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Keywords:

  • geranylgeranylacetone;
  • invasion;
  • lysophosphatidic acid;
  • Rho;
  • geranylgeranylation;
  • ovarian carcinoma

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

Lysophosphatidic acid (LPA) induced a dose-dependent increase of cancer cell invasion by promoting Rho/Rho-associated kinase signaling. Prenylation of Rho is essential for regulating cell growth, motility, and invasion. Geranylgeranylacetone (GGA), an isoprenoid compound, is used clinically as an antiulcer drug. Recent findings suggested that GGA might inhibit the small GTPase activation by suppressing prenylation. The authors hypothesized that the anticancer effects of GGA result from the inhibition of Rho activation.

METHODS

The authors examined the effect of GGA using an in vitro invasion assay in human ovarian carcinoma cells, and analyzed the mechanism of the GGA effect on Rho activation, stress fiber formation and focal adhesion assembly, which are essential processes for cell invasion.

RESULTS

The induction of ovarian carcinoma cell invasion by LPA was inhibited by the addition of GGA in a dose-dependent manner. Treatment of cancer cells with GGA resulted in inactivation of Rho, changes in cell morphology, loss of stress fiber formation and focal adhesion assembly, and the suppression of tyrosine phosphorylation of focal adhesion proteins. The effect of GGA on cancer cells was partially prevented by the addition of geranylgeraniol, which is an intermediate of geranylgeranyl pyrophosphate and compensates geranylgeranylation of Rho.

CONCLUSIONS

The inhibition of LPA-induced invasion by GGA was, at least in part, derived from suppressed Rho activation by preventing geranylgeranylation. Cancer 2005. © 2005 American Cancer Society.

Ovarian carcinoma is a highly metastatic disease characterized by widespread peritoneal dissemination and ascites and is the leading cause of death from gynecologic malignancy.1 This poor outcome appears to be correlated with the peritoneal dissemination of cancer cells.1 Accordingly, one new therapeutic strategy is to clarify the mechanism of metastasis of cancer cells and to identify agents that prevent cancer cells from invading or migrating into the peritoneum. Among many growth-promoting factors known to be present in ovarian carcinoma ascites, lysophosphatidic acid (LPA) is found at significant levels (approximately 10 μM) and may play an important role in the development or progression of ovarian carcinoma.2 LPA has been reported to induce many cellular effects including mitogenesis, the secretion of proteolytic enzymes,3 and migration activity.4

We have reported that the effects of LPA act mainly through Rho/Rho-associated kinase signaling in ovarian carcinoma cells.5

Protein isoprenylation, such as geranylgeranylation and farnesylation, is a posttranslational modification that is essential for membrane localization and the full function of small GTP-binding proteins (G protein), such as Rho, Rac, and Ras.6, 7 Because isoprenylated proteins (e.g., small G proteins) play crucial roles in signal transduction, isoprenoids are of fundamental importance in the control of various cellular functions.8, 9 Recently, several isoprenyl compounds such as farnesol, geranylgeraniol (GGOH), and geranylgeranioic acid have been shown to induce apoptotic cell death10, 11 and modulate cell motility.12, 13 Accordingly, isoprenyl compounds might influence cancer cell invasion.

Geranylgeranylacetone (GGA), an isoprenoid compound, has been used orally as an antiulcer drug developed in Japan. GGA protects the gastric mucosa from various stresses without affecting gastric acid secretion.14 Conversely, GGA suppresses cell growth and induces differentiation or apoptosis by modulating small G-protein activation in leukemia cell lines.15, 16 Moreover, the chemical structure of GGA is similar to that of geranylgeranylpyrophosphate, which is in the metabolic pathway of Rho and is essential for geranylgeranylation of Rho.17 Thus, GGA has the potential to inhibit Rho, which regulates not only cell growth, but also motility and invasion, in the same way as B-Hydroxy-B-Methylglutaryl-CoA (HMG-CoA) reductase inhibitors9, 12, 18 and bisphosphonates.13, 19

Previous reports concerning the effect of GGA on cancer cells were focused mainly on cell viability and apoptosis.15, 16 The exact mechanism of GGA on cancer cell migration and invasion has not yet been examined.

