Downregulation of RhoB GTPase confers resistance to cisplatin in human laryngeal carcinoma cells

Cancer Letters 295 (2010) 182–190

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Downregulation of RhoB GTPase confers resistance to cisplatin in human laryngeal carcinoma cells Tamara Cˇimbora-Zovko a, Gerhard Fritz b, Nevenka Mikac c, Maja Osmak a,* - Boškovic´ Institute, Bijenicˇka 54, 10000 Zagreb, Croatia Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruder Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany c - Boškovic´ Institute, Bijenicˇka 54,10000 Zagreb, Croatia Laboratory for Physical Chemistry of Traces, Division for Marine and Environmental Research, Ruder a

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a r t i c l e

i n f o

Article history: Received 20 November 2009 Received in revised form 23 February 2010 Accepted 25 February 2010

Keywords: Cisplatin Drug-resistance Rho GTPases RhoB

a b s t r a c t Acquired resistance to cisplatin represents a major obstacle to an efficient chemotherapy. We found downregulation of RhoB expression in cisplatin-resistant tumor cell lines from different origin. In cisplatin-resistant laryngeal carcinoma subline overexpression of farnesylated or geranylgeranylated RhoB increased cisplatin-induced cell death, while silencing of RhoB expression diminished sensitivity of parental HEp-2 cells via decreased cellular accumulation of cisplatin. However, since RhoB silencing in additional tumor cell lines did not alter their sensitivity to cisplatin, we can assume that RhoB downregulation does not provide general protective role in cell response to cisplatin. Nevertheless, gene therapy involving restoration of RhoB expression might improve the efficiency of cisplatin treatment, especially in patients with laryngeal carcinoma that acquired resistance to this chemotherapeutic drug. Ó 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The majority of cancer patients are still treated with classical chemotherapy. In this respect, chemotherapeutic drug cis-diamminedichloroplatinum (II) (cisplatin) is one of the most effective and commonly used agents for the treatment of a wide spectrum of solid tumors [1]. However, continuous clinical use of this drug often results in the development of resistance, which represents a major obstacle to a favorable therapeutic outcome. Although the mechanisms that lead to resistance have been discovered on cisplatin-resistant cell lines established in vitro, there is compelling evidence that the treatment failure in clinics is driven by the same mechanisms [2]. These include decreased accumulation and increased detoxification of cisplatin, more efficient removal of cisplatin DNA-

* Corresponding author. Tel.: +385 1 4560 939; fax: +385 1 4561 177. ˇ imbora-Zovko), fritz@ E-mail addresses: [email protected] (T. C uni-mainz.de (G. Fritz), [email protected] (N. Mikac), [email protected] (M. Osmak). 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.02.025

adducts, enhanced capacity to replicate past adducts, inhibition of apoptosis, as well as recently discovered mechanism based on altered expression of cell adhesion proteins (cell-adhesion mediated drug resistance, CAMDR), in particular integrins [1,3–5]. Integrin-mediated signaling regulates small GTPases of the Rho family and vice versa [6], while Rho GTPases mediate key cellular processes in response to various stimuli. For instance, avb3 and avb5 integrins regulate RhoB expression, as well as RhoB activation after exposure to ionizing radiation, in that way conferring radioresistance to glioblastoma cells [7]. Activation of Rho GTPases is a highly regulated process. Posttranslational modification at their C-terminal CAAX motive by isoprenoid lipids targets these proteins to cell membrane, where they cycle between active GTP-bound and inactive GDP-bound forms. Binding of GTP is promoted by guanine nucleotide exchange factors (GEFs) and GTP hydrolysis is catalyzed by GTPase activating proteins (GAPs). An additional regulation is achieved by Rho-GDP dissociation inhibitors (RhoGDIs), which sequester Rho-GDP in the cytoplasm [8]. Among three known RhoGDIs, RhoGDIa is the most ubiquitous and

