Melanoma cell sensitivity to Docetaxel-induced apoptosis is determined by class III β-tubulin levels

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FEBS Letters 582 (2008) 267–272

Melanoma cell sensitivity to Docetaxel-induced apoptosis is determined by class III b-tubulin levels Nizar M. Mhaidata,1, Rick F. Thorneb,1, Charles Edo de Bockc, Xu Dong Zhangd, Peter Herseyd,* a Department of Clinical Pharmacy, Jordan University of Science and Technology, Irbid 22110, Jordan Cancer Research Unit at the University of Newcastle, University Drive, Callaghan Newcastle, NSW 2308, Australia c Queensland Institute of Medical Research, Herston, QLD 4006, Australia Immunology and Oncology Unit, Room 443, David Maddison Clinical Sciences Building, Cnr. King & Watt Streets, Newcastle, NSW 2300, Australia b


Received 25 October 2007; accepted 6 December 2007 Available online 18 December 2007 Edited by Richard Marais

Abstract We have previously shown that Docetaxel-induced variable degrees of apoptosis in melanoma. In this report, we studied the b-tubulin repertoire of melanoma cell lines and show that class III b-tubulin expression correlated with Docetaxelresistance. Sensitive cells showed low levels of class III b-tubulin with little microtubular incorporation, whereas class III b-tubulin expression was higher in resistant cells and was incorporated into the cytoskeleton. As proof of concept, abrogation of class III by siRNA reverted Docetaxel-resistant cells to a sensitive phenotype, restoring the microtubular polymerisation response and promoting high levels of apoptosis through Bax activation. These results suggest that phenotypic expression of b-tubulin class III in melanoma may help identify patients with melanoma that can respond to taxanes.  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Melanoma; Docetaxel; Class III b-tubulin; Apoptosis; Taxanes

inappropriately induce activation of pro-survival signaling pathways such as Ras–Raf–MEK–ERK pathway leading to cancer cell resistance [7–9]. Studies have also shown that alteration in the composition and mutations of b-tubulin isotypes, can lead to resistance to taxanes [10,11]. Expression of mutant tubulin or differential expression of certain tubulin isotypes has been shown to correlate with the resistance profile of taxaneresistant cells [10–12]. In some cases, the induced expression of mutant tubulins [13] and b-tubulin isotypes classes III and V [14,15] were shown to confer resistance to paclitaxel. Purified mutant tubulins also have altered polymerisation characteristics [16] and increased expression of classes I, III, and IVa isotypes was reported in paclitaxel-resistant cells [10]. In the present study, we have explored the relation between tubulin isotypes and sensitivity of melanoma cells to Docetaxel and report that high level of class III b-tubulin can confer resistance to Docetaxel-induced apoptosis. Moreover, a mechanism is suggested by the increased incorporation of class III b-tubulin into the microtubular network in Docetaxel-resistant melanoma cells.

1. Introduction

2. Materials and methods

Given the pivotal importance of microtubules in many cellular functions, they have been the targets for anticancer drugs such as taxanes and vinca alkaloids. By interfering with the dynamics of microtubule assembly, microtubule-binding agents exert profound effects on cellular processes such as gene expression, cell cycle arrest, and apoptosis [1,2]. Taxanes represent an important class of anticancer agents that have anticancer effects in vitro and in vivo against cancers of lung, ovaries, breast, and leukemia [3]. Recently, we have extended these findings showing that Docetaxel induces caspase-dependent apoptosis of some but not all human melanoma cell lines [4]. Resistance of cancer cells to taxanes has been attributed to various mechanisms including over-expression of (multi-drug resistance phenotype) P-glycoprotein that induces efflux of the drug [5,6]. Other studies have reported that taxanes may

2.1. Cell lines The panel of human melanoma cell lines used have been described previously [17].


Corresponding author. Fax: +61 2 49236184. E-mail address: [email protected] (P. Hersey). 1

These authors made an equal contribution towards the work.