In the current study, we analyzed the cellular effects of GGA. We showed that GGA markedly inhibited LPA-induced invasion of human ovarian carcinoma cells by attenuating the activation of Rho. This resulted in changes in cell morphology, loss of stress fiber formation and focal adhesion assembly, and the suppression of phosphorylation of focal adhesion proteins, which are essential processes for cell migration.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Materials

GGA was supplied by the Eisai Co. (Tokyo, Japan). Bovine serum albumin (BSA), collagen (type I), and LPA were purchased from Sigma Chemical Company (St. Louis, MO). Growth factor-reduced basement membrane proteins (Matrigel®) was purchased from BD Biosciences (Bedford, MA). Anti-focal adhesion kinase (FAK) polyclonal antibody, anti-phospho-FAK (Tyr 397) polyclonal antibody, anti-paxillin monoclonal antibody, anti-phospho-paxillin (Tyr 118) polyclonal antibody, and Rho activation assay kit were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-RhoA polyclonal antibody, horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene difluoride membrane (Hybond-P) and enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham (Arlington Heights, IL). Hoechst 33354, rhodamine-labeled phalloidin, and Alexa Fluor 488-labeled goat anti-rabbit and anti-mouse antibodies were purchased from Molecular Probes (Eugene, OR). The CellTiter 96AQ kit to monitor cell proliferation was purchased from Promega (Madison, WI). Dulbecco modified Eagle medium (DMEM) was purchased from Gibco BRL (Gaithersburg, MD).

Cell Culture

The human ovarian carcinoma cell lines, Caov-3 and SKOV-3, were purchased from American Type Culture Collection (Rockville, MD). Both cell lines were grown in DMEM, supplemented with 10% (volume/volume) fetal bovine serum and penicillin (10 U/mL)-streptomycin (10 U/mL) in 95% air and 5 % CO2 at 37 °C, and used within 15 passages after the initiation of culture.

Evaluation of Apoptosis by Fluorescence Microscopy

Cells were plated in 8-well chambers under slides coated with type I collagen and allowed to attach for 4.5 hours. The cells were then cultured under serum-free conditions (DMEM containing 0.1% (w/v) BSA) with or without 100 μM GGA for 24 hours. After incubation, cells were fixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes and permeabilized with 0.5% Triton X-100, and cell nuclei were stained with the specific chromatin dye Hoechst 33354 at 37 °C for 30 minutes. After washing, samples were observed using a Nikon photomicroscope Eclipse TE2000-u (Nikon, Tokyo, Japan).

Cell Viability Assessment

Cell viability was assessed by the MTT assay using a CellTiter 96AQ kit (Promega) according to the manufacturer's instructions. Briefly, the cells (3 × 103 per well) were plated in 96-well plates and allowed to attach for 4.5 hours, and then cultured under serum-free conditions with various concentrations of GGA for 24 hours. The number of surviving cells was determined by measuring the absorbance at 590 nm (A 590 nm) of the dissolved formazan product after addition of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt for 1 hour as described by the manufacturer. All experiments were carried out in quadruplicate, and viability was expressed as the ratio of the absorbance of GGA-treated cells to the absorbance of control cells.

In Vitro Invasion Assay

Chemotactic directional invasion was evaluated using a modified Boyden chamber (Kurabo Industries, Ltd., Tokyo, Japan). Porous filters (8-μm pores) were coated with Matrigel. Cells (4.5 × 105 per well) were plated in the upper chamber in medium containing various concentrations of GGA, LPA, and GGOH as indicated, and invasion was allowed for 24 hours. Noninvading cells were removed from the upper chamber with a cotton swab and invaded cells adherent to the underside of the filter were fixed and stained with Mayer's hematoxylin solution and counted using an ocular micrometer. All experiments were performed in triplicate, and at least five fields per filter were counted.