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binds with high affinity to RhoA, Rac1 and Cdc42, which are the best studied members of the family and commonly recognized as major regulators of the actin cytoskeleton. The three Rho isoforms, RhoA, RhoB and RhoC, are highly homologous, and all induce stress fibers. Rac1 stimulates lamellipodium and membrane ruffle formation, while Cdc42 has a conserved role in regulating cell polarity and formation of filopodia [9]. Apart from regulating cytoskeleton, Rho GTPases are also involved in cell growth and cell cycle progression, cell adhesion formation and maintenance, membrane trafficking, transcriptional regulation and apoptosis [9]. Consequently, their deregulation has been linked to malignant transformation and tumor progression [8]. However, unlike RhoA, RhoC, Rac1 and Cdc42, which stimulate oncogenesis and tumor dedifferentiation, RhoB is proposed to function as a tumor suppressor. Namely, its expression is reduced in various tumor cell lines [10], its overexpression inhibits cell growth, invasion and metastasis [11,12], while RhoB null mice have increased susceptibility to carcinogen-induced skin tumors [10]. Rho GTPases are also mediators of cell response to stress, including treatment with chemotherapeutic drugs. Moreover, caspase-3 mediated cleavage of Rac1 is required for maximal drug-induced apoptosis [13]. Although RhoA overexpression conferred cisplatin resistance in human osteosarcoma cells [14], in cisplatin-resistant human epidermoid carcinoma KB-3-1 and liver carcinoma BEL-7404 cells Rac1 and RhoA expression were downregulated on transcriptional level [15]. In mouse fibroblasts expression of RhoB mRNA was induced by various genotoxins, including cisplatin [16], and stable transfection with wild type RhoB increased their sensitivity to alkylating agents and cisplatin [17]. Furthermore, silencing of RhoGDIa in breast carcinoma cells increased their sensitivity to doxorubicin and etoposide, while reintroduction of RhoGDIa protein expression restored resistance to apoptosis [18]. In our laboratory, we have developed cisplatin-resistant CA3ST and CK2 sublines that exhibit altered morphology and formation of cell–cell adherence junctions, comparing to their parental cisplatin-sensitive human laryngeal carcinoma HEp-2 cells [19]. In addition, cisplatin-resistant cells display numerous stress fibers and focal adhesions (unpublished data), while CA3ST subline has increased expression of avb3 integrin [20]. Bearing in mind the key role of Rho GTPases in the regulation of cell morphology and adhesion, these observations prompted us to investigate their potential role in the development of cisplatin resistance.

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platin-resistant MeWocis1 subline was derived from human melanoma MeWo cell line [23]. The extent of cisplatin resistance in these sublines is presented in Supplementary Table S1. Breast adenocarcinoma MCF7, pancreatic carcinoma MIA PaCa-2, colorectal carcinoma SW480, SW620, HCT 116 and Caco-2 cells, and NIH/3T3 mouse fibroblasts were obtained from American Type Culture Collection (ATCC; Manassas, VA), whereas urinary bladder carcinoma RT-112 and T-24 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Braunschweig, Germany). Cells were grown as a monolayer culture in Dulbecco’s modified Eagle’s medium, DMEM (GIBCO), supplemented with either 10% bovine serum (GIBCO) for NIH/3T3, HEp2 and HeLa cell lines and their respective sublines, or 10% fetal bovine serum (GIBCO) for all other cell lines, at 37 °C with 5% CO2. 2.2. Semiquantitative RT-PCR analysis Total cellular RNA was extracted from cells, reverse transcribed to cDNA and amplified by PCR as previously described [19], using the specific primers for rhoA, rhoB, rhoC, rac1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [24] or b-catenin [19]. The amplified products were resolved by electrophoresis in 1.2% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. 2.3. Western blot analysis

2. Materials and methods

Crude membranes were obtained as previously published [19]. Total cellular extracts were obtained by lysing the cells in lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 1 mM PMSF) on ice. Protein concentration was determined by Bradford and equal amounts of protein were separated by SDS–PAGE (12.5%) and blotted onto nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany). After overnight incubation at 4 °C in blocking buffer (5% milk (w/v) in TBS buffer with 0.1% Tween20 (v/v)), the membranes were probed with monoclonal antibodies against Cdc42, Rac1, RhoGDI, N-cadherin (Transduction Laboratories, San Diego, CA) and PARP (Pharmingen, San Diego, CA) or polyclonal antibodies against RhoA, RhoB and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA). Primary antibodies were detected with corresponding horseradish-peroxidase conjugated secondary antibodies (GE Healthcare, Piscataway, NJ), followed by Western Blot Chemiluminescence Reagent Plus detection according to the protocol provided by the manufacturer (PerkinElmer Life Science, Boston, MA).

2.1. Cell lines and culture conditions

2.4. Plasmid construction and transfection

Human laryngeal carcinoma (HEp-2) cells were obtained from cell culture bank (GIBCO, Grand Island, NY). Development of cisplatin-resistant CA3ST and CK2 sublines from HEp-2 cells has been published previously [19,21]. Cisplatin-resistant HeLa CK subline was derived from human cervical carcinoma HeLa cell line [22], while cis-

The plasmid vector pEGFP-RhoB was constructed by the insertion of SacI fragment of rat RhoB cDNA (according to HomoloGene, RhoB from rat shares complete homology to the human counterpart) into pEGFP-C3 vector (Clontech, Palo Alto, CA). Upon transfection into mammalian cells, it gives rise to the fusion protein in which N-terminus of

