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FTY720, a fungus metabolite, inhibits invasion ability of androgen-independent prostate cancer cells through inactivation of RhoA-GTPase
Cancer Letters 233 (2006) 36–47 www.elsevier.com/locate/canlet
FTY720, a fungus metabolite, inhibits invasion ability of androgen-independent prostate cancer cells through inactivation of RhoA-GTPase* Chun Zhoua, Ming-Tat Linga, Terence Kin-Wah Leeb, Kwan Manb, Xianghong Wanga,*, Yong-Chuan Wonga,* a
Cancer Biology Group, Department of Anatomy, Laboratory Block, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, SAR, China b Cancer Biology Group, Department of Surgery, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, SAR, China Received 19 January 2005; received in revised form 20 February 2005; accepted 25 February 2005
Abstract The failure of controlling androgen-independent and metastatic prostate cancer growth is the main cause of death in prostate cancer patients. In this study, we have demonstrated evidence on the inhibitory effects of a fungus metabolite, FTY720, on the clonogenesity as well as invasion ability of androgen-independent prostate cancer cells. First, using colony forming assay, we found that FTY720 treatment led to decreased colony forming ability of androgen-independent prostate cancer cell lines DU145 and PC3, indicating its negative role on cancer cell survival. In addition, treatment with relatively low dose of FTY720 (i.e. inhibitory concentration of 50% cell survival) resulted in suppression of prostate cancer cell migration and invasion abilities demonstrated by Wound closure, 3D collagen gel invasion assays and stress fiber staining. Furthermore, we found that the inhibitory effect of FTY720 on prostate cancer invasion was associated with down-regulation of GTP-bound active form of RhoA. Transfection of a dominant-active RhoA vector in DU145 and PC3 cells conferred resistance to FTY720. Since activation of RhoA-GTPase is associated with metastasis in many types of malignancies, our results not only suggest a new agent for the treatment of advanced prostate cancer, but also implicate a possible novel anticancer drug especially against metastatic cancers. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: FTY720; Prostate cancer; Invasion; RhoA
1. Introduction *
This work was supported by RGC grants to Y. C. Wong (HKU7490/03M) and to X. Wang (HKU7478/03M). * Corresponding authors. Tel.: C86 852 2819 9226; fax: C86 852 2817 0857. E-mail addresses: [email protected] (X. Wang), [email protected] (Y.-C. Wong).
Prostate cancer is one of the most frequently diagnosed cancers in the Western countries. In America, it is the most commonly diagnosed cancer in men representing one-third of all new cancer cases
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.02.039
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each year [1]. The incidence of prostate cancer is also increasing in Asia including in Hong Kong in recent years [2]. Currently, androgen-depletion therapy is the predominant treatment which is effective for androgen-dependent prostate cancer. However, after a period of 1–3 years most tumors reoccur and eventually develop into androgen-independent growth [3]. It is estimated that over 50% of prostate cancer patients are eventually refractory to hormonal therapy [4]. Unfortunately, at this stage, androgen-depletion therapy is no longer effective. Therefore, development of alternative treatment strategies is crucial for improving the survival of androgen-independent prostate cancer. Unlike its effect on majority of human cancers, chemotherapy has not shown satisfactory results for prostate cancer, probably due to its relatively slow growth rate of the prostate cancer cells or the lower tolerance of elderly patients. Recently, alternative approaches have been reported and shown promising results in the treatment of hormone refractory prostate cancer. For example, administration of non-steroidal anti-inflammatory drugs (NSAID) as well as several antioxidants such as vitamin E in prostate cancer patients have shown effect in controlling prostate cancer growth in clinical trials [5,6]. In addition, the a1-adrenoceptor antagonist terazosin is found to suppress androgen-independent prostate cancer cell growth through induction of apoptosis [7]. The great advantage of these agents is that they are not designed to target fast growing cells and have been widely used clinically. Recently, one of these agents, FTY720, a derivative of fungus Isaria sinclairii [8], has been shown a dramatic anticancer effect in both in vitro and in vivo studies [9,10]. FTY720 was originally identified as an immunosuppressant. It has been shown to prevent rejection of transplanted grafts in organ transplantation patients by directing lymphocytes away from peripheral bloodstream [11–14]. In some cases, it selectively induces apoptosis of mature T-lymphocytes through downregulation of Bcl-2 expression [15]. Administration of FTY720 in these patients is not associated with renal, gastrointestinal or bone marrow toxicity [16,17] and as a result, it is currently undergoing Phase II clinical trials for treatment of transplantation patients [15]. Recently, FTY720 was reported to induce apoptosis in several types of cancer cells such as bladder, breast
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and glioma cancer cells [9,10,18]. In addition, two recent in vivo studies showed that FTY720 prevented tumor growth and metastasis of breast and bladder xenografts in nude mice without causing detectable toxicity in vital organs [9,10]. These results raised a hypothesis that FTY720 may be a potential anticancer drug. However, the underlying mechanisms responsible for its action are not clear. In prostate cancer, treatment of FTY720 on androgen-independent DU145 cells reduced cell viability and induced apoptosis [19], indicating that it may be an effective agent for suppression of androgen-independent prostate cancer growth. In this study, we investigated if FTY720 was able to suppress invasion ability of androgen-independent prostate cancer cells and studied the molecular mechanisms responsible. To achieve this, we first treated two androgen-independent prostate cancer cell lines, DU145 and PC3, with different doses of FTY720 and tested its effect on colony formation, migration and invasion abilities. Since Rho-GTPase family proteins play positive roles in cancer cell motility and invasion [20–22], we then studied if RhoA-GTPase was involved in the FTY720-induced suppression of prostate cancer invasion ability. Our results showed that FTY720 treatment in prostate cancer cells led to suppression of tumor cell invasion and motility through inactivation of RhoA-GTPase pathway, indicating its potential role as an effective agent in the treatment for metastatic prostate cancer.
