RhoGDI2 confers gastric cancer cells resistance against cisplatin-induced apoptosis by upregulation of Bcl-2 expression

Cancer Letters 311 (2011) 48–56

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

RhoGDI2 confers gastric cancer cells resistance against cisplatin-induced apoptosis by upregulation of Bcl-2 expression Hee Jun Cho a,1, Kyoung Eun Baek a,1, Sun-Mi Park a, In-Kyu Kim a, In-Koo Nam a, Yeong-Lim Choi a, Seung-Ho Park a, Min-Ju Im a, Jungil Choi b, Jinhyun Ryu b, Jae Won Kim a, Chang Won Lee a, Sang Soo Kang b,⇑, Jiyun Yoo a,⇑ a

Department of Microbiology/Research Institute of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju, Republic of Korea Department of Anatomy and Neurobiology, Institute of Health Science, and Medical Research Center for Neural Dysfunction, School of Medicine, Gyeongsang National University, Jinju, Republic of Korea

b

a r t i c l e

i n f o

Article history: Received 11 March 2011 Received in revised form 7 June 2011 Accepted 16 June 2011

Keywords: RhoGDI2 Cisplatin Gastric cancer Bcl-2

a b s t r a c t Rho GDP dissociation inhibitor (RhoGDI)2 has been identified as a regulator of Rho family GTPase. Recently, we suggested that RhoGDI2 could promote tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that RhoGDI2 contributes to another important feature of aggressive cancers, i.e., resistance to chemotherapeutic agents such as cisplatin. Forced expression of RhoGDI2 attenuated cisplatin-induced apoptosis, whereas RhoGDI2 depletion showed opposite effects in vitro. Moreover, the increased anti-apoptotic effect of RhoGDI2 on cisplatin was further validated in RhoGDI2-overexpressing SNU-484 xenograft model in nude mice. Furthermore, we identified Bcl-2 as a major determinant of RhoGDI2-mediated cisplatin resistance in gastric cancer cells. Depletion of Bcl-2 expression significantly increased cisplatin-induced apoptosis in RhoGDI2-overexpressing gastric cancer cells, whereas overexpression of Bcl-2 blocked cisplatin-induced apoptosis in RhoGDI2-depleted gastric cancer cells. Overall, these findings establish RhoGDI2 as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk in gastric cancer. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Although the incidence and mortality of gastric cancer have declined steadily over the past several decades, it remains one of the most important malignancies because of its relatively high incidence and mortality rates. Chemotherapy is commonly used for this type of cancer, however, the prognosis of advanced gastric cancer is still poor and treatment is usually unsuccessful. Cisplatin is a common anti-tumor agent that is used to treat various cancers [1–5]. The initial tumor response to cisplatin treatment is robust; however, the treatment efficacy decreases as the ⇑ Corresponding authors. Tel.: +82 55 7515947; fax: +82 55 7595199 (J. Yoo), tel.: +82 55 7518733; fax: +82 55 7516227 (S.S. Kang). E-mail addresses: [email protected] (S.S. Kang), [email protected] (J. Yoo). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.06.024

duration and number of therapy cycles increases. Resistance is acquired rapidly, contributing to therapy failure [6]. However, the mechanisms by which cells develop resistance to cisplatin are not fully understood. Rho GDP dissociation inhibitors (RhoGDIs) were originally identified as negative regulator of Rho GTPases because they bind the majority of Rho GTPases in the cytoplasm, maintaining Rho in an inactive form in which it cannot interact with effector target proteins [7,8]. On the other hand, recent reports suggest that RhoGDIs may also act as positive regulator of Rho GTPases because they are also associated with active forms of Rho, Rac, and Cdc42 [9,10]. This interaction results in an inhibition of the intrinsic and GTPase-activating protein-stimulated GTPase activities of the Rho GTPases, maintaining Rho in an active form. Three human RhoGDIs have been identified thus far: RhoGDI1 (also referred to as RhoGDI or RhoGDI-a),