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RhoB is fused to C-terminus of EGFP. Mutations introduced into CAAX box were according to Chen et al. [25], where Cterminal CKVL amino acid sequence found in wild type RhoB, was replaced by CAIM to obtain farnesylated RhoB (RhoB-F) and CLLL to get geranylgeranylated RhoB (RhoBGG). CAAX box mutants were sequenced and found to have correct mutations. pEGFP-C3 vector served as control. Plasmids were transfected into CA3ST cells using Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions. The day after transfection cisplatin was added and 48 h later cells were stained with propidium iodide and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). In parallel, transfected cells were plated on degreased glass coverslips and on the next day observed on an Axiovert 35 epifluorescence microscope (Opton, Germany) to visualize localization of exogenous EGFP proteins. 2.5. RhoB silencing To achieve RhoB silencing, cells were transiently transfected with 25 nM SilencerÒ Select Predesigned siRNA catalog No. 4390817, locus ID 388 (Ambion, Austin, TX) using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocol for forward transfection. As a negative control, cells were transfected with a nontargeting control siRNA SilencerÒ Select Predesigned siRNA Negative control #1, catalog No. 4390844. Silencing of RhoB expression was verified by Western blot on whole cell lysates.

many). Platinum values were normalized to the number of cells per each well. 2.8. Statistical analysis Each experiment was repeated at least three times. All data were analyzed by unpaired Student’s t-test and expressed as means ± standard deviation. Data were considered statistically significant at p-value of < 0.05.

3. Results 3.1. Cisplatin-resistant human laryngeal carcinoma cells have decreased expression of RhoB on both mRNA and protein level We have previously observed that cisplatin-resistant CA3ST and CK2 cells display substantial alterations in cell morphology, adhesion and cytoskeleton organization, comparing to parental human laryngeal carcinoma HEp-2 cells. Since members of Rho family of small GTPases act as major regulators of actin cytoskeleton [9], we analyzed the expression of several representative Rho GTPases in HEp-2 model of cisplatin resistance. As determined by semiquantitative RT-PCR depicted on Fig. 1A, rhoA, rhoC and rac1 are expressed at the similar level in all cell lines, while the expression of rhoB was strongly downregulated in both cisplatinresistant sublines. Densitometrical analysis confirmed that the difference in rhoB mRNA expression between the resistant and parental cells is approximately twofold (Fig. 1D). Downregulation of RhoB in cisplatin-

2.6. Cytotoxicity assay The sensitivity of cells to cisplatin was determined by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well tissue culture plate (1500 cells per well) and incubated for 24 h. Cisplatin (Sigma–Aldrich, Taufkirchen, Germany), dissolved in saline, was added and after 96 h of incubation MTT assay was performed. The absorbance of the formazan product was measured on a microplate reader (Awareness Technology Inc., Palm City, FL) at 545 nm. 2.7. Determination of platinum accumulation by HR-ICPMS Platinum accumulation was determined according to previously published method [26]. In brief, 24 h after siRNA transfection, cells were seeded in 6-well tissue culture plates (100,000 cells per well). On the next day cisplatin was added and, after 5 h of incubation at 37 °C, plates were placed on ice and cells were quickly rinsed twice with icecold PBS and then 430 ll of 70% nitric acid was added into each well. The cell lysates were then transferred into 15 ml plastic tubes, capped and placed at 65 °C for 2 h, after which the samples were diluted with mQH2O to a final concentration of 5% nitric acid and 1 lg/l of indium was added as internal standard. Concentrations of platinum in samples were determined by high resolution inductively coupled plasma mass spectrometry (HR-ICPMS) on the instrument Element 2 (Thermo Finnigan, Bremen, Ger-

Fig. 1. Expression of Rho GTPases in human laryngeal carcinoma HEp-2 cells and their cisplatin-resistant CA3ST and CK2 sublines. (A) Semiquantitative RT-PCR analysis of rhoA, rhoB, rhoC, rac1, b-catenin and GAPDH mRNA expression. (B) Western blot analysis on whole cell lysates for RhoA, RhoB, Rac1, Cdc42 and RhoGDIa expression. ERK2 expression was used as the control for equal loading. (C) Western blot analysis on crude membranes for RhoA, RhoB, Rac1, Cdc42 and RhoGDIa expression. N-cadherin expression was used as the control for equal loading of membrane proteins. (D) Ethidium-bromide stained gels and immunoblots were densitometrically analyzed, and the area determined is given as the means ± SD. The data shown are representative of at least three independent experiments.