2. Materials and methods 2.1. Cell lines and cell culture conditions Two androgen-independent human prostate cancer cell lines DU145 and PC3, obtained from American Type Culture Collection (Rockville, MD, USA), were maintained in RPMI1640 medium (Sigma, MO, USA) supplemented with 5% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37 8C, 5% CO2. 2.2. Colony forming assay Eighty percent confluent cells were trypsinized and single-cell suspensions were obtained. Two hundred
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viable cells were seeded per well in 12-well plates. Twenty-four hours later cells were treated with five different doses of FTY720. Two wells were used for each drug concentration and two wells treated with solvent only were set up as controls. Ten days later, cells were fixed with 70% ethanol and stained with 10% (v/v) Giemsa (MERCK, Damstadit, Germany). Colonies consisting of more than 50 cells were scored. The percentage of colony formed after FTY720 treatment was calculated as the ratio between the number of colonies in the treated wells and the untreated controls multiplied by 100. Cell survival curves were plotted and the inhibitory concentrations of 10, 50 and 90% cell survival (IC10, IC50 and IC90) were determined. 2.3. Wound closure assay Cells were plated in six-well-plates. When the cells grew into full confluency, a wound was induced on the monolayer cells by scraping a gap using a micropipette tip and then FTY720 was added immediately after wound induction. The speed of wound closure was compared between FTY720 treated and untreated cells. Photographs were taken under 100! magnifications using phase-contrast microscopy immediately after wound incision and at later time points. 2.4. 3D collagen colony forming assay Two-hundred microliters of cell suspension (3!104 cells/ml) was mixed with 200 ml of cold rat tail collagen, type I (3.60 mg/ml, BD Biosciences, MA, USA). The mixed cell solution in collagen was plated as droplets in 60 mm Petri-dish and air dried at room temperature. RPMI1640 medium with 3% FCS with or without FTY720 was added in each dish containing the semisolid collagen-cell droplets. FTY720 treated and control samples were incubated at 37 8C for 3–5 days. Cell morphology was observed under phase-contrast microscopy and pictures were captured under 200! magnifications. Total of 500 colonies were counted in each experiment and percentage of colonies that consisted of elongated morphology was calculated. Each experiment was repeated at least three times and error bars indicate SD.
2.5. Stress fiber assay Thirty-five microliters of cell suspension (2!105 cells/ml) was seeded per well in eight-well chamber slides (ICN Biomedicals, OH, USA) and incubated over night. Cells were then treated with FTY720 and incubated for 24 h. Cells were incubated with serum free RMPI1640 medium for further 24 h. Two wells were set up for each treatment and the untreated control. At the end of incubation, each well was treated with 30 ml of lysophosphatidic acid (100 ng/ml, SIGMA, MO, USA) and incubated for 10 min at 37 8C. Cells were then washed with 1!PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with 1!PBS, cells were permeabilized in 1% Trion X-100 for 15 min at room temperature and blocked in 1% BSA (in 1!PBS) for 30 min. Cells were incubated with fluorescein phalloidin (Molecular Probes, Eugen, Oregon, USA) in 1% BSA (dilution factor, 1:50) at 37 8C for 1 h. After incubation, cells were washed with 1!PBS and mounted. Stress fiber was visualized under confocal microscopy and photographs were taken under 600! magnifications. 2.6. RhoA-GTP pull down assay Cells were plated in T-75 flasks and grown to 70–80% confluence. After serum free treatment for 24 h, cells were exposed to FTY720 for different time periods and then the cells were lysed. GTP-RhoA protein was pulled down using Rho Activation Assay Biochem Kit, BK036 (Cytoskeleton, CO, USA). The GTP-RhoA protein and total RhoA protein were assayed by Western-blot, using an antibody against RhoA provided in the Rho Activation Assay Kit (Cytoskeleton, CO, USA). Detailed Western-blot procedures have been described in our previous publications [23]. 2.7. Transfection Plasmids expressing constitutively active RhoA [pcDNA remobilized with enhanced GFP (EGFP)RhoA Q63L] have been described [24] and were kindly provided by Professor G. Bokoch (The Scripps Research Institute, La Jolla, CA). Cells were transfected using FuGENE 6 transfection reagent
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inhibiting colony forming ability of prostate cancer cells, which is the main target of chemotherapy.
(Roche, IN, USA) according to the manufacturer’s instructions. The empty pcDNA vector was transfected to cells as a control. G418 (550 mg/ml for DU145, 600 mg/ml for PC3) was added 48 h after transfection. After 10 days selection, a pool of transfectant clones was obtained.
3.2. Effect of FTY720 on prostate cancer migration Increased migration rate is one of the characteristics of metastatic cancer cells [25,26]. To study if FTY720 had any effect on migration ability of DU145 and PC3 cells, we performed Wound closure assay. As shown in Fig. 2A, similar sized wounds were introduced in monolayer DU145 cells at 0 h. In the control cells, the gap of the wound was filled gradually by migrating cells and 32 h after wound induction, the gap was almost closed (solid arrow). In contrast, after exposure to FTY720 (IC50Z1.5 mM), the speed of wound closure was much slower and the wound was still widely open at 32 h postexposure time (dotted arrow). Similar results were observed in PC3 cells except the shorter experimental time points (Fig. 2B). As the speed of wound closure reflects the migration ability of cancer cells, these results indicate that FTY720 treatment is able to inhibit cell migration. This effect of FTY720 was not due to inhibition of proliferation as we did not observe any difference in proliferation rates between the treated and untreated cells by BrdU staining (data not shown).
3. Results 3.1. Effect of FTY720 on colony forming ability of prostate cancer To study the effect of FTY720 on prostate cancer cell survival, we performed colony forming assay on two androgen-independent metastatic prostate cancer cell lines DU145 and PC3. As shown in Fig. 1A, FTY720 was able to inhibit clonogenesity of both cell lines. These two cell lines showed similar sensitivity to FTY720 evidenced by the similarity in their inhibitory concentrations of 10, 50 and 90% of cell survival (IC10, IC50 and IC90, Fig. 1B). We also noted that the FTY720 concentrations required for inhibiting colony forming ability in this study were much lower (i.e. IC50Z1.5 mM) than previously reported (i.e. 25 mM) for inhibiting similar percentage of cell viability on these two cell lines [19]. These results indicate that FTY720 is much more effective in
Colony Forming Ability (% of untreated control)
A
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B
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Inhibition concentrations (IC) of FTY720 on prostate cancer cells
DU145 PC3
DU145
PC3
IC10 (µM)
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IC50 (µM)
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IC90 (µM)
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10
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0.1 0
1
2
3
4
5
Note: IC10,50 and 90 represent inhibition concentration of 10,50 or 90% cell survival respectively.
FTY720 concentration (µM) Fig. 1. Effect of FTY720 on clonogenesity of androgen-independent prostate cancer cells. Colony forming assays were performed on DU145 and PC3 cells after continuous exposure to FTY720. (A) Survival curves of DU145 and PC3 after FTY720 treatment. (B) Summary of inhibitory concentrations (IC) of FTY720 on DU145 and PC3 cells. Note that both cell lines showed similar sensitivity to FTY720.
