Preclinical Development

Contrasting Effects of Nutlin-3 on TRAIL- andDocetaxel-Induced Apoptosis Due to Upregulationof TRAIL-R2 and Mcl-1 in Human Melanoma Cells

Hsin-Yi Tseng1, Chen Chen Jiang1, Amanda Croft1, Kwang Hong Tay1, Rick Francis Thorne2,Fan Yang1, Hao Liu1, Peter Hersey1, and Xu Dong Zhang1

AbstractWild-type p53 is commonly expressed in melanoma but does not appear to be effective in the induction of

apoptosis. One explanation is that p53 is targeted for degradation by the E3 ligaseMDM2. However, we found

in this study that blockade of the interaction of p53 andMDM2 by theMDM2 antagonist nutlin-3 inmelanoma

cells did not induce apoptosis, even though it upregulated p53 and its proapoptotic targets. Nevertheless,

nutlin-3 enhanced TRAIL-induced apoptosis as a result of p53-mediated upregulation of TRAIL-R2. Unex-

pectedly, nutlin-3 upregulatedMcl-1, which attenuated apoptotic signaling triggered by TRAIL, and inhibited

apoptosis induced by the microtubule-targeting drug docetaxel. The increase in Mcl-1 was related to a p53-

independent transcriptional mechanism, but stabilization of the Mcl-1 protein played a dominant role, as

nutlin-3 upregulated theMcl-1 protein to amuch greater extent than theMcl-1mRNA, and this was associated

with prolonged half-life time and reduced ubiquitination of the protein. Knockdown of p53 blocked the

upregulation of the Mcl-1 protein, indicating that p53 plays a critical role in the stabilization of Mcl-1. The

contrasting effects of nutlin-3 on TRAIL- and docetaxel-induced apoptosis were confirmed in freshmelanoma

isolates. Collectively, these results show that nutlin-3 may be a useful agent in combination with TRAIL and,

importantly, uncover a novel regulatory effect of p53 on the expression of Mcl-1 in melanoma cells on

treatment with nutlin-3, which may antagonize the therapeutic efficacy of other chemotherapeutic drugs in

addition to docetaxel in melanoma. Mol Cancer Ther; 9(12); 3363–74. �2010 AACR.

Introduction

The tumor suppressor p53 is a transcriptional factorthat is activated in response to various cellular stressesleading to cell-cycle arrest or apoptosis, thus protectingcells from malignant transformation (1–3). Inactivatingmutations in the gene-encoding p53 occur in about 50% ofhuman cancers, which play an important role in cancerdevelopment, progression, and resistance to treatment(2, 3). In addition, other mechanisms, such as overexpres-sion of murine double minute 2 (MDM2), an E3 ubiquitinligase that targets p53 for proteasomal degradation (4),

are also involved in the suppression of wild-type p53.There has been considerable interest in the developmentof small-molecule antagonists of MDM2 to restore p53function in the treatment of cancer. Nutlin-3, a cis-imi-dazoline derivative, was identified as a compound thatbinds MDM2 in the p53-binding pocket and interfereswith MDM2-mediated p53 degradation, thus leading toaccumulation of p53 (5). Nutlin-3 has been shown toinduce p53-mediated cell-cycle arrest and apoptosis invarious types of cancer cells (6–8).

Unlike many other cancers, melanoma commonly har-bors wild-type p53 (9, 10). However, as judged from itsmalignant nature and resistance to treatment, p53 does notseem to function effectively as a tumor suppressor inmelanoma. Although the mechanism(s) remains to be elu-cidated, somedownstream targets of p53 have been shownto be dysregulated (11, 12). This puts forward a questionabout whether the application of agents that stabilize p53such as nutlin-3 may induce apoptosis in melanoma cells.Moreover, it is unknown whether nutlin-3 affects sensitiv-ity of melanoma cells to other apoptosis-inducing agents.

TNF-related apoptosis-inducing ligand (TRAIL) is apromising candidate for cancer therapeutics (13, 14). Itinduces apoptosis by interaction with 2 death domain–containing receptors, TRAIL-R1 and TRAIL-R2 (13, 14).Previous studies have shown that sensitivity ofmelanoma

Authors' Affiliations: 1Immunology and Oncology Unit, Calvary MaterNewcastle Hospital, Newcastle; and 2Cancer Research Unit, School ofBiomedical Science, University of Newcastle, Callaghan, New SouthWales, Australia

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

X.D. Zhang is a Cancer Institute NSW fellow (07/CDF/1-39).