T. Cˇimbora-Zovko et al. / Cancer Letters 295 (2010) 182–190 resistant sublines was also established on protein level by Western blot analysis of the whole cell lysates, while the expression of RhoA, Rac1, Cdc42 and RhoGDIa was similar in all cell lines (Fig. 1B). Membrane localization is a prerequisite for the activation of Rho GTPases after stimulation with integrins or growth factors [9]. Therefore, we analyzed crude membrane fractions isolated from cisplatin-resistant and parental cells. Western blot analysis revealed similar membrane expression of RhoA, Rac1 and Cdc42 (Fig. 1C). In accordance to the data from the whole cell lysates, membrane expression of RhoB was decreased in CA3ST and CK2 cells (Fig. 1D). Thus, among all the Rho GTPases analyzed, only RhoB expression is altered in cisplatin-resistant CA3ST and CK2 cells, specifically, strongly downregulated on both mRNA and protein level. 3.2. Cisplatin does not induce RhoB expression in human laryngeal carcinoma cells It is known for over a decade that rhoB is robustly activated after cell exposure to various stimuli, especially after treatment with mitogens [27] and DNA damaging agents, among others cisplatin [16,28,29]. Considering the differences in RhoB expression between parental and cisplatinresistant CA3ST and CK2 cells, obtained by acute and chronic exposure to cisplatin, respectively, we sought to determine whether there are any differences in RhoB induction between resistant and sensitive cells after a single cisplatin treatment. Initially, we wanted to establish time-course of RhoB induction. For that reason, we treated HEp-2 cells with 7.4 lM cisplatin, which corresponds to pharmacologically relevant cisplatin concentrations (0.5–10 lM) observed in patients [30], and evaluated protein expression at several time points, ranging from 1 h until 24 h of treatment. Namely, in this time range other authors observed RhoB induction [12,16,29]. However, we did not observe any upregulation of RhoB (Fig. 2A). There were also no alterations in the expression level of other Rho GTPases analyzed. Zalcman et al. observed that the expression of RhoB is cell cycle dependent in HeLa cells, with the highest level of RhoB detected in S-phase [31]. Since it is well-known that cisplatin DNA-adducts block transcription and DNA replication leading to cell cycle arrest in the S-phase, we checked the cell cycle distribution after treatment of HEp-2 and CA3ST cells with several concentrations of cisplatin for 24 h. However, although over 70% of HEp-2 and 80% of CA3ST cells were blocked in the S-phase after treatment with 2.4 and 7.4 lM cisplatin, respectively (Supplementary Fig. S1), RhoB induction failed to appear (Fig. 2B). Thus, cisplatin does not induce RhoB expression neither in cisplatin-resistant CA3ST nor in parental HEp-2 cells.

Fig. 2. Expression of Rho GTPases after a single dose of cisplatin. (A) Western blot analysis of RhoA, RhoB, Rac1 and Cdc42 expression. HEp-2 cells were treated with 7.4 lM cisplatin and the whole cell lysates were collected at several time points. (B) Western blot analysis of RhoA, RhoB, Rac1 and Cdc42 expression. HEp-2 and CA3ST cells were treated for 24 h with the indicated concentrations of cisplatin (cDDP) and the whole cell lysates were collected. The data shown are representative of at least three independent experiments.