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A
DU145 0h
8h
24h
32h
0h
4h
8h
12h
Untreated control
FTY720 (IC50)
B
PC3
Untreated control
FTY720 (IC50)
Fig. 2. Effect of FTY720 on the migration ability of androgen-independent prostate cancer cells. Similar sized wounds were introduced in confluent monolayer cells and IC50 concentration of FTY720 was added. The speed of wound closure was monitored at different time points. Note that the speed of wound closure was suppressed by FTY720 in DU145 (A) and PC3 (B) cells in a time dependent manner. Significant difference in the speed of wound closure between treated (dotted arrows) and untreated (solid arrows) cells are demonstrated at 32 (DU145) and 12 h (PC3) postexposure, respectively.
3.3. Effect of FTY720 on invasion ability in 3D collagen gel Another characteristic of metastatic cancer cells is their ability to invade into extracellular-matrix [27,28]. To study whether FTY720 could inhibit the invasion ability of DU145 and PC3, we performed 3D collagen gel assay. As shown in Fig. 3, the untreated cells grew extensively inside the semi-solid collagen gel and displayed an elongated morphology as early as 2 days after plating. After 5 days, high percentage of DU145
(56.2G15%) and PC3 (69.3G6%) cells formed elongated colonies, indicating their ability to invade into extracellular-matrix. In contrast, after FTY720 treatment, the percentage of colonies showing elongated morphology was reduced to 4.4G6% for DU145 and 9.3G5% for PC3 cells. Majority of the cells lost their invasive ability and remained round morphology in the collagen gel. These results suggest that FTY720 could effectively suppress prostate cancer cell’s ability to invade in collagen gel, therefore, indicating its inhibitory effect on cell invasion.
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A
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DU145 Day 3
Day 5 % of colonies showing elongated structure
Day 2
Untreated control
FTY720 (IC50)
80 Control
60
FTY720
40 20 0 Untreated control
PC3 Day 2
Day 3
Untreated control
FTY720 (IC50)
Day 5 % of colonies showing elongated structure
B
FTY720 (IC50)
80 Control
60
FTY720
40 20 0 Untreated control
FTY720 (IC50)
Fig. 3. Effect of FTY720 on the invasion ability of androgen-independent prostate cancer cells. Single prostate cancer cells were plated in semisolid collagen gel and FTY720 was then added in culture medium. The invasive ability was studied by monitoring the morphology of colonies. Note that FTY720 treatment suppressed the invasive growth in collagen gel.
3.4. Effect of FTY720 on cytoskeleton reorganization Cell motility is dependent on rearrangements of actin cytoskeleton in invasive cells to facilitate metastasis [29]. Since the actin cytoskeleton is composed of actin filaments, we used fluorescent phalloidin to detect alterations of actin structure of these cancer cells before and after FTY720 treatment. In the untreated DU145 and PC3 cells, bundles of actin filaments were well organized evidenced by the strong staining of stress fiber formation, intact network and protruding morphology (Fig. 4, panels A and C). However, after exposure to FTY720, a remarkable cytoskeletal changes with reduction of actin stress fiber network, relatively round morphology, and lack of filopodia were present in both cell lines (Fig. 4, panels B and D). These results demonstrate that FTY720 treatment led to severe
damage of cytoskeleton network, further suggesting its negative role on cancer cell invasion. 3.5. Inactivation of RhoA-GTPase by FTY720 in prostate cancer cells RhoA is one of the members of Rho-GTPase proteins which are involved in cell motility and actin cytoskeleton reorganization [30]. Like the other members of this protein family, RhoA protein switches between the active GTP-bound form and inactive GDP-bound form [31]. Activation of RhoAGTPase has been reported to associate with invasive cancers and it has been suggested to be one of the molecular mechanisms responsible for tumor metastasis [32,33]. In order to study whether the inhibitory effect of FTY720 on prostate cancer cell migration and invasion was mediated through the inactivation of
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Untreated control A
FTY720 treated B
generated. Western-blot assay showed that in addition to the endogenous RhoA protein, the EGFP-bound high molecular weight RhoA was detected in the EGFP-RhoA Q63L transfected cells (Fig. 6).
DU145 A DU145 0
C
2
4
8
Time (h)
D RhoA-GTP
PC3 Total RhoA
B PC3 Fig. 4. Effect of FTY720 on stress fiber formation. FTY720 (IC50, 24 h) treated and untreated cells were stained using FITC-labeled phalloidin and the morphology of stress fibers was examined. Photos were taken under 600! magnifications. Note that FTY720 treatment destroyed the actin network and reduced the protrusion ability of prostate cancer cells.
Rho-GTPase pathway, we examined the expression level of active form RhoA-GTPase under the effect of FTY720 (IC50Z1.5 mM) with different treatment periods by Western-blot. We found that the active form GTP-bound RhoA was down-regulated after exposure to FTY720 in a time dependent manner in both DU145 and PC3 cells (Fig. 5). In contrast, the expression level of total RhoA did not show any detectable changes. These results demonstrate that the concentration of FTY720 (IC50) that was effective in suppressing migration and invasion was also able to reduce the level of GTP-bound RhoA, suggesting that the inhibitory effect of FTY720 on metastatic ability of prostate cancer cells may be regulated through inactivation of RhoA-GTPase pathway.
0
2
4
8
Time (h) RhoA-GTP Total RhoA
Fig. 5. Effect of FTY720 on RhoA-GTPase. GTP-pull down assay was performed on the FTY720 (IC50) treated and untreated cells at different time points and the levels of GTP-bound RhoA proteins were measured. Total RhoA was measured using Western-blot as an internal control. Note that FTY720 down-regulated active form of RhoA in a time dependent manner.
3.6. Role of constitutively active RhoA on the inhibitory effect of FTY720 on prostate cancer cell invasion To further confirm the involvement of RhoAGTPase pathway in the inhibitory effect of FTY720 on metastatic ability of prostate cancer cells, we transfected DU145 and PC3 cells with p-EGFP-RhoA Q63L, an expression plasmid containing the constitutively active RhoA [24]. After G418 selection for 10 days, a pool of more than 50 transfectant clones was
Fig. 6. Generation of transfectants expressing constitutively active RhoA-GTPase. DU145 and PC3 cells were transfected with pcDNA3-EGFP-RhoA Q63L vector and then selected in G418 for 10 days. Western-blot analysis was carried out on cells transfected with pcDNA and pcDNA3-EGFP-RhoA Q63L vectors, respectively. Note that in addition to the endogenous RhoA protein, the RhoA Q63L transfected cells showed a higher molecular weight EGFP-bound RhoA, but it was absent in the vector transfected cells.
