effects on cell invasion, we next determined their roles in cell motile activity. Our data indicated that a change in p160ROCK activity or its expression could significantly affect the motility of the ovarian cancer cells. The Boyden chamber assay clearly revealed that the p160ROCKΔ3 strongly potentiated both chemotaxis and chemokinesis of ovarian cancer cells, and p160ROCK ASODN inhibited both of these activities of the cancer cells significantly (Figure 3B and 3C). Furthermore, we found that the p160ROCKΔ3 mutant was able to significantly increase migration of the cells from the edge of the wound into the open space of the wound, while p160ROCK ASODN repressed the cell migration (Figure 4). These data indicated that exogenous p160ROCK activity and the change in endogenous p160ROCK expression level were sufficient to affect the migration of the ovarian cancer cells. These results were coincident with the changes in actin stress fibers and morphology of the cells as discussed above.

During cell migration, after attachment of the lamellipodium at the leading edge, the cell body is moved forward, and the tail of the cell detaches from the substratum by disrupting focal adhesions, which indicates that the focal adhesions are a fundamental component affecting cell motility [28]. Changes in the activities of the components regulating focal adhesions could impair cancer invasion. p160ROCK has been shown to contribute to the formation of focal adhesions of N1E-115 neuroblastoma and Hela cells [15, 36]. Our results showed that both the p160ROCKΔ3 and p160ROCK ASODN had no statistically significant effect on the adhesion of the ovarian cancer cells on Matrigel (Figure 5A). The difference in these results might be a result of differences in the cell type used or the conditions of the adhesion assay used in this study. It will be interesting to see under what conditions or at which stage of cell migration, (i.e., temporal and spatial regulation), p160ROCK is involved in adhesion. Tumor cells must complete a complex series of steps to metastasize, and the most basic step is an increase in cell proliferation. Rho GTPase affects several aspects of growth control [37]. Therefore, it is possible that p160ROCK affects tumor metastasis by regulating cell proliferation. In our study, Caov-3 cells transfected with p160ROCKΔ 3 p160ROCK ASODN were measured for in vitro proliferation. Significant differences in proliferation were not seen between the cells transfected with p160ROCKΔ3 or p160ROCK ASODN and their controls (Figure 5B). Taken together, it indicated that p160ROCK regulated invasion and metastasis by controlling cell motility rather than cell proliferation and cell adhesion. It remains to be determined whether regulation of the organization of actin by p160ROCK during cytokinesis is distinct from the role of p160ROCK in migration.

In conclusion, stimulation or inhibition of cell motility by treatment with p160ROCKΔ3 and p160ROCK ASODN, respectively, resulted in alteration of invasion the phenotype of the ovarian cancer cells. This study may provide the basis for new clinically applicable therapies to control the metastasis of ovarian cancer.

ACKNOWLEDGEMENTS

Grant supporters: National Science Foundation of China (No. 30025017, 30271358) and the “973” Program of China (No. 2002CB513100)