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3.3. RhoB downregulation is one of the determinants of cisplatin resistance in laryngeal carcinoma cells To assess whether modulation of RhoB expression level in cisplatinresistant cells was related to cisplatin sensitivity, we transfected CA3ST cells with pEGFP-RhoB plasmid vector that expresses RhoB fused to the C-terminal end of EGFP. The EGFP-RhoB fusion protein was detected in whole cell lysates of transiently transfected CA3ST cells by Western blot with anti-rhoB antibody (Supplementary Fig. S2A). We also verified that RhoB in conjunction to EGFP is still functional by performing gene reporter assay for NF-jB inhibition in NIH/3T3 mouse fibroblasts (Supplementary Fig. S2B), as previously reported [32]. Plasmid vector pEGFP-C3 expressing full-length EGFP protein served as control. Cellular localization of EGFP-RhoB fusion protein was visualized under the fluorescence microscope. The perceived pattern (Fig. 3A) confirmed previously published observations in which EGFP-RhoB was detected at plasma membrane and discrete perinuclear structure that the authors identified as Golgi [33]. Transiently transfected cells were treated with cisplatin for 48 h, after which the percentage of dead propidium-iodide positive transfectants was determined by flow cytometry. As depicted on Fig. 3B, untreated CA3ST cells transfected with EGFP-RhoB had higher fraction of propidium-iodide positive cells than mock-transfected cells, but this difference was not statistically significant. However, CA3ST cells that expressed EGFP-RhoB were much more prone to cisplatin-induced cell death than the cells transfected with control vector. Additionally, after the samples underwent FACS analysis, we evaluated nuclear morphology of the propidium-iodide stained cells under the fluorescence microscope. Majority of the dead cells in the EGFP-RhoB transfected samples had apoptotic morphology, with extensive chromatin condensation and appearance of apoptotic bodies (Supplementary Fig. S3), implying that RhoB augments cisplatin-induced apoptosis. Lipid modification is essential for the correct localization and function of Rho GTPases. However, unlike most Rho GTPases that are substrates for geranylgeranylation, RhoB has been reported to be either farnesylated or geranylgeranylated [34]. Moreover, some publications suggested antiapopoptotic function of farnesylated RhoB (RhoB-F) and proapoptotic function of geranylgeranylated RhoB (RhoB-GG) [12,35,36]. Therefore, to resolve whether RhoB-F or RhoB-GG is involved in cisplatin-induced cell death, we used PCR mutagenesis to create plasmid vectors that express preferentially farnesylated or geranylgeranylated EGFP-RhoB fusion protein. Both constructs showed inhibitory effect on NF-jB transcriptional activity (Supplementary Fig. S2B) and the cellular localization resembling the one of EGFP-RhoB wild type construct (Fig. 3A). Moreover, both EGFPRhoB-F and EGFP-RhoB-GG constructs significantly increased the percentage of dead cells in untreated population, as well as substantially increased the extent of cell death after cisplatin treatment (Fig. 3B). Although EGFP-RhoB-F gave the highest percentage of propidium-iodide positive transfectants, there was no statistically significant difference as compared to EGFP-RhoB-GG or EGFP-RhoB constructs. Apparently, both farnesylated and geranylgeranylated RhoB are able to amplify cisplatininduced death. In order to confirm the data obtained with exogenously expressed RhoB protein in cisplatin-resistant cells, which point to protective role of RhoB downregulation in cellular response to cisplatin, we decided to use the opposite approach. Namely, we silenced endogenous RhoB expression in parental HEp-2 cells with specific siRNA and analyzed cisplatin-induced cytotoxicity by MTT assay after 96 h treatment. Since RhoB is a short-lived protein [31], we checked RhoB expression level by Western blot on whole cell lysates at the time when cisplatin was added to the cells (48 h), and the day when the MTT assay was performed (144 h). Silencing of RhoB was confirmed at 48 h, whereas at 144 h after transfection, the expression of RhoB protein returned to the basal level (Fig. 4A). At the same time, the expression of RhoA protein that shows 84% identity with RhoB protein was not altered, confirming the specificity of silencing (Fig. 4A). As depicted on Fig. 4B, silencing of RhoB expression in HEp-2 cells significantly decreased their sensitivity to cisplatin. Literature data and our transient transfections with EGFP-fused RhoB imply that RhoB is localized in a discrete perinuclear compartment that resembles Golgi apparatus [33], which is known to be involved in intracellular trafficking of cisplatin [26]. Therefore, to gain insight into potential mechanism of RhoB downregulation-induced cisplatin resistance we determined cellular accumulation of cisplatin in siRNA transfected HEp2 cells after 5 h of cisplatin treatment. In a preliminary experiment, we evaluated cell response to cisplatin after this short treatment and found

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Fig. 3. Increased sensitivity of CA3ST cells transiently transfected with EGFP-RhoB plasmid vectors to cisplatin. (A) Cellular localization of EGFP and EGFPRhoB fusion proteins after transient transfection of the appropriate plasmid vectors in CA3ST cells, as viewed under the epifluorescence microscope. (B) Sensitivity of the transfectants to cisplatin. The day after transfection, cells were treated with different concentrations of cisplatin for 48 h, after which they were stained with propidium iodide and analyzed by flow cytometry. Each point represents the mean ± standard deviation of three independent experiments. p < 0.05. p < 0.001.

that 5 h exposure provides comparable toxicity to continuous cisplatin treatment (Supplementary Fig. S4). As determined by high resolution inductively coupled plasma mass spectrometry (HR-ICPMS) cellular platinum content was around 20% decreased in siRhoB transfected cells as compared to siContr transfected cells (Fig. 4C). It is essential to point out that cisplatin-resistant CK2 subline, which shows fourfold higher resistance factor than siRhoB transfected cells, in the same experimental settings accumulates 60% less cisplatin than HEp-2 cells (unpublished data). Thus, we suggest that RhoB downregulation contributes to cisplatin resistance of laryngeal carcinoma cells through reduced cellular accumulation of cisplatin. 3.4. RhoB downregulation does not provide general protective role in cell response to cisplatin To test whether RhoB downregulation is a general characteristic acquired during the development of cisplatin resistance in different tumor cell lines, we analyzed RhoB expression in cisplatin-resistant HeLa CK subline, derived from human cervical carcinoma HeLa cells, and cisplatin-resistant MeWocis1 subline, derived form human melanoma MeWo cells. Western blot on whole cell lysates revealed decreased expression of RhoB in both cisplatin-resistant sublines, while the expression of RhoA and Rac1 was similar (Fig. 5A). Densitometric analysis of Western blots established that this downregulation of RhoB expression was more than twofold in HeLa CK cells and slightly less in MeWocis1 subline (Fig. 5B). However, when we performed cytotoxicity assay after silencing of RhoB expression in HeLa cells (Fig. 5C), we did not observe any difference in response to cisplatin (Fig. 5D). We were not able to perform the same experiment on MeWo cell line, due to poor plating efficiency and long period necessary for complete attachment of these cells to tissue culture plates, which is a disadvantage when having in mind short half-life of RhoB mRNA and protein [31]. In order to explore the general role of RhoB downregulation in cell response to cisplatin, additional cell types were analyzed. Initially, we screened the level of RhoB expression on a panel of eight human tumor cell lines of different origin (Fig. 6A) and for further experiments chose mammary adenocarcinoma MCF7, colorectal carcinoma SW480 and urinary bladder carcinoma RT-112 cells, because they expressed RhoB in a reasonably high level so that we can easily achieve RhoB silencing (Fig. 6B). However, as depicted on Fig. 6C, silencing of RhoB expression in these cell lines did not alter their sensitivity to cisplatin.