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DU145 Untreated DU145
FTY720 (IC50) DU145
DU145-pcDNA
DU145-RhoA-Q63L
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12h
32h
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PC3 Untreated PC3
FTY720 (IC50) PC3
PC3-pcDNA
PC3-RhoA-Q63L
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12h
Fig. 7. Constitutively active RhoA expression leads to resistance to FTY720-induced suppression in cell migration. Similar sized wounds were introduced on confluent monolayer cells and the speed of wound closure was monitored. Note that transfection of pEGFP-RhoA Q63L confers resistance to the inhibitory effect of FTY720 on cell migration (dotted arrows) compared to the parental and vector controls (solid arrows).
In contrast, the corresponding band was absent in the control vector pcDNA3 transfected cells. We then studied if ectopic expression of constitutively active RhoA had any effect on the function of
FTY720. As shown in Fig. 7, FTY720 treatment was able to prevent cell migration in the vector control, however, the dominant active RhoA transfected cells showed similar migration rate as the untreated control
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vector cells was destroyed after FTY720 treatment (Fig. 9). In contrast, well organized actin filaments were clearly stained in cells expressing constitutively active RhoA. In addition, there was no obvious difference in actin structures and morphology in constitutively active RhoA transfected cells before and after FTY720 treatment. These results further indicate that over-expression of active RhoA confers prostate cancer cells resistance to the FTY720induced destruction of stress fiber network.
4. Discussion
Fig. 8. Constitutively active RhoA confers resistance to FTY720induced suppression in invasion. Cells were plated in semi-solid collagen gel as described in Fig. 3. The percentage of elongated colonies was counted in the pEGFP-RhoA Q63L and the controls. Note that over-expression of RhoA Q63L in DU145 and PC3 cells reversed the effect of FTY720 on cell invasion.
(dotted arrows) in both DU145 and PC3 cells. In addition, the dominant active RhoA transfected cells showed increased invasion ability in 3D collagen gel compared to the vector control after FTY720 treatment, evidenced by the fact that the percentage of elongated colonies in cells expressing constitutively active RhoA was similar to the untreated control (47.7G3 versus 44.9G11%, Fig. 8A). Similar results were observed in PC3 transfectants (Fig. 8B). These results indicate that cells transfected with constitutively active RhoA are resistant to inhibitory effect of FTY720 on migration and invasion. Furthermore, as previously observed in the parental cells, the actin stress fiber formation in parental cells and control
The evidence presented in this study have demonstrated that relatively low dose of FTY720 is able to suppress colony forming (Fig. 1), migration (Fig. 2) and invasion abilities (Figs. 3 and 4) of androgenindependent prostate cancer cells. In combination with previous results that much higher concentrations of FTY720 (i.e. 25 mM) did not induce detectable biological effects on non-malignant fibroblast cells [19] and in transplant patients [34], our results suggest a potential novel agent in the treatment of androgenindependent metastatic prostate cancer with minimal toxicity on normal cells. In addition, the results that the suppression of metastatic ability by FTY720 was mediated through inactivation of RhoA-GTPase provide an insight into molecular mechanisms responsible for its action. Since activation of GTPases is frequently found in many types of metastatic cancers [22,35], our results also indicate that FTY720 may be a potential new agent in the treatment of advanced cancers. Once prostate cancer progresses to androgenindependence, the prognosis is very poor. This is because the commonly used androgen-depletion therapy is no longer efficient. In this study, we have demonstrated that FTY720 is able to inhibit colony forming ability of androgen-independent prostate cancer cells (Fig. 1). In addition, the FTY720 doses required for suppression of colony forming ability in this study were much lower (up to 13-fold lower, Fig. 1B) than previously reported on the same cell lines for its effect on short-term cell viability [19,36]. These results suggest that FTY720 is more effective on clonogenesity, which is the target of anticancer therapy. Previously, FTY720 was also reported to
C. Zhou et al. / Cancer Letters 233 (2006) 36–47
DU145
DU145-pcDNA
45
DU145-RhoA-Q63L
A
B
C
D
E
F
Untreated control
FTY720 (IC50)
Fig. 9. Effect of constitutively active RhoA expression on FTY720-induced cytoskeleton reorganization. The parental, pCDNA3 and pEGFPRhoA Q63L transfected cells were treated with FTY720 as described in the legend of Fig. 4. Note that the cells expressing constitutively active RhoA protein are resistant to FTY720-induced stress fiber disruption.
inhibit tumor growth in two recent in vivo studies on breast and bladder cancer xenografts in nude mice with minimal toxicity on normal organs [9,10]. In combination with these results, our results suggest that FTY720 may be a new promising anticancer agent for the treatment of cancers that show poor response to conventional chemotherapy. Once prostate cancer has developed metastatic growth, the survival rate is extremely low [4,37]. This is because to date very few agents have shown effective results on suppression of metastatic ability of prostate cancer cells. In this study, our results demonstrated that after exposure to a relatively low dose (IC50) of FTY720, the migration rate of DU145 and PC3 cells was significantly inhibited (Fig. 2), which indicates the suppression of tumor cell movement by FTY720. In addition, the tumor cell’s ability to invade into extracellular-matrix was also reduced after FTY720 treatment (Fig. 3). Furthermore, the cytoskeleton reorganization required for cell motility such as stress fiber formation was also significantly inhibited after exposure to FTY720 (Fig. 4). These results demonstrate that FTY720 is effective in suppressing the metastatic ability of androgen-independent prostate cancer cells. In addition, the antimetastatic effect was regulated through inactivation of
RhoA-GTPase as we found that after FTY720 treatment, the RhoA-GTPase was significantly decreased with increased exposure time (Fig. 5). Expression of constitutively active RhoA-GTPase, on the other hand, led to resistance to the inhibitory effect of FTY720 on cell migration, invasion and motility (Figs. 6–9). Recently, several studies also reported that FTY720 was able to suppress cancer invasion to other tissues and organs in nude mice bearing metastatic breast and bladder cancer xenografts [9, 10]. In this study, our evidence has provided a biological and mechanistic basis for the inhibitory role of FTY720 on cancer cell invasion. Since activation of RhoA-GTPases has been found in many metastatic cancers [20,22,35] and shown to promote metastasis in prostate cancer [38], our results in combination with previous in vivo studies [9,10,19] suggest that in addition to androgen-independent metastatic prostate cancer, FTY720 may also be an effective agent in the treatment of other metastatic human cancers. However, further investigations are required to confirm this hypothesis. In summary, we have demonstrated that FTY720, a fungus metabolite, is able to suppress both clonogenesity and metastatic ability of androgen-independent prostate cancer cells. In addition, we found that
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the function of FTY720 may be mediated through down-regulation of the RhoA-GTPase pathway. As currently FTY720 is being used clinically in transplant patients with minimal side effects, it could be served as an alternative strategy in the treatment of androgen-independent metastatic prostate cancer patients who are resistant to conventional therapy. Currently, we are investigating if the anti-metastasis role of FTY720 can be observed in in vivo systems.