Abbreviation: MMP, mitochondrial membrane potential

2.2. Antibodies and other reagents Docetaxel (Taxotere), kindly provided by Aventis Pharma S.A (France), was stored as a 100 mM solution in absolute ethanol at 80 C and diluted immediately prior to use. The polyclonal antiBax antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The 107.3 mouse IgG1 antibody was purchased from PharMingen (San Diego, CA). Monoclonal and polyclonal anti-atubulin, classes I, III, and IV b-tubulin and b-actin were purchased from Sigma–Aldrich (Castle Hill, NSW, Australia). 2.3. Apoptosis Quantitation of apoptotic cells by measurement of sub-G1 DNA content using the PI method was carried out as described elsewhere [18] under conditions established from previous work [4]. 2.4. Measurement of tubulin polymerisation Cytosolic and polymerised fractions of tubulin were separated by differential centrifugation as previously described [15]. Briefly cells were lysed in microtubule-stabilizing buffer (20 mM Tris–HCl (pH 6.8), 0.14 M NaCl, 0.5% NP40, 1 mM MgCl2, 2 mM EGTA, and 10 ll/ml protease inhibitor cocktail (Sigma), with 4 lg/ml Docetaxel). The polymerised fraction was obtained by collecting the supernatant

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.12.014

268 fraction after centrifuging the lysate at 10,000 · g for 20 min. The pellet (polymerised tubulin) was solubilised before electrophoresis in SDS–PAGE sample buffer. 2.5. Indirect immunofluorescence and confocal microscopy Melanoma cells seeded onto glass coverslips were fixed after the indicated treatments before permeation with 0.1% Triton X-100. After blocking with 3% bovine serum albumin, cells were immunostained with primary antibodies before sequential detection with Alexa-488 anti-mouse IgG and or Alexa-594 anti-rabbit IgG (Invitrogen, Australia). In some experiments nuclei were visualised by DAPI staining. After mounting in SlowFade Gold reagent cells were examined using a Zeiss Axioplan 2 epifluorescence microscope or Zeiss Axiovert 100 M fitted with the LSM510 confocal system (Oberkochem, Germany). 2.6. Flow cytometry and mitochondrial membrane potential (DWm) Flow cytometric analysis of permeabilised cells for Bax [19] and mitochondrial membrane using JC-1 staining under established Docetaxel treatment conditions as previously described [4]. 2.7. Western blot and protein expression analysis Western blots were performed as described previously [19]. Relative expression was determined against control proteins (GAPDH or actin) using densitometric analysis. 2.8. Small RNA interference (siRNA) Transfections were performed for 48–72 h as previously described [4] prior to each assay using either SiConTRol Non-targeting SiRNA pool (D-001206-13-20) or the siGENOME SMARTpool for class III b-tubulin (TUBB3) siRNA (Dharmacon, Lafayette, CO). 2.9. Cell viability assays Cell viability assays were performed using the MTT method. After siRNA treatment cells were seeded at 500 cells/well in 96 well plates and allowed to adhere overnight before addition of Docetaxel. After

N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272 a further 72 h 30 ll of 5 mg/ml MTT (3-(4,5-dimethyl thiazolyl-2)2,5-diphenyl tetrazolium bromide; Sigma) was added to each well for 2 h. The supernatant was removed, the MTT formazan crystals dissolved in 100 ll of DMSO and the optical density read at 550 nm. 2.10. Statistical analysis The statistical significance of intergroup differences was determined using StudentÕs t-test. P values 60.05 and 60.001 are indicated by * and **, respectively. Regression analyses were performed using the StatView program.

3. Results 3.1. Docetaxel-resistant melanoma cells have low levels of polymerized tubulin Previous studies have shown that Docetaxel binds to the btubulin subunit of the microtubules resulting in their polymerisation [20]. We studied the Docetaxel-induced polymerisation response of microtubules in the IgR3 and MM200 human melanoma cell lines that have been shown previously to be differentially sensitive to this agent [4]. Lysates were prepared from untreated and Docetaxel treated cells and the samples were fractionated into soluble and polymerized tubulin fractions as described in Section 2. Western blotting analysis showed that under normal growth conditions, MM200 cells have a lower level of polymerized tubulin compared to IgR3 cells (Fig. 1A). Densitometric analysis showed that the percentage of polymerized tubulin before and after treatment with Docetaxel was changed from 47% to 86% in IgR3 (P 6 0.05) whereas a slight increase from 22% to 31% was seen in

Fig. 1. Docetaxel induces tubulin polymerisation in melanoma cells. (A) Soluble and polymerized fractions of tubulin were separated from cells treated with or without Docetaxel at 20 nM for 3 h and the relative amounts were determined by Western blotting using antibodies against a-tubulin. (B) Data collected from three individual experiments shown in (A) was analysed by densitometry (Columns, mean and bars, S.E.M.). (C) Cells were also analysed by immunofluorescence microscopy for a-tubulin (green) and nuclear staining (DAPI; blue) under the treatment conditions described for (A). bar = 20 lm.

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Fig. 2. Expression of different b-tubulin isotypes in melanoma cells. (A) Total cellular protein from a panel of melanoma cells was subjected to Western blots for classes I, III, and IV b-tubulins. (B) The relative expression of each class was determined by dividing the densitometric value against a b-actin control. Columns, mean of three individual experiments; bars, S.E.. (C) Correlation between the relative expression of class III b-tubulin and the levels of apoptosis induced by 20 nM Docetaxel after 48 h.

MM200 cells (Fig. 1B). These results were confirmed by immunofluorescent staining showing in IgR3 cells that Docetaxel treatment greatly intensified the microtubular network. In contrast, the microtubular network of MM200 cells was largely unchanged with Docetaxel treatment (Fig. 1C). 3.2. Human melanoma cells have variable expression of classes III and IV b-tubulin isotypes The ability of taxanes to bind and polymerize tubulin was reported to depend upon the expression of specific b-tubulin isotypes [21,22]. The expression of various classes of b-tubulin was examined in a panel of melanoma cell lines by immunoblotting. Fig. 2A shows that melanoma cells express classes I, III, and IV b-tubulin. Expression of class I b-tubulin was similar between different melanoma cells whereas classes III and IV b-tubulin varied in expression levels (Fig. 2A and B). Since we have shown previously that Docetaxel induces variable degrees of apoptosis in melanoma cells [4] we examined whether this variable expression of classes III and IV b-tubulin tubulin levels correlated with sensitivity to Docetaxel. Fig. 2C indicates that the expression of class III b-tubulin was inversely correlated with the degree of Docetaxel-induced apoptosis. No significant correlations were seen between expression of class IV b-tubulin and Docetaxel-induced apoptosis (R2 = 0.1699) or with an index considering the combined effects of class III plus class IV b-tubulin expression versus apoptosis (data not shown). Next, we examined the cellular distribution of class III btubulin in both the sensitive IgR3 and the resistant MM200 cells before and after treatment with Docetaxel (Fig. 3). Before treatment, the staining for class III b-tubulin in the IgR3 cells revealed a largely cytoplasmic distribution whereas a proportion of class III b-tubulin was clearly incorporated into the microtubular network in the MM200 cells. Treatment with Docetaxel caused little alteration in MM200 cells with much of the class III b-tubulin retained in microtubules. In contrast,

Docetaxel caused significant disruption of IgR3 cells with class III b-tubulin localised in membranous blebs and it did not appear to contribute to the microtubular network. The incorporation of class III b-tubulin into the microtubular network therefore appears to phenotypically correlate to the mechanism of melanoma resistance to Docetaxel. 3.3. Silencing of class III b-tubulin sensitized melanoma cells to Docetaxel-induced apoptosis The results suggested that resistance of melanoma cells to Docetaxel-induced apoptosis was due to high expression of class III b-tubulin. We therefore examined whether modulation of expression levels of class III b-tubulin altered Docetaxel-resistance in melanoma cells. As shown in Fig. 4A, silencing of class III b-tubulin using siRNA inhibited expression by 79% in MM200 cells compared to the cells transfected with a control siRNA. Expression levels of classes I and IV btubulins were not affected emphasising the specificity of the class III b-tubulin siRNA. Knockdown of class III b-tubulin expression significantly enhanced microtubule polymerisation (P 6 0.05) and this was accompanied by significant increases in apoptosis induced by Docetaxel (Fig. 4B). This was particularly dramatic in the MM200 cells reverting these resistant cells to a sensitive phenotype (P 6 0.001). The increased Docetaxel-induced apoptosis observed after class III b-tubulin silencing was also accompanied by enhanced Bax activation and increases in mitochondrial membrane potential (MMP) changes (Fig. 4C; P 6 0.001). Furthermore, class III b-tubulin siRNA increased the anti-proliferative effects of Docetaxel in both MM200 and IgR3 cells, reducing the IC50 from 30 to 10.5 nM and 9 to 2 nM respectively (Fig. 4D). In order to determine if class III b-tubulin transfection could increase the levels of resistance of IgR3 cells to Docetaxel, we derived permanent transfectants of these cells over-expressing class III b-tubulin (Suppl. Fig. 1A). Although these cells displayed expression of class III b-tubulin at levels similar to


N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272

Fig. 3. Comparative distribution of a-tubulin with class III b-tubulin in IgR3 and MM200 melanoma cells before and after treatment with 20 nM Docetaxel for 3 h. Results were recorded by dual colour confocal microscopy using monoclonal a-tubulin and class III b-tubulin polyclonal antibodies, bar = 20 lm.

Fig. 4. Inhibition of class III b-tubulin expression sensitised melanoma cells to Docetaxel-induced apoptosis. (A) MM200 cells were transfected with control or class III b-tubulin-specific siRNA (TUBB3) and whole cell lysates subjected to Western blot analysis for classes I, III, IV b-tubulins and a control protein, b-actin. Data shown are representative of three individual experiments. (B) Measurements of apoptosis and tubulin polymerisation in siRNA transfected IgR3 and MM200 cells were performed with or without 20 nM Docetaxel treatment after 48 and 3 h, respectively. (C) Measurements of Bax activation and DWm by JC-1 staining in siRNA transfected IgR3 and MM200 cells were performed after 16–24 h with or without 20 nM Docetaxol treatment. Data in B and C represent the means ± S.E. of three individual experiments. (D) IgR3 and MM200 cells were treated with control or TUBB3 siRNA were subjected to the indicated dose titration of Docetaxel. After 72 h cell viability was measured using the MTT assay. Data were averaged from triplicate determinations and normalised to the control condition, bars, S.E.M.

MM200 cells (refer Fig. 2B), they were not more resistant to Docetaxel in assays measuring apoptosis or cell viability (Suppl. Fig. 1C and D). However permanent transfection of class

III b-tubulin did result in a modest increase in the expression of a-tubulin (Suppl. Fig. 1B) that may account for this discrepancy (see Section 4).

N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272

4. Discussion In the present study, we have explored for the first time the relationship between microtubular polymerisation, the expression of tubulin isotypes and the resistance of human melanoma cell lines to Docetaxel. We have chosen to use a relatively large panel of cell lines that have different intrinsic properties rather than deriving resistant tumour sublines by selection with increasingly higher doses of drugs. The latter method is often used to mimic events that lead to acquired drug resistance but we believe our approach, which has been far more comprehensive than many of the cited studies, may be advantageous in minimising the likelihood of in vitro artefacts. The results showed that there was generally a low rate of tubulin polymerisation in resistant melanoma cells and that sensitive cells responded by increasing the microtubular mass whereas resistant cells did not. This result appeared entirely consistent with the known mode of action of taxanes. Examination of b-tubulin isotype expression revealed there was very little variation in class I tubulin expression. Rather the expression of class III b-tubulin, which varied widely between different melanoma cell lines, appeared to be a major determinant of the degree of tubulin polymerisation and to the resistance to Docetaxel. This concept was confirmed using an siRNA mediated strategy where knockdown of class III b-tubulin enhanced Docetaxel-induced tubulin polymerisation, activation of Bax and downstream apoptotic events that manifested in reduced cell proliferation. In contrast to the results obtained using siRNA against class III b-tubulin, we were unable to demonstrate that high level expression of class III b-tubulin by transfection could promote the resistance of IgR3 melanoma cells to Docetaxel. This somewhat unexpected result has been previously reported. For example, transfection of class III b-tubulin into prostate cancer cells, which is upregulated in these cells by Paclitaxel exposure, was unable to confer resistance to Paclitaxel and other microtubule targeting agents [23]. However, it was determined that stable transfection of class III b-tubulin actually caused compensatory increases in the expression of a-tubulin in conjunction with increases in both class II and class IV btubulins. The authors of this work as well as Hari et al. [15] have both flagged the difficulties encountered when using gene transfection to assay the contribution of individual tubulin isotypes. The mechanism of the compensatory phenomenon is not well studied but appears to be acting through transcriptional regulation [23] and this also appears to be occurring in the IgR3 cells with a modest increase in a-tubulin expression noted to occur. We conclude from our study that siRNA is a preferable technique to demonstrate the functional effects on individual tubulin isotypes. This is probably because its effects generally occur quite rapidly and its actions are effective against the entire cell population under treatment. While our study showed that class III b-tubulin appeared important in unselected melanoma cell lines, studies in other types of cancer cell lines have revealed that alterations in btubulins occurs with resistance to microtubule-targeting drugs although not always associated with a single isotype. For example, the increased expression of classes III and IVa btubulin has also been associated with the Paclitaxel-resistance in non-small lung carcinoma cell lines [10] and in K562 erythroleukemia cells [24]. Docetaxel-resistant MCF-7 breast cancer cells also exhibited increased expression of classes I, II, III and


IV b-tubulin isotypes whereas all isotypes examined except for class IV were decreased in Docetaxel-resistant MB-MDA-231 breast cancer cells [25]. Notwithstanding the differences observed between various cancer cell lines, increased levels of b-tubulins do appear to have significance for clinical outcomes with taxanes. Samples of ovarian tumours from patients treated with Paclitaxel displayed increased levels of classes I, III and IVa b-tubulins when compared to non-treated patients [10]. More recently, acquired expression of class III b-tubulin was not seen in another study of ovarian cancer, however, the pre-treatment level of this isotype did act as an independent prognostic indicator of poor outcome [26]. Similarly, increased expression of classes I and III b-tubulin in breast [27] and class III b-tubulin non-small cell lung cancer [28] also appear to predict poor outcomes with Paclitaxel and Docetaxel, respectively. Questions still remain in regard to the mechanism(s) that underlie the prognostic significance of b-tubulin isotypes. One model proposed by Carbral and colleagues associates unstable or dynamic microtubules with the resistance of cells to microtubule targeting agents [29,30]. In accordance, when class III b-tubulin is assembled into microtubules it has been reported to result in more unstable microtubules [30] that are less sensitive to Paclitaxel [31]. In our study we observed that Docetaxel-resistance in melanoma cells coincided with increased class III b-tubulin incorporation into the microtubular network. High levels of class III b-tubulin have also been reported to reduce the assembly of microtubules [15] and this is also consistent with our results measuring the percentages of microtubular polymerisation. Together these data suggest that the large monomeric pool of class III b-tubulin acts to promote dynamic/unstable microtubules that in turn limits the polymerised microtubular mass in response to Docetaxel. Towards an understanding of how this process may be regulated, we have recently reported that PKCe is associated with pro-survival signaling through ERK in melanoma cells treated with Docetaxel [32]. Moreover PKCe was also shown to coimmunoprecipitate with cytosolic class III b-tubulin suggesting it may also be influencing the incorporation rates of class III btubulin into microtubules. To our knowledge no clinical data currently exists on the expression of b-tubulin isotypes in melanoma in vivo. In light of the work presented here there is now a need determine if expression of class III b-tubulin can be correlated with tumour responses in xenograft models and in melanoma patients treated with taxanes. It will also be important to better understand the complex mechanisms governing the cellular regulation of the tubulin repertoire that is clearly very important in determining how cells respond to taxanes. Acknowledgements: This work was supported by the NSW State Cancer Council and National Health and Medical Research Council of Australia. X.D. Zhang is a Cancer Institute NSW Fellow. The authors would also like to thank Mr. Tiongsun Chia for his invaluable technical assistance.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet. 2007.12.014.


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