Corresponding Author: Xu Dong Zhang, David Maddison ClinicalSciences Building, Cnr. King & Watt Streets, Newcastle 2300, New SouthWales, Australia. Phone: 61-2-49138174; Fax: 61-2-49138184. E-mail:xu.zhang@newcastle.edu.au

doi: 10.1158/1535-7163.MCT-10-0646

�2010 American Association for Cancer Research.

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cells to TRAIL-induced apoptosis is in general correlatedwith the levels of the cell surface expression of thesereceptors, in particular TRAIL-R2 (13, 15). However, mel-anoma cells in vivo express low levels of TRAIL deathreceptors (16, 17), suggesting that melanoma may notrespond to treatment with TRAIL unless given withagents that increase the cell surface expression of TRAILdeath receptors (18, 19).

Taxanes, such as paclitaxel and docetaxel, represent aclass of anticancer agents that target microtubules (20).Past studies have shown that docetaxel induces apop-tosis of melanoma cells by the mitochondrial apoptoticpathway (21, 22), which is regulated by pro- and anti-apoptotic Bcl-2 family proteins (21, 22). ProapoptoticBcl-2 family proteins are divided into 2 subgroups, BH3-only proteins, such as PUMA and Noxa, and multi-domain proapoptotic proteins, including Bax and Bak.Activation of BH3-only proteins leads to the activationof Bax and Bak, resulting in the release of mitochondrialapoptogenic proteins (23, 24). Antiapoptotic Bcl-2family proteins may also constitute more than 1 func-tional classes that possess different potency in antag-onizing apoptotic signaling (25, 26). For example, Mcl-1is of particular importance in the protection of mela-noma cells from apoptosis (27).

Wehave examined the effect of nutlin-3 on survival andgrowth of melanoma cells and its potential interactionswith TRAIL and docetaxel. We show in this report thatnutlin-3 alone does not killmelanoma cells, even though itupregulates p53 and its proapoptotic targets. Neverthe-less, nutlin-3 enhances TRAIL-induced apoptosis byp53-mediated upregulation of TRAIL-R2. In contrast, itinhibits apoptosis induced by docetaxel, which is largelydue to an increase in Mcl-1 that is primarily mediated byp53-related posttranslational stabilization of the Mcl-1protein.

Materials and Methods

Cell culture and reagentsHuman melanoma cell lines ME4405, IgR3, Mel-FH,

Mel-RM, Mel-CV, Mel-JD, Sk-Mel-28, Sk-Mel-110, andMM200 have been described previously (15). No furtherauthentication of the cell lines was done. Nutlin-3 waspurchased fromSigma-Aldrich anddissolved in dimethylsulfoxide to make up a stock solution of 12.5 mmol/L.Recombinant human TRAIL, the TRAIL-R2/Fc chimera,andmousemonoclonal antibodies (mAbs) against TRAILreceptors were supplied by Genentech Inc. Docetaxel wasprovided byAventis Pharma SA. The cell-permeable pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F (z-VAD-FMK) and the caspase-8 specific inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (z-IETD-FMK) were pur-chased fromCalbiochem.The rabbit polyclonal antibodies(pAbs) against caspase-3 and -9 and the mouse mAbsagainst caspase-8 were from Stressgen. The mouse mAbagainst cleaved form of PARP was from BD Pharmingen

(Bioclone). The mouse mAbs against Bcl-2, Mcl-1, Apaf-1,and Bax were from Santa Cruz Biotechnology, Inc. Themouse mAb against Noxa was from Imgenex. The mousemAbs against p21 and p53were fromUpstate (Millipore).The rabbit pAb against PUMA and Bid were purchasedfrom Cell Signaling Technology.

Fresh melanoma isolatesIsolation of melanoma cells from fresh surgical speci-

mens was carried out as described previously (16).

ApoptosisQuantitation of apoptotic cells was carried out by mea-

surement of sub-G1 DNA content with propidium iodide(PI) on a flow cytometer as described elsewhere (15).

Flow cytometryImmunostaining on intact and permeabilized cells was

carried out as described previously (15).

Mitochondrial membrane potentialMitochondrial membrane potential (DYm) was quanti-

tated with JC-1 dye staining in flow cytometry as pre-viously described (28).

Western blot analysisWestern blot analysis was carried out as described

previously (27, 29). Labeled bands were detected byImmun-Star HRP Chemiluminescent kit, and imageswere captured and the intensity of the bands was quan-titated with the Bio-Rad VersaDoc image system (Bio-Rad, Regents Park).