[12]
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FTY720, a fungus metabolite, inhibits invasion ability of androgen-independent prostate cancer cells through inactivation of RhoA-GTPase* Chun Zhoua, Ming-Tat Linga, Terence Kin-Wah Leeb, Kwan Manb, Xianghong Wanga,*, Yong-Chuan Wonga,* a
Cancer Biology Group, Department of Anatomy, Laboratory Block, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, SAR, China b Cancer Biology Group, Department of Surgery, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, SAR, China Received 19 January 2005; received in revised form 20 February 2005; accepted 25 February 2005
Abstract The failure of controlling androgen-independent and metastatic prostate cancer growth is the main cause of death in prostate cancer patients. In this study, we have demonstrated evidence on the inhibitory effects of a fungus metabolite, FTY720, on the clonogenesity as well as invasion ability of androgen-independent prostate cancer cells. First, using colony forming assay, we found that FTY720 treatment led to decreased colony forming ability of androgen-independent prostate cancer cell lines DU145 and PC3, indicating its negative role on cancer cell survival. In addition, treatment with relatively low dose of FTY720 (i.e. inhibitory concentration of 50% cell survival) resulted in suppression of prostate cancer cell migration and invasion abilities demonstrated by Wound closure, 3D collagen gel invasion assays and stress fiber staining. Furthermore, we found that the inhibitory effect of FTY720 on prostate cancer invasion was associated with down-regulation of GTP-bound active form of RhoA. Transfection of a dominant-active RhoA vector in DU145 and PC3 cells conferred resistance to FTY720. Since activation of RhoA-GTPase is associated with metastasis in many types of malignancies, our results not only suggest a new agent for the treatment of advanced prostate cancer, but also implicate a possible novel anticancer drug especially against metastatic cancers. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: FTY720; Prostate cancer; Invasion; RhoA
1. Introduction *
This work was supported by RGC grants to Y. C. Wong (HKU7490/03M) and to X. Wang (HKU7478/03M). * Corresponding authors. Tel.: C86 852 2819 9226; fax: C86 852 2817 0857. E-mail addresses: [email protected] (X. Wang), [email protected] (Y.-C. Wong).
Prostate cancer is one of the most frequently diagnosed cancers in the Western countries. In America, it is the most commonly diagnosed cancer in men representing one-third of all new cancer cases
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.02.039
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each year [1]. The incidence of prostate cancer is also increasing in Asia including in Hong Kong in recent years [2]. Currently, androgen-depletion therapy is the predominant treatment which is effective for androgen-dependent prostate cancer. However, after a period of 1–3 years most tumors reoccur and eventually develop into androgen-independent growth [3]. It is estimated that over 50% of prostate cancer patients are eventually refractory to hormonal therapy [4]. Unfortunately, at this stage, androgen-depletion therapy is no longer effective. Therefore, development of alternative treatment strategies is crucial for improving the survival of androgen-independent prostate cancer. Unlike its effect on majority of human cancers, chemotherapy has not shown satisfactory results for prostate cancer, probably due to its relatively slow growth rate of the prostate cancer cells or the lower tolerance of elderly patients. Recently, alternative approaches have been reported and shown promising results in the treatment of hormone refractory prostate cancer. For example, administration of non-steroidal anti-inflammatory drugs (NSAID) as well as several antioxidants such as vitamin E in prostate cancer patients have shown effect in controlling prostate cancer growth in clinical trials [5,6]. In addition, the a1-adrenoceptor antagonist terazosin is found to suppress androgen-independent prostate cancer cell growth through induction of apoptosis [7]. The great advantage of these agents is that they are not designed to target fast growing cells and have been widely used clinically. Recently, one of these agents, FTY720, a derivative of fungus Isaria sinclairii [8], has been shown a dramatic anticancer effect in both in vitro and in vivo studies [9,10]. FTY720 was originally identified as an immunosuppressant. It has been shown to prevent rejection of transplanted grafts in organ transplantation patients by directing lymphocytes away from peripheral bloodstream [11–14]. In some cases, it selectively induces apoptosis of mature T-lymphocytes through downregulation of Bcl-2 expression [15]. Administration of FTY720 in these patients is not associated with renal, gastrointestinal or bone marrow toxicity [16,17] and as a result, it is currently undergoing Phase II clinical trials for treatment of transplantation patients [15]. Recently, FTY720 was reported to induce apoptosis in several types of cancer cells such as bladder, breast
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and glioma cancer cells [9,10,18]. In addition, two recent in vivo studies showed that FTY720 prevented tumor growth and metastasis of breast and bladder xenografts in nude mice without causing detectable toxicity in vital organs [9,10]. These results raised a hypothesis that FTY720 may be a potential anticancer drug. However, the underlying mechanisms responsible for its action are not clear. In prostate cancer, treatment of FTY720 on androgen-independent DU145 cells reduced cell viability and induced apoptosis [19], indicating that it may be an effective agent for suppression of androgen-independent prostate cancer growth. In this study, we investigated if FTY720 was able to suppress invasion ability of androgen-independent prostate cancer cells and studied the molecular mechanisms responsible. To achieve this, we first treated two androgen-independent prostate cancer cell lines, DU145 and PC3, with different doses of FTY720 and tested its effect on colony formation, migration and invasion abilities. Since Rho-GTPase family proteins play positive roles in cancer cell motility and invasion [20–22], we then studied if RhoA-GTPase was involved in the FTY720-induced suppression of prostate cancer invasion ability. Our results showed that FTY720 treatment in prostate cancer cells led to suppression of tumor cell invasion and motility through inactivation of RhoA-GTPase pathway, indicating its potential role as an effective agent in the treatment for metastatic prostate cancer.