Immunocytochemical Study

Cells were plated on 8-well chamber slides coated with type I collagen, and allowed to attach for 4.5 hours, and then cultured under serum-free conditions with or without various concentrations of GGA and GGOH for 24 hours. After pretreatment, cells were stimulated with 25 μM LPA for 30 minutes and fixed with 3.7% paraformaldehyde in PBS for 30 minutes, permeabilized with 0.3% Triton X-100, and stained with anti-paxillin (1:500) at 4 °C overnight. After washing, samples were incubated with Alexa Fluor 488-labeled goat anti-mouse IgG (1:1000). Specimens were double stained with rhodamine-labeled phalloidin (1:200) for 1 hour at room temperature. The images were recorded and analyzed using a Zeiss confocal photomicroscope LSM510 (Zeiss, Thornwood, NY).

Western Blot Analysis

Cells (1.5 × 105 per well) were plated in 35-mm dishes and allowed to attach for 4.5 hours. The cells were then cultured in serum-free conditions with or without GGA and GGOH for 24 hours. After the treatment, cells were stimulated with 25 μM LPA for 30 minutes, washed with ice-cold PBS, and lysed in sample buffer. Equal amounts of samples were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to Hybond-P. The transferred samples were incubated with the antibody indicated in the text and then incubated with HRP-conjugated IgG. The immunoblotted proteins were visualized with ECL reagents.

Rho Pull-Down Assay

The Rho pull-down assay was performed using a Rho activation assay kit according to the manufacturer's instructions (Upstate Biotechnology). Briefly, cells (3 × 105/mL) were plated and allowed to attach for 4.5 hours and then cultured under serum-free conditions with or without various agents for 24 hours. After incubation, cells were stimulated with 25 μM LPA for 1 minute, washed twice with cold PBS, and lysed in Mg2+ lysis buffer. Cell lysates were clarified by centrifugation, and equal volumes of lysates were incubated with Rhotekin RBD-agarose beads (60 μg) at 4 °C for overnight. The beads were washed three times with wash buffer. Bound Rho proteins were detected by Western blotting using a polyclonal antibody against Rho-A. Western blotting of the total amount of Rho in cell lysates was performed for the comparison of Rho activity (level of GTP-bound Rho) in the same samples.

Statistical Analysis

Results are presented as means ± standard deviations. Data were analyzed using one-way analysis of variance followed by an unpaired Student t test for comparison between groups. Differences between groups were considered statistically significant at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Effects of GGA on Cell Morphology and Viability.

We first studied the effect of GGA on the morphology of human ovarian carcinoma cells. The typical shape of Caov-3 cells is shown in Figure 1Aa. Untreated Caov-3 cells were flat and well spread, but treatment with 100 μM GGA induced cell retraction from the substratum and loss of contacts between neighboring cells, resulting in a spindle-shaped morphology (Fig. 1Ac). These morphologic changes were reversed 24 hours after the removal of GGA. The cells treated with GGA in the presence of GGOH (10 μM) were flat, like the control cells (Fig. 2d). These results are consistent with the possibility that GGA changes the well-spread morphology of Caov-3 cells by inhibiting geranylgeranylation of Rho. To investigate whether GGA induces apoptosis in ovarian carcinoma cells, morphologic evaluation of apoptosis was performed by nuclear staining of Caov-3 cells. Apoptotic nuclei, as indicated by chromatin aggregation, marginalization, or clumping, were not seen in great amounts in GGA-treated cells (Fig. 1Ad) and the morphology and condensation of nuclei of GGA-treated cells did not differ from those of untreated cells (Fig. 1Ab). To confirm that these morphologic data were in accordance with the viability of Caov-3 cells, an MTT assay was performed. Figure 1B shows that ≤ 100 μM GGA did not affect cell viability. These results suggest that the change of morphology induced by GGA does not require apoptosis of cells in ovarian carcinoma cells.