H.J. Cho et al. / Cancer Letters 311 (2011) 48–56

RhoGDI2 (Ly-GDI or D4GDI or RhoGDI-b), and RhoGDI3 (RhoGDI-c) [11–13]. RhoGDI2 is preferentially expressed in hematopoietic cells, RhoGDI3 is expressed in the brain, lungs, kidneys, testes, and pancreas, and RhoGDI1 is ubiquitously expressed in all mammalian organs [14,15]. RhoGDI1 has been known to bind to and regulate most of the Rho GTPases, including RhoA, Rac and Cdc42 [8]. However, RhoGDI2 appears to have a narrow selectivity and lower binding affinity for Rho GTPases [7]. RhoGDI2 associates with Rac1 and Rac3 in breast cancer cells, but not with RhoA, Cdc42, and RhoC, and negatively regulates their activities [16]. On the other hand, RhoGDI2 was also described as an activator of Rac1 in human bladder cancer cells [17]. Thus, RhoGDI2 may regulate cellular function by interacting primarily with Rac GTPase. Several reports have assessed the expression levels of RhoGDI2 in a variety of cancer cells, revealing opposite patterns depending on the tumor type. For instance, RhoGDI2 expression has been shown to be inversely correlated with invasive capacity in bladder cancer cell lines [18]. Furthermore, the reduced expression of the RhoGDI2 protein has been associated with a poor prognosis of patients with advanced bladder cancer [19]. In contrast, the mRNA level of RhoGDI2 has been known to be significantly higher in ovarian adenocarcinoma than in benign adenoma [20]. Consistent with this, RhoGDI2 is overexpressed in human breast cancer cell lines and increases cancer cell invasion and motility in vitro [21]. We also recently showed that RhoGDI2 expression is positively correlated with tumor progression and metastasis potential in gastric cancer [22]. There are many reports showing the function of RhoGDI2 in tumor growth and malignant progression in many cancer types, however, the role of RhoGDI2 in resistance to genotoxic anticancer reagents has not been defined yet. In this study, we provide the first evidence to our knowledge that RhoGDI2 contributes to the resistance of gastric cancer cells to chemotherapy-induced apoptosis. Functional characterization of RhoGDI2 reveals its dual functions in promoting chemoresistance and metastasis of gastric cancer cells.

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instruction. The morphology of apoptotic cell death was observed using In Situ Cell Death Detection Kit, Fluorescein (Roche). Cells were treated with cisplatin, etoposide, or staurosporine for different times. Cells were washed with cold PBS and fixed with 4% paraformaldehyde. Fixed cells were permeabilized and stained using the TUNEL reaction mixture in the dark. Samples were observed under a fluorescence microscope. 2.3. Antibodies and western blotting Rabbit anti-RhoGDI2 antibody was from Spring Bioscience. Rabbit anti-caspase-3 and anti-PARP antibodies were from Cell Signaling Technology. Mouse anti-a-tubulin antibodies were purchased from Sigma. Rabbit antiBcl-XL, anti-Bax, anti-Bak, and mouse anti-Bcl-2 antibodies were Santa Cruz Biotechnology. Cell lysates were separated by 7.5–15% SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Amersham Bioscience). Subsequently, the membrane was incubated in TBST supplemented with 5% non-fat dry milk and probed with the appropriate primary antibodies. The bound antibodies were visualized with a suitable secondary antibody conjugated with horseradish peroxidase using enhanced chemiluminescence reagents (ECL, Amersham Bioscience).

2.4. RNA interference experiments Two different siRNA oligo duplexes for targeting Bcl-2 were purchased from Samchully (Seoul, Korea). Transient transfection of each siRNA oligo duplex (siBcl-2-1; 50 -GTT CATTCCATTATTAGAC-30 and siBcl-2-2; 50 -ACTGGATGTTT ACAGACAA-30 ) was accomplished using siLentFect™ Reagent (BIO-RAD). After incubation for 48 h, the cells were harvested and efficiency of each siRNA oligo duplex was confirmed by western blot or RT-PCR analysis.