2. Materials and methods 2.1. Cell lines and cell culture conditions Two androgen-independent human prostate cancer cell lines DU145 and PC3, obtained from American Type Culture Collection (Rockville, MD, USA), were maintained in RPMI1640 medium (Sigma, MO, USA) supplemented with 5% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37 8C, 5% CO2. 2.2. Colony forming assay Eighty percent confluent cells were trypsinized and single-cell suspensions were obtained. Two hundred
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viable cells were seeded per well in 12-well plates. Twenty-four hours later cells were treated with five different doses of FTY720. Two wells were used for each drug concentration and two wells treated with solvent only were set up as controls. Ten days later, cells were fixed with 70% ethanol and stained with 10% (v/v) Giemsa (MERCK, Damstadit, Germany). Colonies consisting of more than 50 cells were scored. The percentage of colony formed after FTY720 treatment was calculated as the ratio between the number of colonies in the treated wells and the untreated controls multiplied by 100. Cell survival curves were plotted and the inhibitory concentrations of 10, 50 and 90% cell survival (IC10, IC50 and IC90) were determined. 2.3. Wound closure assay Cells were plated in six-well-plates. When the cells grew into full confluency, a wound was induced on the monolayer cells by scraping a gap using a micropipette tip and then FTY720 was added immediately after wound induction. The speed of wound closure was compared between FTY720 treated and untreated cells. Photographs were taken under 100! magnifications using phase-contrast microscopy immediately after wound incision and at later time points. 2.4. 3D collagen colony forming assay Two-hundred microliters of cell suspension (3!104 cells/ml) was mixed with 200 ml of cold rat tail collagen, type I (3.60 mg/ml, BD Biosciences, MA, USA). The mixed cell solution in collagen was plated as droplets in 60 mm Petri-dish and air dried at room temperature. RPMI1640 medium with 3% FCS with or without FTY720 was added in each dish containing the semisolid collagen-cell droplets. FTY720 treated and control samples were incubated at 37 8C for 3–5 days. Cell morphology was observed under phase-contrast microscopy and pictures were captured under 200! magnifications. Total of 500 colonies were counted in each experiment and percentage of colonies that consisted of elongated morphology was calculated. Each experiment was repeated at least three times and error bars indicate SD.
2.5. Stress fiber assay Thirty-five microliters of cell suspension (2!105 cells/ml) was seeded per well in eight-well chamber slides (ICN Biomedicals, OH, USA) and incubated over night. Cells were then treated with FTY720 and incubated for 24 h. Cells were incubated with serum free RMPI1640 medium for further 24 h. Two wells were set up for each treatment and the untreated control. At the end of incubation, each well was treated with 30 ml of lysophosphatidic acid (100 ng/ml, SIGMA, MO, USA) and incubated for 10 min at 37 8C. Cells were then washed with 1!PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with 1!PBS, cells were permeabilized in 1% Trion X-100 for 15 min at room temperature and blocked in 1% BSA (in 1!PBS) for 30 min. Cells were incubated with fluorescein phalloidin (Molecular Probes, Eugen, Oregon, USA) in 1% BSA (dilution factor, 1:50) at 37 8C for 1 h. After incubation, cells were washed with 1!PBS and mounted. Stress fiber was visualized under confocal microscopy and photographs were taken under 600! magnifications. 2.6. RhoA-GTP pull down assay Cells were plated in T-75 flasks and grown to 70–80% confluence. After serum free treatment for 24 h, cells were exposed to FTY720 for different time periods and then the cells were lysed. GTP-RhoA protein was pulled down using Rho Activation Assay Biochem Kit, BK036 (Cytoskeleton, CO, USA). The GTP-RhoA protein and total RhoA protein were assayed by Western-blot, using an antibody against RhoA provided in the Rho Activation Assay Kit (Cytoskeleton, CO, USA). Detailed Western-blot procedures have been described in our previous publications [23]. 2.7. Transfection Plasmids expressing constitutively active RhoA [pcDNA remobilized with enhanced GFP (EGFP)RhoA Q63L] have been described [24] and were kindly provided by Professor G. Bokoch (The Scripps Research Institute, La Jolla, CA). Cells were transfected using FuGENE 6 transfection reagent
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inhibiting colony forming ability of prostate cancer cells, which is the main target of chemotherapy.
(Roche, IN, USA) according to the manufacturer’s instructions. The empty pcDNA vector was transfected to cells as a control. G418 (550 mg/ml for DU145, 600 mg/ml for PC3) was added 48 h after transfection. After 10 days selection, a pool of transfectant clones was obtained.
3.2. Effect of FTY720 on prostate cancer migration Increased migration rate is one of the characteristics of metastatic cancer cells [25,26]. To study if FTY720 had any effect on migration ability of DU145 and PC3 cells, we performed Wound closure assay. As shown in Fig. 2A, similar sized wounds were introduced in monolayer DU145 cells at 0 h. In the control cells, the gap of the wound was filled gradually by migrating cells and 32 h after wound induction, the gap was almost closed (solid arrow). In contrast, after exposure to FTY720 (IC50Z1.5 mM), the speed of wound closure was much slower and the wound was still widely open at 32 h postexposure time (dotted arrow). Similar results were observed in PC3 cells except the shorter experimental time points (Fig. 2B). As the speed of wound closure reflects the migration ability of cancer cells, these results indicate that FTY720 treatment is able to inhibit cell migration. This effect of FTY720 was not due to inhibition of proliferation as we did not observe any difference in proliferation rates between the treated and untreated cells by BrdU staining (data not shown).
3. Results 3.1. Effect of FTY720 on colony forming ability of prostate cancer To study the effect of FTY720 on prostate cancer cell survival, we performed colony forming assay on two androgen-independent metastatic prostate cancer cell lines DU145 and PC3. As shown in Fig. 1A, FTY720 was able to inhibit clonogenesity of both cell lines. These two cell lines showed similar sensitivity to FTY720 evidenced by the similarity in their inhibitory concentrations of 10, 50 and 90% of cell survival (IC10, IC50 and IC90, Fig. 1B). We also noted that the FTY720 concentrations required for inhibiting colony forming ability in this study were much lower (i.e. IC50Z1.5 mM) than previously reported (i.e. 25 mM) for inhibiting similar percentage of cell viability on these two cell lines [19]. These results indicate that FTY720 is much more effective in
Colony Forming Ability (% of untreated control)
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FTY720 concentration (µM) Fig. 1. Effect of FTY720 on clonogenesity of androgen-independent prostate cancer cells. Colony forming assays were performed on DU145 and PC3 cells after continuous exposure to FTY720. (A) Survival curves of DU145 and PC3 after FTY720 treatment. (B) Summary of inhibitory concentrations (IC) of FTY720 on DU145 and PC3 cells. Note that both cell lines showed similar sensitivity to FTY720.