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Figure 1. The effect of geranylgeranylacetone (GGA) on (A) Caov-3 cell morphology, (B) cell viability, and (C) lysophosphatidic Acid (LPA)-induced invasion. (A) Cells were incubated for 24 hours in Dulbecco modified Eagle medium containing 0.1% bovine serum albumin in the absence (a and b) or presence (c and d) of 100 μM of GGA. After fixation, cell nuclei were stained with the specific chromatin dye Hoechst 33354 (b and d). Scale bar = 100 μm. (B) Culturing of Caov-3 cells with ≤ 100 μM GGA had no effect on their viability. Cell viability was assessed after the treatment of the indicated concentrations of GGA for 24 hours. (C) Serum-starved Caov-3 cells were seeded onto porous filters coated with basement membrane proteins (Matrigel®). After incubation for 24 hours with various agents described in the figure to permit penetration of the filters, nonpenetrated cells were removed from the upper chamber, and migrating cells adherent to the underside of the filters were counted in a minimum of 5 high-power fields. GGOH: geranylgeraniol. Bars indicate the standard deviations. *P < 0.01 relative to LPA-stimulated cells.

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Figure 2. Lysophosphatidic acid (LPA)-induced formation of stress fibers and focal adhesions by LPA is inhibited by geranylgeranylacetone (GGA), and this inhibition is prevented by the addition of geranylgeraniol (GGOH). Caov-3 cells were seeded in collagen-coated wells and incubated in the (a) absence or (b) presence of LPA (25 μM) with the addition of (c) 100 μM GGA, (d) 100 μM GGA and 10 μM GGOH for 30 minutes, and double stained with rhodamine-labeled phalloidin (red) and anti-paxillin antibody followed by Alexa 488-labeled anti-mouse IgG (green). Fluorescence microscopy images focused near the bottom of the cells are shown. These results are representative of triplicate experiments. Scale bar = 10 μm for panels a–d.

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LPA-Induced Invasion was Inhibited by GGA, and the Inhibition was Prevented by the Addition of GGOH

The effect of GGA on the invasion capacity of ovarian carcinoma cells was assessed using an in vitro invasion assay (Fig. 1C). Previously, we reported that LPA promoted the migration of Caov-3 cells in a dose-dependent manner at concentrations of ≤ 25 μM,5 and we therefore adopted 25 μM as the concentration of LPA in the current study. GGA significantly suppressed the LPA-induced invasion in a dose-dependent manner, suggesting that inhibition of Rho geranylgeranylation plays an important role in GGA-induced suppression, in the same way as bisphosphonates.12, 18 To confirm this hypothesis, we applied GGOH (which is metabolized to geranylgeranylpyrophosphate in the cells and forms geranylgeranylated Rho) and examined the effect on GGA-induced suppression of cancer invasion. The inhibitory effect of GGA on the LPA-induced invasion was prevented partially by the addition of GGOH (Fig. 1C). The profiles of the responses to LPA and the modulation by GGA and GGOH were also the same in SKOV-3 cells (data not shown). LPA-induced invasion has been reported to be regulated by Rho-mediated activation of actomyosin contractility in fibroblasts20 and in cancer cells.5, 21 Thus, these results suggest that GGA suppresses LPA-induced invasion, at least in part, by suppressing the geranylgeranylation of Rho in ovarian carcinoma cells.

LPA-Induced Rho Activation was Inhibited by GGA

To confirm that the inhibitory effect of GGA on LPA-induced invasion is due to the inactivation of Rho, we measured the intracellular levels of the GTP-bound, active form of Rho using the pull-down assay system. Because the activation of Rho by LPA was reported to reach a peak after 1 minute,5, 22 we compared the activation of Rho after a 1-minute treatment with LPA or other agents. As shown in Figure 3, the level of the active form of Rho was elevated after the addition of LPA, and GGA inhibited the elevation induced by LPA. The addition of GGOH in the presence of GGA restored the activation of Rho. Similar changes of Rho activation under each of these conditions also were observed in SKOV-3 cells (data not shown). These results suggest that Rho activation by LPA is suppressed by GGA via the inhibition of geranylgeranylation.