2.5. Tumorigenicity in nude mice 2. Materials and methods 2.1. Reagents and cell culture Cisplatin, etoposide, staurosporin, 5-fluorouracil, paclitaxel and doxorubicin were purchased from Sigma. Human gastric cancer cell lines SNU-484, SNU-638, SNU-719 and MKN-28 were obtained from the Korean Cell Line Bank (Seoul, Korea) and maintained in RPMI-1640 medium (Gibco) supplemented with 10% FBS and antibiotics. The SNU484 cells stably transfected with RhoGDI2 and MKN-28 cells stably transfected with shRNA-expressing lentiviral vector for targeting RhoGDI2 were described in our previous report [22].

For tumorigenicity experiments, six-week-old male BALB/cSlc-nude mice were injected subcutaneously with 5  106 SNU-484(GDI2-4) or SNU-484(Mock) cells. When tumors measured an average volume of 100 mm3, the mice (10 per group) were treated with cisplatin (5 mg/ kg, 2 times a week) or physiological saline for three weeks. Tumors were measured with calipers to estimate volume (0.5  width2  length). All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of Gyeongsang National University and followed National Research Council Guidelines.

2.6. Statistical analysis 2.2. Cell viability and apoptosis detection Cell viability was determined using the cell proliferation reagent MTS (Promega) following the manufacturer’s

We performed statistical analysis using the unipolar, paired Student t-test. The significance of the data was accepted when the P value was less than 0.05.

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H.J. Cho et al. / Cancer Letters 311 (2011) 48–56 significantly increased compared to control cells (Fig. 1D), which has been previously reported [22]. Cisplatin significantly inhibited tumor growth in mice injected with control cells, which was not seen in mice injected with SNU-484(GDI2-4) cells (Fig. 1D). These findings indicate that RhoGDI2 contributes to cisplatin resistance in gastric cancer cells in xenograft tumor models.

3. Results 3.1. RhoGDI2 expression confers resistance to cisplatin-induced apoptosis in gastric cancer cells

GDI2-7

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To investigate whether RhoGDI2 modifies gastric cancer chemosensitivity, we used SNU-484 and SNU-719 cells in which RhoGDI2, which is not normally expressed [22], was ectopically overexpressed. Cisplatin treatment markedly decreased the viability of SNU-484(Mock) and SNU-719(Mock) cells in a dose-dependent manner, whereas RhoGDI2overexpressing cells were significantly more resistant to cisplatin than control cells (Fig. 1A and Supplementary Fig. 1A). To determine whether RhoGDI2 affects cisplatin-induced apoptosis, cells were analyzed by TUNEL staining. Control cells were highly sensitive to cisplatin-induced apoptosis. However, cisplatin-induced apoptosis was significantly attenuated in RhoGDI2-overexpressing cells (Fig. 1B and Supplementary Fig. 1B), and RhoGDI2 attenuated cisplatin-induced activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) (Fig. 1C and Supplementary Fig. 1C). These results suggest that RhoGDI2 expression confers resistance to cisplatin-induced apoptosis in gastric cancer cells. To determine if RhoGDI2 expression affects chemosensitivity in vivo, SNU-484(Mock) and SNU-484(GDI2-4) cells were injected subcutaneously into the flanks of nude mice. Growth of the SNU-484(GDI2-4) cell-injected tumor was

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3.2. Depletion of RhoGDI2 expression enhances cisplatin-induced apoptosis in gastric cancer cells To further elucidate the effect of endogenous RhoGDI2 on cisplatinsensitivity in human gastric cancer cells, we used MKN-28 and SNU638 cells in which RhoGDI2, which is highly expressed [22], was depleted by shRNA transfection. Cisplatin markedly decreased the viability of RhoGDI2-depleted MKN-28 and SNU-638 cells, whereas control shRNA (shCon)-transfected cells were considerably more resistant to cisplatin (Fig. 2A and Supplementary Fig. 2A). Furthermore, RhoGDI2-depleted MKN-28 and SNU-638 cells exhibited markedly increased apoptosis (Fig. 2B and C and Supplementary Fig. 2B), activation of caspase-3, and PARP cleavage (Fig. 2D and Supplementary Fig. 2C) compared to control cells in response to cisplatin. These findings suggest that the resistance of gastric cancer cells to cisplatin-induced apoptosis could be modulated by RhoGDI2 expression.

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cisplatin treatment Fig. 1. RhoGDI2 attenuates cisplatin-induced apoptosis in gastric cancer cells. (A) SNU-484 cells stably transfected with empty (Mock) or RhoGDI2expressing vector (GDI2-4 and GDI2-7) were treated with varying concentrations of cisplatin for 24 h. Cell viability was determined by MTS analysis. (B) Representative images for TUNEL staining of RhoGDI2-overexpressing SNU-484(GDI2-4 and GDI2-7) cells after treatment with the indicated concentrations of cisplatin for 24 h (left). Percentage of TUNEL-positive RhoGDI2-overexpressing cells after treatment with cisplatin for 24 h (right). Data are mean ± SD of three individual experiments, each in triplicate. P < 0.01 as determined by paired Student t test. (C) Representative immunoblot for caspase-3 and PARP cleavage after cisplatin treatment in RhoGDI2-overexpressing cells. (D) In vivo chemoresistance assay of RhoGDI2-overexpressing cells. Xenograft tumor volumes in mice treated with cisplatin or PBS are shown. Tumor volume is presented as the mean ± SD (n = 10 mice in each group).

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shGDI2-2

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H.J. Cho et al. / Cancer Letters 311 (2011) 48–56

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Cisplatin (ug/ml) Fig. 2. Depletion of RhoGDI2 expression enhances cisplatin-induced apoptosis in gastric cancer cells. (A) MKN-28 cells transfected with control shRNA (shCon) or two RhoGDI2-specific shRNAs (shGDI2-1 and shGDI2-2) were treated with varying concentrations of cisplatin for 24 h. Cell viability was determined by MTS analysis. (B) Representative images for TUNEL staining of RhoGDI2-depleted cells after treatment with the indicated concentrations of cisplatin for 24 h. (C) Percentage of TUNEL-positive RhoGDI2-depleted cells after treatment with cisplatin for 24 h. Data are mean ± SD of three individual experiments, each in triplicate. P < 0.01 as determined by paired Student t test. (D) Representative immunoblot for caspase-3 and PARP cleavage after cisplatin treatment in RhoGDI2-depleted cells.

3.3. RhoGDI2 confers resistance to etoposide- and staurosporin-induced apoptosis in gastric cancer cells To examine whether RhoGDI2 regulates cellular response to multiple classes of chemotherapeutic drugs, we first used etoposide and staurosporin to treat the RhoGDI2-overexpressing SNU-484 cells. Overexpression of RhoGDI2 resulted in increased cell viability and markedly blocked the apoptosis, activation of caspase-3, and PARP cleavage induced by these agents (Fig. 3). In contrast, depletion of RhoGDI2 resulted in hypersensitivity to etoposide and staurosporine in MKN-28 cells (Fig. 4). We also found that RhoGDI2 expression markedly inhibits the cytotoxic effect of 5-fluorouracil, paclitaxel, and doxorubicin in RhoGDI2-overexpressing SNU-484 cells, however, depletion of RhoGDI2 expression reverses this effect in RhoGDI2-depleted MKN-28 cells (Supplementary Fig. 3). Overall, these results have established an important role of RhoGDI2 in regulating cellular response to a wide range of clinically important therapeutic agents in gastric cancer cells. 3.4. Bcl-2 is upregulated in RhoGDI2-overexpressing cells and confers RhoGDI2-mediated resistance to cisplatin-induced apoptosis Elevated expression of antiapoptotic Bcl-2 family proteins has been associated with resistance to chemotherapy [23]. We therefore examined whether the expression of Bcl-2 family proteins is altered by RhoGDI2 expression. The expression of Bcl-2 was markedly increased in RhoGDI2-overexpressing SNU-484 and SNU-719 cells and decreased in RhoGDI2-depleted MKN-28 and SNU-638 cells, whereas the expression of the other Bcl-2 family proteins remained fairly constant (Fig. 5A and Sup-

plementary Figs. 1A and 2A). To determine whether Bcl-2 mediates resistance to the cisplatin-induced apoptosis promoted by RhoGDI2, SNU484(GDI2-4) cells were transfected with two different Bcl-2-specific siRNAs. Downregulation of Bcl-2 significantly increased cisplatin-induced apoptosis (Fig. 5B and C) and cleavage of PARP (Fig. 5D) in RhoGDI2-overexpressing SNU-484 cells. To further confirm the effect of Bcl-2 on RhoGDI2-mediated cisplatin resistance, we investigated whether ectopic overexpression of Bcl-2 could enhance cisplatin resistance in RhoGDI2-depleted MKN-28 cells. In Bcl-2overexpressing RhoGDI2-depleted MKN-28 cells, cisplatin-induced apoptosis and cleavage of PARP were markedly inhibited compared to control cells (Fig. 6). All of these results suggest that Bcl-2 is a key mediator for resistance to cisplatin-induced apoptosis in RhoGDI2-expressing gastric cancer cells.

4. Discussion RhoGDI2 has been known to be preferentially expressed in hematopoietic tissues [12,24]. However, accumulating evidence suggests that RhoGDI2 is also frequently expressed in multiple types of human cancers and can function as positive (in breast cancer) or negative (in bladder cancer) regulator of cancer progression [21,25,26]. We recently demonstrated that RhoGDI2 expression is increased in advanced stage gastric tumors and significantly

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-tubulin Fig. 3. RhoGDI2 attenuates etoposide- and staurosporin-induced apoptosis in gastric cancer cells. (A) Viability of RhoGDI2-overexpressing SNU-484(GDI2-4 and GDI2-7) cells after treatment with the indicated concentrations of etoposide (left) or staurosporin (right) for 24 h. (B) Representative image for TUNEL staining of RhoGDI2-overexpressing cells after treatment with etoposide (10 lg/ml) or staurosporin (1 lM) for 24 h (left). Percentage of TUNEL-positive RhoGDI2-overexpressing cells after treatment with etoposide or staurosporin for 24 h (right). Data are mean ± SD of three individual experiments, each in triplicate. P < 0.01 as determined by paired Student t test. (C) Representative immunoblot for caspase-3 and PARP cleavage in RhoGDI2-overexpressing cells after etoposide (left) or staurosporin (right) treatment.

associated with lymph node metastasis [22]. However, whether RhoGDI2 may be involved in the development of resistance to genotoxic anticancer reagents in cancer cells is not known yet. In this study, we provided evidence suggesting that RhoGDI2 can protect gastric cancer cells against apoptosis induced by chemotherapeutic agents. Forced expression of RhoGDI2 increased resistance of SNU-484 gastric cancer cells to the induction of apoptosis by three chemotherapeutic agents (cisplatin, etoposide, and staurosporin) and attenuated the inhibition of tumor growth by cisplatin in a xenograft model. Conversely, RhoGDI2 depletion sensitized MKN-28 gastric cancer cells to cisplatin-, etoposide- and staurosporin-induced apoptosis. These results suggest that RhoGDI2 confers a survival mechanism to gastric cancer cells that protects them from these chemotherapeutic agents.