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Fig. 2. Effect of FTY720 on the migration ability of androgen-independent prostate cancer cells. Similar sized wounds were introduced in confluent monolayer cells and IC50 concentration of FTY720 was added. The speed of wound closure was monitored at different time points. Note that the speed of wound closure was suppressed by FTY720 in DU145 (A) and PC3 (B) cells in a time dependent manner. Significant difference in the speed of wound closure between treated (dotted arrows) and untreated (solid arrows) cells are demonstrated at 32 (DU145) and 12 h (PC3) postexposure, respectively.
3.3. Effect of FTY720 on invasion ability in 3D collagen gel Another characteristic of metastatic cancer cells is their ability to invade into extracellular-matrix [27,28]. To study whether FTY720 could inhibit the invasion ability of DU145 and PC3, we performed 3D collagen gel assay. As shown in Fig. 3, the untreated cells grew extensively inside the semi-solid collagen gel and displayed an elongated morphology as early as 2 days after plating. After 5 days, high percentage of DU145
(56.2G15%) and PC3 (69.3G6%) cells formed elongated colonies, indicating their ability to invade into extracellular-matrix. In contrast, after FTY720 treatment, the percentage of colonies showing elongated morphology was reduced to 4.4G6% for DU145 and 9.3G5% for PC3 cells. Majority of the cells lost their invasive ability and remained round morphology in the collagen gel. These results suggest that FTY720 could effectively suppress prostate cancer cell’s ability to invade in collagen gel, therefore, indicating its inhibitory effect on cell invasion.
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Fig. 3. Effect of FTY720 on the invasion ability of androgen-independent prostate cancer cells. Single prostate cancer cells were plated in semisolid collagen gel and FTY720 was then added in culture medium. The invasive ability was studied by monitoring the morphology of colonies. Note that FTY720 treatment suppressed the invasive growth in collagen gel.
3.4. Effect of FTY720 on cytoskeleton reorganization Cell motility is dependent on rearrangements of actin cytoskeleton in invasive cells to facilitate metastasis [29]. Since the actin cytoskeleton is composed of actin filaments, we used fluorescent phalloidin to detect alterations of actin structure of these cancer cells before and after FTY720 treatment. In the untreated DU145 and PC3 cells, bundles of actin filaments were well organized evidenced by the strong staining of stress fiber formation, intact network and protruding morphology (Fig. 4, panels A and C). However, after exposure to FTY720, a remarkable cytoskeletal changes with reduction of actin stress fiber network, relatively round morphology, and lack of filopodia were present in both cell lines (Fig. 4, panels B and D). These results demonstrate that FTY720 treatment led to severe
damage of cytoskeleton network, further suggesting its negative role on cancer cell invasion. 3.5. Inactivation of RhoA-GTPase by FTY720 in prostate cancer cells RhoA is one of the members of Rho-GTPase proteins which are involved in cell motility and actin cytoskeleton reorganization [30]. Like the other members of this protein family, RhoA protein switches between the active GTP-bound form and inactive GDP-bound form [31]. Activation of RhoAGTPase has been reported to associate with invasive cancers and it has been suggested to be one of the molecular mechanisms responsible for tumor metastasis [32,33]. In order to study whether the inhibitory effect of FTY720 on prostate cancer cell migration and invasion was mediated through the inactivation of
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Untreated control A
FTY720 treated B
generated. Western-blot assay showed that in addition to the endogenous RhoA protein, the EGFP-bound high molecular weight RhoA was detected in the EGFP-RhoA Q63L transfected cells (Fig. 6).
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B PC3 Fig. 4. Effect of FTY720 on stress fiber formation. FTY720 (IC50, 24 h) treated and untreated cells were stained using FITC-labeled phalloidin and the morphology of stress fibers was examined. Photos were taken under 600! magnifications. Note that FTY720 treatment destroyed the actin network and reduced the protrusion ability of prostate cancer cells.
Rho-GTPase pathway, we examined the expression level of active form RhoA-GTPase under the effect of FTY720 (IC50Z1.5 mM) with different treatment periods by Western-blot. We found that the active form GTP-bound RhoA was down-regulated after exposure to FTY720 in a time dependent manner in both DU145 and PC3 cells (Fig. 5). In contrast, the expression level of total RhoA did not show any detectable changes. These results demonstrate that the concentration of FTY720 (IC50) that was effective in suppressing migration and invasion was also able to reduce the level of GTP-bound RhoA, suggesting that the inhibitory effect of FTY720 on metastatic ability of prostate cancer cells may be regulated through inactivation of RhoA-GTPase pathway.
0
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Fig. 5. Effect of FTY720 on RhoA-GTPase. GTP-pull down assay was performed on the FTY720 (IC50) treated and untreated cells at different time points and the levels of GTP-bound RhoA proteins were measured. Total RhoA was measured using Western-blot as an internal control. Note that FTY720 down-regulated active form of RhoA in a time dependent manner.
3.6. Role of constitutively active RhoA on the inhibitory effect of FTY720 on prostate cancer cell invasion To further confirm the involvement of RhoAGTPase pathway in the inhibitory effect of FTY720 on metastatic ability of prostate cancer cells, we transfected DU145 and PC3 cells with p-EGFP-RhoA Q63L, an expression plasmid containing the constitutively active RhoA [24]. After G418 selection for 10 days, a pool of more than 50 transfectant clones was
Fig. 6. Generation of transfectants expressing constitutively active RhoA-GTPase. DU145 and PC3 cells were transfected with pcDNA3-EGFP-RhoA Q63L vector and then selected in G418 for 10 days. Western-blot analysis was carried out on cells transfected with pcDNA and pcDNA3-EGFP-RhoA Q63L vectors, respectively. Note that in addition to the endogenous RhoA protein, the RhoA Q63L transfected cells showed a higher molecular weight EGFP-bound RhoA, but it was absent in the vector transfected cells.