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Figure 3. The activation of Rho by lysophosphatidic acid (LPA) is attenuated by geranylgeranylacetone (GGA) and restored by the addition of geranylgeraniol (GGOH) in Caov-3 cells. Cells were plated and allowed to attach for 4.5 hours, and then cultured under serum-free conditions with or without various agents described in the figure for 24 hours. After incubation, cells were stimulated with 25 μM LPA for 1 minute. After lysis, cell lysates were incubated with Rhotekin RBD-agarose beads (60 μg) at 4 °C overnight. The beads were washed, and bound Rho proteins were detected by Western blot analysis using polyclonal antibody against RhoA (top panel). Western blot analysis of the total amount of Rho in cell lysates (bottom panel) was performed for the comparison of Rho activity (level of GTP-bound Rho) in the same lysates. Blots are representative of four experiments. The concentrations of agents that were used in this figure are as follows: LPA, 25 μM; GGA, 100 μM; GGOH, 10 μM.

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LPA-Induced Formation of Stress Fibers and Focal Adhesions were Inhibited by GGA, and this Inhibition was Prevented by the Addition of GGOH

Cell migration begins with an initial protrusion or extension of the plasma membrane at the leading edge of the cell. The protrusion is driven by the polymerization of a network of cytoskeletal actin filaments and is stabilized through the formation of adhesive complexes.4 To investigate the mechanism of the inhibitory effect of GGA on LPA-induced invasion, actin stress fibers and paxillin, one of the major focal adhesion proteins, were visualized (Fig. 2). LPA treatment caused a drastic increase of actin bundles and changed the localization of paxillin to the edge of the actin bundles (Fig. 2b). Treatment with GGA significantly decreased the LPA-induced formation of stress fibers and focal adhesions (Fig. 2c). The inhibitory effect of GGA on the formation of stress fibers and focal adhesions was almost prevented by the addition of GGOH (Fig. 2d). These results suggest that in ovarian carcinoma cells, GGA inhibits the formation of stress fibers and focal adhesions by suppressing the prenylation of Rho.

Inhibitory effect of GGA on LPA-stimulated Tyrosine Phosphorylation of Paxillin and FAK

Cell migration is regulated by a combination of different processes, such as the contraction of actomyosin, the formation of stress fibers, and the turnover of focal adhesions.4 Especially, tyrosine phosphorylation of focal adhesion proteins such as paxillin or FAK is an essential process in LPA-induced cell migration.23 Therefore, we analyzed the effect of GGA on the phosphorylation of these proteins by Western blotting (Fig. 4). We assessed the time course of the tyrosine phosphorylation of focal adhesion proteins induced by LPA. Those experiments indicated that tyrosine phosphorylation of focal adhesion proteins reached a peak after 30 minutes of treatment.5 Therefore, we compared the phosphorylation of these proteins after treatment with LPA for 30 minutes. GGA inhibited the tyrosine phosphorylation of paxillin and FAK (Fig. 4). These inhibitory effects were prevented partially by the addition of GGOH. The results of Western blotting showed that GGA inhibited the Rho-mediated activation of tyrosine phosphorylation of focal adhesion proteins, at least in part, by suppressing the geranylgeranylation of Rho.