4. Discussion Greater insight into the molecular mechanisms regarding modulation of the cellular response to cisplatin should help to develop and optimize therapeutic strategies in tu-

mor treatment. Since our cisplatin-resistant CA3ST and CK2 cells displayed alterations in cell adhesion and cytoskeleton organization, as compared to their parental cisplatin-sensitive human laryngeal carcinoma HEp-2 cells, we screened the expression of several members of the family of Rho GTPases in this model of cisplatin resistance. We identified decreased expression of RhoB as distinctive feature associated with prolonged and recurring cisplatin exposure in tumor cell lines from different origin. In addition, we established, by overexpression of exogenous RhoB in cisplatin-resistant subline and silencing of endogenous RhoB in cisplatin-sensitive cell line, that in the laryngeal carcinoma cells RhoB downregulation confers resistance to cisplatin toxicity. Cisplatin-resistant CA3ST and CK2 cells displayed strong downregulation of RhoB, on both mRNA and protein level. The fact that these sublines were obtained by different resistance induction treatments, i.e. acute and chronic treatment, respectively, with different concentrations of cisplatin (see Section 2), argues against the possibility of random clonal selection. Supporting this notion, the expression of RhoA, rhoC, Rac1, Cdc42 and RhoGDIa was similar between resistant and sensitive cells. These findings are not surprising, since it is known that the RhoB expression level, unlike the expression of RhoA, Rac1 and Cdc42, is highly cell- and tissue-type dependent [28]. Decreased RhoB expression was also found in cisplatin-resistant human cervical carcinoma HeLa CK subline and melanoma MeWocis1 subline, indicating that downregulation of RhoB that is associated with cisplatin resistance is not restricted to a single tumor type and could be a broad-spectrum phenomenon. In accordance with the tumor-suppressor function of RhoB, tumor cells in general have lower expression of RhoB than normal surrounding tissue [10]. Interestingly, in head and neck squamous cell carcinomas the expression of RhoB further declines with the progression of disease [37]. Therefore, we can assume that in cisplatin-resistant laryngeal carcinoma cells RhoB downregulation could be accompanied by some other properties of progressive stage cancer. To our knowledge,

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Fig. 4. Silencing of endogenous RhoB expression in HEp-2 cells diminishes their sensitivity to cisplatin. (A) Western blot analysis of RhoA and RhoB expression in whole cell lysates after transient transfection of HEp-2 cells with 25 nM siRNA. ERK2 expression was used as the control for equal loading. (B) Sensitivity of transfected cells to cisplatin. The day after transfection cells were seeded in 96-well tissue culture plates and 24 h later cisplatin was added. Cytotoxicity assay was performed with a modified MTT assay after 96 h treatment with cisplatin and the results obtained were expressed as percent of untreated cells. (C) Platinum accumulation in siRNA transfected HEp-2 cells after 5 h of treatment with the indicated concentrations of cisplatin. Cellular platinum content was determined by HR-ICPSM, and the obtained data are presented as total amount of platinum per 105 cells or percent of platinum accumulation in siRhoB transfected as compared to siContr transfected cells. Each point represents the mean ± standard deviation of three independent experiments. p < 0.05. p < 0.001.

only one paper reported on RhoB expression in cells resistant to DNA damaging agents. However, the authors did not observe any difference in RhoB expression level between the subline resistant to ionizing radiation and parental HeLa cell line [38]. Nevertheless, RhoB expression is significantly decreased in osteoarthritic chondrocytes, which could be a reason for sustained pre- or para-apoptotic state of these chondrocytes, in spite of high level of DNA damage [39]. Even though rhoB is recognized as an early-inducible gene, quickly upregulated after various stimuli including growth factors [27,31] and stress [16,28], several reports suggest that RhoB induction is cell type and agent specific. Namely, untransformed cells like NIH/3T3 mouse fibroblasts can easily upregulate RhoB mRNA in different condi-