RhoGDI1 has been known to protect breast cancer cells from drug-induced apoptosis through the inhibition of Rac1 cleavage mediated by capase-3 [27]. Although RhoGDI1 is completely resistant to degradation during apoptosis, RhoGDI2 is well characterized as a substrate for caspases and is cleaved during apoptosis in various cells [28]. Therefore, RhoGDI2 may act as an antiapoptotic molecule via a distinct mechanism from RhoGDI1 and may do so prior to caspase activation during drug-induced apoptosis. Although the mechanism by which RhoGDI2 inhibits apoptosis remains to be fully established, one plausible mechanism is that RhoGDI2 inhibits drug-induced apoptosis through positive regulation of Rac1 activity. In many cell types, Rac1 has been known to play a key role in controlling cell survival [29]. RhoGDIs were originally identified as a negative regulator of Rho GTPases.

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-tubulin Fig. 4. Depletion of RhoGDI2 expression increases etoposide- and staurosporin-induced apoptosis in gastric cancer cells. (A) Viability of RhoGDI2-depleted MKN-28(shGDI2-1 and shGDI2-2) cells after treatment with varying concentrations of etoposide (left) or staurosporin (right) for 24 h. (B) Percentage of TUNEL-positive RhoGDI2-depleted cells after treatment with etoposide (left) or staurosporin (right) for 24 h. Data are mean ± SD of three individual experiments, each in triplicate. P < 0.01 as determined by paired Student t test. (C) Representative immunoblot for caspase-3 and PARP cleavage in RhoGDI2-depleted cells after etoposide (left) or staurosporin (right) treatment.

However, recent evidences point to a positive role for RhoGDIs in Rho GTPases function. RhoGDIs could act as an escort protein directing Rho GTPases to membrane, which is crucial for coupling the GTPases to their downstream effector proteins [30,31]. These results have led us to attempt to verify the activation status of RhoA, Rac1, and cdc42 in RhoGDI2-expressing and -depleting cells, and found that RhoGDI2 specifically activates Rac1, but not RhoA or cdc42 in gastric cancer cells (data not shown). It is conceivable that RhoGDI2 may function as a positive regulator of Rho GTPases signaling in gastric cancer cells. Consistent with our results, a recent report suggests that RhoGDI2 could preferentially bind and activate Rac1 in bladder cancer cells [17]. However, how RhoGDI2 causes activation of Rac1 and whether activated Rac1 plays a critical role for RhoGDI2-induced drug resistance in gastric cancer cells requires further analyses. Another plausible scenario is that RhoGDI2 influences other signal pathway. Groysman et al. showed that Rho-

GDI2 associates with Vav1 (the guanine nucleotide exchange factor of Rho GTPases) and enhances, rather than counteracting, the effects of Vav1, the induction of phospholipase (PLC)-c phosphorylation during T cell activation by T cell receptor (TCR) [32]. We also found that phosphorylation of PLC-c is marked increased and inhibition of PLC-c activation by chemical inhibitor markedly increases cisplatin-induced apoptosis in RhoGDI2-overexpressing gastric cancer cells (data not shown). Although, how RhoGDI2 regulates the phosphorylation status of PLC-c requires further analysis, PLC-c may be, at least in part, an important mediator for RhoGDI2-induced drug resistance. In this study, we found that Bcl-2, an important antiapoptotic protein that inhibits anticancer drug-induced apoptosis [23], expression is upregulated in RhoGDI2-overexpressing gastric cancer cells and downregulated in RhoGDI2-depleted gastric cancer cells. We also showed that downregulation of Bcl-2 significantly increases

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H.J. Cho et al. / Cancer Letters 311 (2011) 48–56

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Fig. 5. Bcl-2 expression is critical for RhoGDI2-mediated resistance to cisplatin. (A) Representative immunoblot for Bcl-2, Bcl-XL, Bax, and Bak in RhoGDI2overexpressing SNU-484(GDI2-4 and GDI2-7) cells (left). Representative immunoblot for Bcl-2 in RhoGDI2-depleted MKN-28(shGDI2-1 and shGDI2-2) cells (right). (B) Bcl-2 expression in RhoGDI2-overexpressing SNU-484(GDI2-4) cells transiently transfected with control siRNA (siCon) or Bcl-2 siRNA (si-Bcl2-1 and si-Bcl2-2). (C) Percentage of TUNEL-positive Bcl-2-depleted RhoGDI2-overexpressing SNU-484(GDI2-4/siBcl2-1 and GDI2-4/siBcl2-2) or control (GDI24/siCon) cells after cisplatin treatment for 24 h. Data are mean ± SD of three individual experiments, each in triplicate. P < 0.01 as determined by paired Student t test. (D) Representative immunoblot for PARP cleavage in Bcl-2-depleted RhoGDI2-overexpressing SNU-484 cells after cisplatin treatment (10 lg/ ml) for 24 h.

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Fig. 6. Overexpression of Bcl-2 blocks cisplatin-induced apoptosis in RhoGDI2-depleted MKN-28(shGDI2-1) cells. (A) Percentage of TUNEL-positive Bcl-2overexpressing RhoGDI2-depleted MKN-28(shGDI2-1/Bcl2-1 and shGDI2-1/Bcl2-2) or control (shGDI2-1/Mock) cells after treatment with cisplatin for 24 h. Data are mean ± SD of three individual experiments, each in triplicate. P < 0.01 as determined by paired Student t test. (B) Representative immunoblot for PARP cleavage in Bcl-2-overexpressing MKN-28(shGDI2-1) cells after cisplatin treatment.

cisplatin-induced apoptosis in RhoGDI2-overexpressing SNU-484 cells and upregulation of Bcl-2 markedly inhibits cisplatin-induced apoptosis in Bcl-2-overexpressing RhoGDI2-depleted MKN-28 cells. These results suggest that Bcl-2 is a key mediator for cisplatin resistance in RhoGDI2-expressing gastric cancer cells. There are many reports demonstrating the importance of Bcl-2 in cisplatin resistance [33–35] and the importance of Bcl-2 in cisplatin resistance makes Bcl-2 an important target for the prevention of the development of resistance [36–38]. We also found that RhoGDI2 expression confers gastric cancer cells resistance to a wide range of clinically important therapeutic agents including etoposide, staurosporin, 5-fluorouracil, paclitaxel, and doxorubicin. Consistent with

our results, a recent report showed that RhoGDI2 is involved in the resistance to 5-fluorouracil in colon cancer cells [39]. The overexpression of Bcl-2 is commonly associated with resistance to a wide variety of chemotherapeutic drugs [40]. Regardless of the primary mode of action, whether single or double-strand DNA breaks, whether microtubule depolymerisation or aggregation, essentially all traditional anticancer drugs appear to depend on Bcl-2-dependent mechanisms for killing cancer cells [41]. Although, how RhoGDI2 upregulates Bcl-2 expression and whether upregulated Bcl-2 could mediate resistance to many other therapeutic drugs-induced apoptosis in RhoGDI2-expressing gastric cancer cells requires further analyses, all of our results suggested an important role of

H.J. Cho et al. / Cancer Letters 311 (2011) 48–56

RhoGDI2 in regulating cellular response to a wide range of clinically important therapeutic agents in gastric cancer cells. In summary, we recently demonstrated that RhoGDI2 expression is increased in advanced stage gastric tumors and significantly associated with lymph node metastasis. In the present study, we suggested for the first time that RhoGDI2 can directly contribute to another important feature of aggressive cancers, i.e., resistance to chemotherapeutic agents such as cisplatin. Thus, RhoGDI2 contributes to tumor progression by inducing multiple changes in cellular physiology. In this context, molecular targeting of RhoGDI2 may not only prevent the seeding of gastric cancer cells to other organs, but also sensitize cancer cells to chemotherapy, thereby preventing the spread of gastric cancer.

[12]

[13]

[14]

[15]

[16]

Conflict of interest [17]

None declared. Acknowledgment

[18]

This work was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080285).

[19]

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2011. 06.024.

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