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Figure 4. Western blot analysis of the effect of geranylgeranylacetone (GGA) on lysophosphatidic acid (LPA)-stimulated tyrosine phosphorylation of paxillin and focal adhesion kinase (FAK). Caov-3 cells were plated and allowed to attach for 4.5 hours, and then cultured under serum-free conditions with or without various agents described in the figure for 24 hours. After incubation, cells were stimulated with 25 μM LPA for the indicated times described in the text. After cell lysis, lysates were resolved by 9% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotted with anti-phosphospecific paxillin or anti-phosphospecific FAK antibody. To demonstrate specificity and equal loading, the membranes were stripped and reprobed with anti-paxillin or anti-FAK antibody. Blots are representative of three experiments.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The mechanism of GGA occurs through the induction of heat shock protein (HSP) and nitric oxide synthase in gastric mucosa24 and other effects have been revealed, e.g., myocardial protection against ischemia/reperfusion injury25 and antivial effect in influenza virus infection.26 We have shown that alendronate, which inhibits osteoclast activity, also inhibits LPA-induced cancer cell invasion by preventing the geranylgeranylation of Rho.13 Thus, geranylgeranylation of small G proteins is essential not only in osteoclast activity, but also in cancer cell invasion. Vitamin K2 (menatetrenone) is also used as an antiosteoporosis drug. It is suggested that the inhibitory effect of menatetrenone on osteoclasts is not due to γ-carboxylation and that the side chain component of menatetrenone plays an important role in this inhibitory effect.27 Furthermore, Hiruma et al.17 reported that GGOH, a side-chain component of menatetrenone at the 3-position of naphthoquinone, inhibited osteoclast formation to the same degree as menatetrenone. GGA, known as teprenone, has almost the same chemical structure as that of the side chain of menatetrenone. We hypothesized that GGA also might attenuate the geranylgeranylation of small G proteins and have an inhibitory effect on cancer cell invasion. This mechanism already has been suggested in noncancerous cells.28, 29

In the current study, GGA markedly inhibited LPA-induced invasion of human ovarian carcinoma cells by attenuating the activation of Rho. This resulted in changes in cell morphology, loss of stress fiber formation and focal adhesion assembly, and the suppression of tyrosine phosphorylation of focal adhesion proteins.

A significant inhibitory effect of GGA on LPA-induced Caov-3 cell invasion was observed at a concentration of 1 μM, and half-maximal inhibition was estimated to occur at approximately 3 μM (Fig. 1C). The in vitro effective concentrations of GGA vary from approximately 1 μM15, 30 to approximately 100 μM31, 32 as reported in previous studies. The concentration of GGA in our study is comparable with these previous reports, although the most effective concentration is rather high compared with the concentration in these reports. Furthermore, a pharmacokinetic study in humans revealed that the serum concentration of GGA after oral administration of the usual dosage is 1–10 μM.33 The actual concentration of GGA in cancer tissue samples when GGA is administered to cancer-bearing animals must be examined.

GGOH restored GGA-induced inhibition of Rho activation and FAK phosphorylation. However, GGOH partially compensated the GGA-induced inhibition of cancer cell invasion. Furthermore, GGA-induced inhibition of paxillin phosphorylation and stress fiber formation were restored partially by GGOH. Partial compensation of inhibited phosphorylation of focal adhesion proteins and stress fiber formation by GGOH is parallel to the effect of GGOH on GGA-induced suppressed invasion. Thus, the inhibitory mechanism of LPA-induced cancer cell invasion by GGA is not only through the inhibition of Rho geranylgeranylation, but also through another unknown mechanism. One possibility is that GGA inhibits the activation of other small GTP proteins (such as Ras). Actually, GGA also inhibited LPA-induced Ras activation in Caov-3 cells (data not shown). Okada et al.15 reported that GGA treatment resulted in the inhibition of processing of small GTP-binding proteins like Rap1 and Ras in human leukemia cells, leading to apoptosis. Longer exposure to GGA might induce apoptosis and inhibit the growth of ovarian carcinoma cells.

The advantages of GGA are that it is already in clinical use and has a proven safety profile. Laboratory data revealed no detrimental events, even at the massive dosage of 500 mg/kg of body weight.26 Whereas GGA may have therapeutic potential in the treatment of ovarian carcinoma, in vivo studies are needed to confirm its efficacy especially in the peritoneal dissemination model.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES