FEBS Letters 582 (2008) 267–272

Melanoma cell sensitivity to Docetaxel-induced apoptosis isdetermined 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, Jordanb Cancer Research Unit at the University of Newcastle, University Drive, Callaghan Newcastle, NSW 2308, Australia

c Queensland Institute of Medical Research, Herston, QLD 4006, Australiad Immunology and Oncology Unit, Room 443, David Maddison Clinical Sciences Building, Cnr. King & Watt Streets, Newcastle, NSW 2300, Australia

Received 25 October 2007; accepted 6 December 2007

Available online 18 December 2007

Edited by Richard Marais

Abstract We have previously shown that Docetaxel-inducedvariable degrees of apoptosis in melanoma. In this report, westudied the b-tubulin repertoire of melanoma cell lines and showthat class III b-tubulin expression correlated with Docetaxel-resistance. Sensitive cells showed low levels of class III b-tubulinwith little microtubular incorporation, whereas class III b-tubu-lin expression was higher in resistant cells and was incorporatedinto the cytoskeleton. As proof of concept, abrogation of classIII by siRNA reverted Docetaxel-resistant cells to a sensitivephenotype, restoring the microtubular polymerisation responseand promoting high levels of apoptosis through Bax activation.These results suggest that phenotypic expression of b-tubulinclass III in melanoma may help identify patients with melanomathat can respond to taxanes.� 2007 Federation of European Biochemical Societies. Pub-lished by Elsevier B.V. All rights reserved.

Keywords: Melanoma; Docetaxel; Class III b-tubulin;Apoptosis; Taxanes

1. Introduction

Given the pivotal importance of microtubules in many cellu-

lar 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 rep-

resent an important class of anticancer agents that have anti-

cancer effects in vitro and in vivo against cancers of lung, ova-

ries, 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

Abbreviation: MMP, mitochondrial membrane potential

*Corresponding author. Fax: +61 2 49236184.E-mail address: Peter.Hersey@newcastle.edu.au (P. Hersey).

1These authors made an equal contribution towards the work.

0014-5793/$32.00 � 2007 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2007.12.014

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 alter-

ation 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 taxane-

resistant 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 characteris-

tics [16] and increased expression of classes I, III, and IVa iso-

types 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 mech-

anism is suggested by the increased incorporation of class III

b-tubulin into the microtubular network in Docetaxel-resistant

melanoma cells.

2. Materials and methods

2.1. Cell linesThe panel of human melanoma cell lines used have been described

previously [17].

2.2. Antibodies and other reagentsDocetaxel (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 anti-Bax antibody was purchased from Upstate Biotechnology (Lake Pla-cid, NY). The 107.3 mouse IgG1 antibody was purchased fromPharMingen (San Diego, CA). Monoclonal and polyclonal anti-a-tubulin, classes I, III, and IV b-tubulin and b-actin were purchasedfrom Sigma–Aldrich (Castle Hill, NSW, Australia).

2.3. ApoptosisQuantitation 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 polymerisationCytosolic and polymerised fractions of tubulin were separated by

differential centrifugation as previously described [15]. Briefly cellswere lysed in microtubule-stabilizing buffer (20 mM Tris–HCl (pH6.8), 0.14 M NaCl, 0.5% NP40, 1 mM MgCl2, 2 mM EGTA, and10 ll/ml protease inhibitor cocktail (Sigma), with 4 lg/ml Docetaxel).The polymerised fraction was obtained by collecting the supernatant

blished by Elsevier B.V. All rights reserved.

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

fraction after centrifuging the lysate at 10,000 · g for 20 min. The pel-let (polymerised tubulin) was solubilised before electrophoresis inSDS–PAGE sample buffer.

2.5. Indirect immunofluorescence and confocal microscopyMelanoma cells seeded onto glass coverslips were fixed after the indi-

cated treatments before permeation with 0.1% Triton X-100. Afterblocking with 3% bovine serum albumin, cells were immunostainedwith primary antibodies before sequential detection with Alexa-488anti-mouse IgG and or Alexa-594 anti-rabbit IgG (Invitrogen, Austra-lia). In some experiments nuclei were visualised by DAPI staining. Aftermounting in SlowFade Gold reagent cells were examined using a ZeissAxioplan 2 epifluorescence microscope or Zeiss Axiovert 100 M fittedwith 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 Doce-taxel treatment conditions as previously described [4].

2.7. Western blot and protein expression analysisWestern 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 SiRNApool (D-001206-13-20) or the siGENOME SMARTpool for class IIIb-tubulin (TUBB3) siRNA (Dharmacon, Lafayette, CO).

2.9. Cell viability assaysCell viability assays were performed using the MTT method. After

siRNA treatment cells were seeded at 500 cells/well in 96 well platesand allowed to adhere overnight before addition of Docetaxel. After

Fig. 1. Docetaxel induces tubulin polymerisation in melanoma cells. (A) Sotreated with or without Docetaxel at 20 nM for 3 h and the relative amounts w(B) Data collected from three individual experiments shown in (A) was analyalso analysed by immunofluorescence microscopy for a-tubulin (green) and nfor (A). bar = 20 lm.