Preparation of mitochondrial and cytosolic fractionsMethods used for subcellular fraction were similar to

the methods previously described (18).

Quantitative reverse transcriptionand real-time PCR

Quantitative real-time PCR (qPCR) was done using theABI Prism 7900 sequence detection system (Applied Bio-systems, Carlsbad, CA) as described previously (27).Gene-specific primers used were TRAIL-R2 forward, 50-CGC TGC ACC AGG TGT GAT T-30; TRAIL-R2 reverse,50-GTG CCT TCT TCG CAC TGA CA-30; p53 forward, 50-TGGAAACTACTTCCTGAAAACAAC-30; p53 reverse,50-GGG TCT TCA GTG AAC CAT TG-30; Mcl-1 forward,50-GGA AGG CGC TGG AGA CCT TA-30; Mcl-1 reverse,50-CAA CGA TTT CAC ATC GTC TTC GT-30; b-actinforward, 50-GGCACCCAGCACAATGAAG-30; b-actinreverse, 50-GCC GAT CCA CAC GGA GTA CT-30. Thefollowing PCR conditions were used: standard, fast cycle95�C for 20 seconds, 40 cycles of 95�C for 1 second, and60�C for 20 seconds, using Fast SYBR Green mastermix(Applied Biosystem). Cycle threshold values for specificgeneswere normalized to the cycle threshold value for thehousekeeping gene, b-actin.

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Small RNA interferenceThe small RNA interference (siRNA) constructs used

were obtained as the siGENOME SMARTpool reagents(Dharmacon), TRAIL-R2 siGENOME SMARTpool (M-004448-00-0010), p53 siGENOME SMARTpool (M-003329-03-0010), and siGENOME Nontargeting siRNApool (D-001206-13-20). Transfection of siRNA poolswas carried out as described previously (27).

Short hairpin RNASigma MISSION Lentiviral Transduction Particles for

short hairpin RNA (shRNA)-mediated knockdown ofMcl-1 were used as described previously (27).

Results

Nutlin-3 does not induce apoptosis, even though itupregulates p53 and its proapoptotic targets inwild-type p53 melanoma cellsOur initial studies in 2 wild-type p53 melanoma cell

lines (Mel-JD and MM200) showed that nutlin-3 caused

rapid and sustained upregulation of p53 and inhibition ofcell growth (Fig. 1A and B) but did not induce significantapoptosis even at 20 mmol/L for 48 hours (Fig. 1C). Theinability of nutlin-3 to induce apoptosis was confirmed ina panel of melanoma cell lines carrying either wild-type(IgR3,ME1007,Mel-RM,Mel-CV) ormutant (Mel-FH andSk-Mel-28) p53 and in a p53-null melanoma cell line(ME4405; ref. 30) (data not shown). Nutlin-3 did notinhibit proliferation of mutant p53 and p53-null mela-noma cells (Supplementary Fig. 1), suggesting that theinhibition of melanoma cell growth is p53 dependent.

We examined whether the failure of nutlin-3 to induceapoptosis is due to the dysregulation of proapoptotictranscriptional targets of p53. As shown in Fig. 1A, theBH3-only proteins Bid, PUMA, and Noxa, the multido-main proapoptotic Bcl-2 family protein Bax, and theessential component of the apoptosome Apaf-1 wereall increased by nutlin-3, with varying kinetics.Figure 1D shows that another transcriptional target ofp53, TRAIL-R2 (31), was increased on the cell surface bynutlin-3. As anticipated, p21 was also upregulated, which

A

D

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Figure 1. Nutlin-3 does not induce apoptosis, even though it upregulates p53 and its proapoptotic targets in wild-type p53 melanoma cells. A,whole-cell lysates from Mel-JD and MM200 cells that harbor wild-type p53 treated with nutlin-3 (10 mmol/L) for indicated periods were subjected toWestern blot analysis. B, Mel-JD and MM200 cells were treated with nutlin-3 at indicated concentrations for 48 hours followed by measurement of cellviability by using MTS assays. Bars, SE (n ¼ 3). C, Mel-JD and MM200 cells were treated with nutlin-3 (20 mmol/L) for 48 hours followed by measurement ofapoptosis by using the PI method in flow cytometry. D, representative flow cytometric histograms of measurement of TRAIL-R2 expression on thesurface of Mel-JD and MM200 cells before and after treatment with nutlin-3 (10 mmol/L) for 24 hours. Filled histograms, isotype controls; thick openhistograms, TRAIL-R2 expression before treatment; thin open histograms, TRAIL-R2 expression after treatment.