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Fig. 5. Expression of Rho GTPases in cisplatin-resistant human cervical carcinoma and melanoma cells. (A) Western blot analysis on whole cell lysates for RhoA, RhoB and Rac1 expression. ERK2 expression was used as the control for equal loading. The data shown are representative of at least three independent experiments. (B) Immunoblots were densitometrically analyzed, and the area determined is given as the means ± SD. (C) Western blot analysis of RhoB expression in whole cell lysates after transient transfection of HeLa cells with 25 nM siRNA. ERK2 expression was used as the control for equal loading. (D) Sensitivity of transfected HeLa cells to cisplatin. The day after transfection cells were seeded in 96well tissue culture plates and 24 h later cisplatin was added. Cytotoxicity assay was performed with a modified MTT assay after 96 h treatment with cisplatin and the results obtained were expressed as percent of untreated cells.

tions [16], while in transformed cell lines RhoB induction is cell type dependent. Specifically, rhoB transcription was induced upon EGF treatment of serum-deprived normal human breast epithelial cells, while this induction did not occur in all malignant mammary epithelial cell lines studied [40]. Given that constitutively active EGFR and ErbB2 silence RhoB expression through Ras [11], cell lines that express oncogenic Ras failed to upregulate RhoB after treatment with 5-FU [12]. Likewise, RhoB induction was not detected in HeLa cells exposed to ionizing radiation [38], nor in glioblastoma cells in the state of hypoxia, where RhoB was activated without any change in its expression level [41]. In line with these observations, we did not detect induction of RhoB after cisplatin treatment in our HEp-2 and CA3ST cells. However, we also could not detect RhoB protein induction in NIH/3T3 cells (data not shown), which were previously shown to upregulate rhoB mRNA after cisplatin treatment [16], suggesting that the induction of RhoB in some cell types could be restricted to mRNA. To test the hypothesis that the difference in RhoB expression level between these cell lines determines the extent of sensitivity to cisplatin we used two different approaches: (1) transfection of EGFP-RhoB expressing plasmid vector in cisplatin-resistant CA3ST cells and (2) silencing of endogenous RhoB expression in cisplatin-sensitive HEp-2 cells; followed by cisplatin treatment. As ex-

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Fig. 6. Silencing of endogenous RhoB expression in various human tumor cell lines does not alter their sensitivity to cisplatin. Western blot analysis on whole cell lysates for RhoB expression in (A) different human tumor cell lines and (B) after transient transfection of MCF7, SW480 or RT-112 cells with 25 nM siRNA. ERK2 expression was used as the control for equal loading. The data shown are representative of at least three independent experiments. (C) Sensitivity of transfected cells to cisplatin. The day after transfection cells were seeded in 96-well tissue culture plates and 24 h later cisplatin was added. Cytotoxicity assay was performed with a modified MTT assay after 96 h treatment with cisplatin and the results obtained were expressed as percent of untreated cells.

pected, obtained results verified protective role of RhoB downregulation in the response of laryngeal carcinoma cells to cisplatin. Moreover, cisplatin-resistant CK2 subline also displayed increase in cisplatin-induced cell death after transient transfection with EGFP-RhoB plasmid in a pattern similar to the one found in CA3ST subline (data not shown). In the literature, there are conflicting data regarding the role of RhoB in cellular response to genotoxic stress. Namely, RhoB was protective in UV-irradiated human keratinocytes [42], as well as stably transfected NIH/3T3 fibroblasts, that were also less sensitive to doxorubicin [17]. Moreover, several papers consent that RhoB confers resistance to ionizing radiation-induced mitotic cell death in both mouse fibroblasts and human glioma cells [7,36,38,43]. On the other hand, NIH/3T3 fibroblasts stably transfected with RhoB were more sensitive to alkylating agents, among others cisplatin [17] and 5-FU [12,35], while transformed (but not normal) RhoB / embryonic mouse fibroblasts are resistant to apoptosis induced by doxorubicin, gamma irradiation and taxol [44]. However, silencing of RhoB in our HEp-2 cells did not provide significant difference in their response to doxorubicin or vincristine, while carboplatin treatment gave the same pattern of resistance as cisplatin (data not shown). In addition, our results on RhoB silencing in several tumor cell lines of different origin do not support a general role of RhoB downregulation in providing cisplatin resistance. Therefore, it seems that the mere involvement, and specifically pro- or antisurvival role, of RhoB is determined by the toxic agent itself, or the cell death pathway induced by that particular agent in a particular cell type. Similar divergence holds for anti- and pro-apoptotic role of differently prenylated RhoB: farnesylated RhoB, which is proposed to have antiapopoptotic function [36], and geranylgeranylated RhoB, that appears to uniformly