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 for2 h. The supernatant was removed, the MTT formazan crystals dis-solved in 100 ll of DMSO and the optical density read at 550 nm.

2.10. Statistical analysisThe statistical significance of intergroup differences was determined

using Student�s t-test. P values 60.05 and 60.001 are indicated by *and **, respectively. Regression analyses were performed using theStatView program.

3. Results

3.1. Docetaxel-resistant melanoma cells have low levels of

polymerized tubulin

Previous studies have shown that Docetaxel binds to the b-

tubulin subunit of the microtubules resulting in their polymer-

isation [20]. We studied the Docetaxel-induced polymerisation

response of microtubules in the IgR3 and MM200 human mel-

anoma cell lines that have been shown previously to be differ-

entially 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 Doce-

taxel was changed from 47% to 86% in IgR3 (P 6 0.05)

whereas a slight increase from 22% to 31% was seen in

luble and polymerized fractions of tubulin were separated from cellsere determined by Western blotting using antibodies against a-tubulin.

sed by densitometry (Columns, mean and bars, S.E.M.). (C) Cells wereuclear staining (DAPI; blue) under the treatment conditions described

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

N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272 269

MM200 cells (Fig. 1B). These results were confirmed by immu-

nofluorescent staining showing in IgR3 cells that Docetaxel

treatment greatly intensified the microtubular network. In con-

trast, 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 re-

ported to depend upon the expression of specific b-tubulin iso-

types [21,22]. The expression of various classes of b-tubulin

was examined in a panel of melanoma cell lines by immuno-

blotting. 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 var-

iable 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 inver-

sely correlated with the degree of Docetaxel-induced apopto-

sis. 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 ef-

fects of class III plus class IV b-tubulin expression versus

apoptosis (data not shown).

Next, we examined the cellular distribution of class III b-

tubulin 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 propor-

tion 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 ap-

pear to contribute to the microtubular network. The incorpo-

ration of class III b-tubulin into the microtubular network

therefore appears to phenotypically correlate to the mecha-

nism 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 modula-

tion of expression levels of class III b-tubulin altered Doce-

taxel-resistance in melanoma cells. As shown in Fig. 4A,

silencing of class III b-tubulin using siRNA inhibited expres-

sion by 79% in MM200 cells compared to the cells transfected

with a control siRNA. Expression levels of classes I and IV b-

tubulins 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 partic-

ularly dramatic in the MM200 cells reverting these resistant

cells to a sensitive phenotype (P 6 0.001). The increased Doce-

taxel-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 dis-

played expression of class III b-tubulin at levels similar to

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

Fig. 4. Inhibition of class III b-tubulin expression sensitised melanoma cells to Docetaxel-induced apoptosis. (A) MM200 cells were transfected withcontrol 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 acontrol protein, b-actin. Data shown are representative of three individual experiments. (B) Measurements of apoptosis and tubulin polymerisationin 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 orwithout 20 nM Docetaxol treatment. Data in B and C represent the means ± S.E. of three individual experiments. (D) IgR3 and MM200 cells weretreated with control or TUBB3 siRNA were subjected to the indicated dose titration of Docetaxel. After 72 h cell viability was measured using theMTT assay. Data were averaged from triplicate determinations and normalised to the control condition, bars, S.E.M.

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

MM200 cells (refer Fig. 2B), they were not more resistant to

Docetaxel in assays measuring apoptosis or cell viability (Sup-

pl. 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 discrep-

ancy (see Section 4).

N.M. Mhaidat et al. / FEBS Letters 582 (2008) 267–272 271

4. Discussion

In the present study, we have explored for the first time the

relationship between microtubular polymerisation, the expres-

sion 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 compre-

hensive 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. Exami-

nation of b-tubulin isotype expression revealed there was very

little variation in class I tubulin expression. Rather the expres-

sion of class III b-tubulin, which varied widely between differ-

ent 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 med-

iated strategy where knockdown of class III b-tubulin en-

hanced Docetaxel-induced tubulin polymerisation, activation

of Bax and downstream apoptotic events that manifested in re-

duced 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 deter-

mined 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 b-

tubulins. 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 iso-

types. 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 prefer-

able technique to demonstrate the functional effects on individ-

ual 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 b-

tubulins 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 b-

tubulin has also been associated with the Paclitaxel-resistance

in non-small lung carcinoma cell lines [10] and in K562 eryth-

roleukemia 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 ob-

served 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 trea-

ted 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 indepen-

dent prognostic indicator of poor outcome [26]. Similarly, in-

creased 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 Doce-

taxel, 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 in-

creased class III b-tubulin incorporation into the microtubular

network. High levels of class III b-tubulin have also been re-

ported 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 regu-

lated, 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 co-

immunoprecipitate with cytosolic class III b-tubulin suggesting

it may also be influencing the incorporation rates of class III b-

tubulin 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 trea-

ted 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 deter-

mining how cells respond to taxanes.

Acknowledgements: This work was supported by the NSW State Can-cer Council and National Health and Medical Research Council ofAustralia. X.D. Zhang is a Cancer Institute NSW Fellow. The authorswould also like to thank Mr. Tiongsun Chia for his invaluable techni-cal 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.

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

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