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is conceivably involved in the inhibition of melanoma cellgrowth (Fig. 1A and B).

Nutlin-3 enhances TRAIL-induced, but protectsagainst docetaxel-triggered apoptosis, in melanomacells harboring wild-type p53

Upregulation of TRAIL-R2 is of particular interest asthe expression of TRAIL-R2 is a major determinant ofsensitivity of melanoma cells to TRAIL-induced apopto-sis (13, 15). We therefore examined whether nutlin-3enhances TRAIL-induced apoptosis. Because nutlin-3also upregulatedmultiple proapoptotic proteins that playimportant roles in apoptosis mediated by the mitochon-drial apoptotic pathway, which is essential in docetaxel-induced apoptosis of melanoma cells (21, 22), we alsostudied whether nutlin-3 affects sensitivity of melanomacells to apoptosis induced by docetaxel. As shown inFig. 2A, nutlin-3 enhanced sensitivity of Mel-JD andMM200 cells to TRAIL-induced apoptosis (P < 0.05, 2-tailed Student’s t test), but surprisingly, it inhibitedapoptosis induced by docetaxel (P < 0.05, 2-tailedStudent’s t test). These contrasting effects were confirmedin Mel-RM and Mel-CV cells that carry wild-type p53(Supplementary Fig. 2). Notably, nutlin-3 did not causesignificant changes in sensitivities of mutant p53 or p53-null melanoma cells to apoptosis induced by TRAIL ordocetaxel (Supplementary Fig. 2).

In association with sensitization of melanoma cells toTRAIL, nutlin-3 enhanced TRAIL-induced activation ofcaspase-8, reduction in DYm, mitochondrial release ofcytochrome c, activation of caspase-9 and -3, and cleavageof PARP (Fig. 2B and Supplementary Fig. 3). Inhibition ofTRAIL-R2 by siRNA markedly blocked TRAIL-inducedapoptosis in the presence of nutlin-3 (P < 0.05, 2-tailedStudent’s t test; Fig. 2C). Similarly, the sensitization wasalso blocked by either the general caspase inhibitor z-VAD-fmk or the caspase-8 specific inhibitor z-IETD-fmk(Supplementary Fig. 4). Along with inhibition of doce-taxel-induced apoptosis, nutlin-3 inhibited docetaxel-induced activation of caspase-9 and -3 and cleavage ofPARP (Fig. 2D). Similarly, it blocked reduction in DYm

and mitochondrial release of cytochrome c induced bydocetaxel (Supplementary Fig. 5), suggesting that nutlin-3 interferes with docetaxel-induced apoptotic signalingupstream of mitochondria.

Upregulation of TRAIL-R2 by nutlin-3 is mediatedby p53

As shown in Fig. 3A and B, nutlin-3 increased the totalprotein levels of TRAIL-R2, as measured in permeabi-lized Mel-JD and MM200 cells, and upregulated thelevels of TRAIL-R2 mRNA. The increase in TRAIL-R2mRNA levels could be inhibited by actinomycin D(Fig. 3C), suggesting that this was due to a transcriptionalincrease rather than a change in the mRNA stability.Nutlin-3 did not alter the levels of TRAIL-R2 mRNA ina p53-null (ME4405) and 2 mutant p53 melanoma celllines (Mel-FH and Sk-Mel-28; Supplementary Fig. 6),

suggesting that wild-type p53 is required for upregula-tion of TRAIL-R2 by nutlin-3. This was further demon-strated in Mel-JD and MM200 cells with p53 knockeddown by siRNA (Fig. 3D).

Nutlin-3-mediated protection of melanoma cellsfrom docetaxel-induced apoptosis involvesupregulation of Mcl-1

We also studied the mechanism(s) by which nutlin-3protects melanoma cells from docetaxel-induced apopto-sis. Because nutlin-3 appeared to act upstream of mito-chondria (Fig. 2D and Supplementary Fig. 5), we testedwhether nutlin-3 impinges on the expression of antiapop-totic Bcl-2 family proteins. Fig. 4A shows that treatmentwith nutlin-3 did not alter the expression levels of Bcl-2 butresulted in upregulation ofMcl-1 with a�4.3-fold increaseinMel-JDanda�3.7-fold increase inMM200cells (Fig. 4A).