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Fig. 7. Constitutively active RhoA expression leads to resistance to FTY720-induced suppression in cell migration. Similar sized wounds were introduced on confluent monolayer cells and the speed of wound closure was monitored. Note that transfection of pEGFP-RhoA Q63L confers resistance to the inhibitory effect of FTY720 on cell migration (dotted arrows) compared to the parental and vector controls (solid arrows).
In contrast, the corresponding band was absent in the control vector pcDNA3 transfected cells. We then studied if ectopic expression of constitutively active RhoA had any effect on the function of
FTY720. As shown in Fig. 7, FTY720 treatment was able to prevent cell migration in the vector control, however, the dominant active RhoA transfected cells showed similar migration rate as the untreated control
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vector cells was destroyed after FTY720 treatment (Fig. 9). In contrast, well organized actin filaments were clearly stained in cells expressing constitutively active RhoA. In addition, there was no obvious difference in actin structures and morphology in constitutively active RhoA transfected cells before and after FTY720 treatment. These results further indicate that over-expression of active RhoA confers prostate cancer cells resistance to the FTY720induced destruction of stress fiber network.
4. Discussion
Fig. 8. Constitutively active RhoA confers resistance to FTY720induced suppression in invasion. Cells were plated in semi-solid collagen gel as described in Fig. 3. The percentage of elongated colonies was counted in the pEGFP-RhoA Q63L and the controls. Note that over-expression of RhoA Q63L in DU145 and PC3 cells reversed the effect of FTY720 on cell invasion.
(dotted arrows) in both DU145 and PC3 cells. In addition, the dominant active RhoA transfected cells showed increased invasion ability in 3D collagen gel compared to the vector control after FTY720 treatment, evidenced by the fact that the percentage of elongated colonies in cells expressing constitutively active RhoA was similar to the untreated control (47.7G3 versus 44.9G11%, Fig. 8A). Similar results were observed in PC3 transfectants (Fig. 8B). These results indicate that cells transfected with constitutively active RhoA are resistant to inhibitory effect of FTY720 on migration and invasion. Furthermore, as previously observed in the parental cells, the actin stress fiber formation in parental cells and control
The evidence presented in this study have demonstrated that relatively low dose of FTY720 is able to suppress colony forming (Fig. 1), migration (Fig. 2) and invasion abilities (Figs. 3 and 4) of androgenindependent prostate cancer cells. In combination with previous results that much higher concentrations of FTY720 (i.e. 25 mM) did not induce detectable biological effects on non-malignant fibroblast cells [19] and in transplant patients [34], our results suggest a potential novel agent in the treatment of androgenindependent metastatic prostate cancer with minimal toxicity on normal cells. In addition, the results that the suppression of metastatic ability by FTY720 was mediated through inactivation of RhoA-GTPase provide an insight into molecular mechanisms responsible for its action. Since activation of GTPases is frequently found in many types of metastatic cancers [22,35], our results also indicate that FTY720 may be a potential new agent in the treatment of advanced cancers. Once prostate cancer progresses to androgenindependence, the prognosis is very poor. This is because the commonly used androgen-depletion therapy is no longer efficient. In this study, we have demonstrated that FTY720 is able to inhibit colony forming ability of androgen-independent prostate cancer cells (Fig. 1). In addition, the FTY720 doses required for suppression of colony forming ability in this study were much lower (up to 13-fold lower, Fig. 1B) than previously reported on the same cell lines for its effect on short-term cell viability [19,36]. These results suggest that FTY720 is more effective on clonogenesity, which is the target of anticancer therapy. Previously, FTY720 was also reported to
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FTY720 (IC50)
Fig. 9. Effect of constitutively active RhoA expression on FTY720-induced cytoskeleton reorganization. The parental, pCDNA3 and pEGFPRhoA Q63L transfected cells were treated with FTY720 as described in the legend of Fig. 4. Note that the cells expressing constitutively active RhoA protein are resistant to FTY720-induced stress fiber disruption.
inhibit tumor growth in two recent in vivo studies on breast and bladder cancer xenografts in nude mice with minimal toxicity on normal organs [9,10]. In combination with these results, our results suggest that FTY720 may be a new promising anticancer agent for the treatment of cancers that show poor response to conventional chemotherapy. Once prostate cancer has developed metastatic growth, the survival rate is extremely low [4,37]. This is because to date very few agents have shown effective results on suppression of metastatic ability of prostate cancer cells. In this study, our results demonstrated that after exposure to a relatively low dose (IC50) of FTY720, the migration rate of DU145 and PC3 cells was significantly inhibited (Fig. 2), which indicates the suppression of tumor cell movement by FTY720. In addition, the tumor cell’s ability to invade into extracellular-matrix was also reduced after FTY720 treatment (Fig. 3). Furthermore, the cytoskeleton reorganization required for cell motility such as stress fiber formation was also significantly inhibited after exposure to FTY720 (Fig. 4). These results demonstrate that FTY720 is effective in suppressing the metastatic ability of androgen-independent prostate cancer cells. In addition, the antimetastatic effect was regulated through inactivation of
RhoA-GTPase as we found that after FTY720 treatment, the RhoA-GTPase was significantly decreased with increased exposure time (Fig. 5). Expression of constitutively active RhoA-GTPase, on the other hand, led to resistance to the inhibitory effect of FTY720 on cell migration, invasion and motility (Figs. 6–9). Recently, several studies also reported that FTY720 was able to suppress cancer invasion to other tissues and organs in nude mice bearing metastatic breast and bladder cancer xenografts [9, 10]. In this study, our evidence has provided a biological and mechanistic basis for the inhibitory role of FTY720 on cancer cell invasion. Since activation of RhoA-GTPases has been found in many metastatic cancers [20,22,35] and shown to promote metastasis in prostate cancer [38], our results in combination with previous in vivo studies [9,10,19] suggest that in addition to androgen-independent metastatic prostate cancer, FTY720 may also be an effective agent in the treatment of other metastatic human cancers. However, further investigations are required to confirm this hypothesis. In summary, we have demonstrated that FTY720, a fungus metabolite, is able to suppress both clonogenesity and metastatic ability of androgen-independent prostate cancer cells. In addition, we found that
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the function of FTY720 may be mediated through down-regulation of the RhoA-GTPase pathway. As currently FTY720 is being used clinically in transplant patients with minimal side effects, it could be served as an alternative strategy in the treatment of androgen-independent metastatic prostate cancer patients who are resistant to conventional therapy. Currently, we are investigating if the anti-metastasis role of FTY720 can be observed in in vivo systems.
[12]
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