act as a proapoptotic factor [12,35]. However, these data were obtained on NIH/3T3 mouse fibroblasts that are clearly different in RhoB downstream functions, since in these cells RhoB causes NF-jB inhibition [32], rather than activation, as found in multiple cell types [45,46]. Our results, showing that both RhoB-F and RhoB-GG have antisurvival function, are in accordance with the ones derived on human pancreatic cancer cell line Panc-1 [25]. Thus, in our cisplatin-resistant cells both prenylated forms of exogenously expressed RhoB have similar subcellular localization, which coincides with their comparable antisurvival role. Untreated CA3ST cells transfected with RhoB-expressing plasmids had higher fraction of propidium-iodide positive cells than mock-transfected cells, which is in accordance with previously published data [25]. This observation suggests that exogenous RhoB primes cells to cisplatin-induced cell death. However, we also established that silencing of RhoB expression reduced cisplatin accumulation in HEp-2 cells, which indicates that endogenous RhoB acts upstream of cisplatin-induced DNA damage, probably already on the level of intracellular trafficking of cisplatin. In this context it should be noted that in the literature RhoB was detected in various endosomal compartments, Golgi apparatus or plasma membrane, and has an established role in endosomal trafficking of EGF receptor [10]. However, although we put a lot of effort to determine cellular localization of endogenous RhoB by confocal microscopy, and used siRhoB transfected cells as an appropriate negative control, we were unsuccessful due to unsuitability of anti-rhoB antibody for immunocytochemistry. In the preclinical ovarian cancer models, transduction of the recombinant adenovirus expressing the gene coding for wild type RhoB induced apoptosis in ovarian carcinoma cells in vitro, whereas viral transduction into nude mice

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abolished tumor cell proliferation in vivo [47]. Hence, these recent findings in conjunction with the data presented in this paper imply that the gene therapy with the wild type RhoB might improve the efficiency of cisplatin treatment, particularly in patients that acquired resistance to this chemotherapeutic drug. However, one has to bear in mind that the available results do not support a general role of RhoB in the response to genotoxic stress and that its regulatory function in cellular response to cisplatin apparently depends on the biological context. Conflict of interest Actual or potential conflicts of interest do not exist. Acknowledgements We thank Prof. Dirk Schadendorf from German Cancer Research Center and University Hospital Mannheim, Heidelberg, Germany, for providing us with MeWo and MeWocis1 cell lines. We also thank Ljiljana Krajcar for technical assistance. This work was supported by funds of the Ministry of Science and Technology of the Republic of Croatia (Project No. 098-0982913-2748) and DFG FR 1241/5-3. The funding sources had no role in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.canlet.2010.02.025. References [1] L. Kelland, The resurgence of platinum-based cancer chemotherapy, Nat. Rev. Cancer 7 (2007) 573–584. [2] G. Giaccone, Clinical perspectives on platinum resistance, Drugs 59 (2000) 9–17. [3] A. Ambriovic´-Ristov, M. Osmak, Integrin-mediated drug resistance, Curr. Signal. Transduct. Ther. 1 (2006) 227–237. [4] A. Brozovic, M. Osmak, Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance, Cancer Lett. 251 (2007) 1–16. [5] D.J. Stewart, Mechanisms of resistance to cisplatin and carboplatin, Crit. Rev. Oncol. Hematol. 63 (2007) 12–31. [6] M.A. Schwartz, S.J. Shattil, Signaling networks linking integrins and rho family GTPases, Trends Biochem. Sci. 25 (2000) 388–391. [7] S. Monferran, N. Skuli, C. Delmas, G. Favre, J. Bonnet, E. CohenJonathan-Moyal, C. Toulas, Fvb3 and avb5 integrins control glioma cell response to ionising radiation through ILK and RhoB, Int. J. Cancer 123 (2008) 357–364. [8] F.M. Vega, A.J. Ridley, Rho GTPases in cancer cell biology, FEBS Lett. 582 (2008) 2093–2101. [9] S. Etienne-Manneville, A. Hall, Rho GTPases in cell biology, Nature 420 (2002) 629–635. [10] M. Huang, G.C. Prendergast, RhoB in cancer suppression, Histol. Histopathol. 21 (2006) 213–218. [11] K. Jiang, F.L. Delarue, S.M. Sebti, EGFR, ErbB2 and Ras but not Src suppress RhoB expression while ectopic expression of RhoB antagonizes oncogene-mediated transformation, Oncogene 23 (2004) 1136–1145. [12] K. Jiang, J. Sun, J. Cheng, J.Y. Djeu, S. Wei, S. Sebti, Akt mediates Ras downregulation of RhoB, a suppressor of transformation, invasion, and metastasis, Mol. Cell. Biol. 24 (2004) 5565–5576.

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