As shown in Fig. 4B, inhibition of Mcl-1 by shRNAreversed nutlin-3–mediated protection against docetaxel-induced apoptosis. Similarly, it enhanced docetaxel-induced activation of capase-9, and -3 and cleavage ofPARP in the presence of nutlin-3 (Fig. 4C). Fig. 4D showsthat deficiency in Mcl-1 resulted in a further increase insensitivities of Mel-RM and MM200 cells to TRAIL-induced apoptosis in the presence of nutlin-3. Notably,nutlin-3 alone induced moderate levels of apoptosis inmelanoma cells when Mcl-1 was knocked down (Fig. 4Cand D), indicating that the failure of nutlin-3 to induceapoptosis is partially due to upregulation of Mcl-1.

Upregulation of Mcl-1 by nutlin-3 in melanoma cellsinvolves both transcriptional and posttranslationalmechanisms

We studied the mechanism of upregulation of Mcl-1 bynutlin-3. As shown in Fig. 5A, nutlin-3 caused a�1.5-foldincrease in Mcl-1 in both Mel-JD and MM200 cells, whichwas efficiently blocked by actinomycin D, indicating thatit is a consequence of enhanced Mcl-1 transcription(Fig. 5A). However, the increase in Mcl-1 mRNA wasnot adequate to account for the greater increase in itsprotein levels (Figs. 4 and 5A). Because Mcl-1 is a short-lived protein (32), we sought to determine whether theincrease in the protein levels involves changes in itsstability. Figure 5B shows that exposure to nutlin-3 pro-longed the half-life time of Mcl-1. This was associatedwith reduced ubiquitination of Mcl-1 (Fig. 5C).

Nutlin-3 also increased the Mcl-1 mRNA levels inmutant p53 (Mel-FH) and p53-null (ME4405) melanomacells to a similar extent, as it did in wild-type p53 mela-noma cells (Fig. 5A and D), indicating that transcriptionalupregulation of Mcl-1 by nutlin-3 in melanoma cells isindependent of wild-type p53. This was also shown bysiRNAknockdownofp53 inwild-typep53melanomacells(Supplementary Fig. 7). Strikingly, knockdown of p53inhibited the upregulation of Mcl-1 at the protein levelin wild-type p53melanoma cells (Fig. 5D), suggesting thatwild-type p53 is involved in nutlin-3-mediated stabiliza-tion of the Mcl-1 protein in melanoma cells.

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A

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Figure 2. Nutlin-3 enhances TRAIL-induced apoptosis, but protects against docetaxel-induced apoptosis in wild-type p53 melanoma cells. A, Mel-JDand MM200 cells were treated with nutlin-3 (10 mmol/L), TRAIL (200 ng/mL), or the combination of nutlin-3 and TRAIL for 24 hours (left panel), or nutlin-3 (10mmol/L), docetaxel (20 nmol/L), or the combination of nutlin-3 and docetaxel for 48 hours (right panel). Apoptosis was quantitated by the PI method.B, upper panel, whole-cell lysates fromMel-JD and MM200 cells treated with nutlin-3 (10 mmol/L), TRAIL (200 ng/mL), or the combination of both for 16 hourswere subjected to Western blot analysis. Low panel, mitochondrial and cytosolic fractions of Mel-JD and MM200 cells before and after treatment withnutlin-3 (10 mmol/L), TRAIL (200 ng/mL), or the combination of both for 16 hours were subjected to Western blot analysis. Western blot analysis of COX IV andb-actin was included as controls for the relative purity of mitochondrial and cytosolic fractions, respectively. C, upper panel, mel-JD and MM200 cellstransfected with the control or TRAIL-R2 siRNA. Twenty-four hours later, total RNA was extracted and subjected to real-time qPCR analysis of TRAIL-R2 andTRAIL-R1 mRNA expression. Lower panel, Mel-JD and MM200 cells transfected with the control or TRAIL-R2 siRNA. Twenty-four hours later, cellswere treated with nutlin-3 (10 mmol/L), TRAIL (200 ng/mL), or the combination of both for a further 24 hours before apoptosis was measured by the PI method.D, whole-cell lysates from Mel-JD and MM200 cells treated with nutlin-3 (10 mmol/L), docetaxel (20 nmol/L), or the combination of both for 48 hourswere subjected to Western blot analysis. Bars, SE (n ¼ 3).

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Nutlin-3 differentially regulates sensitivities offresh melanoma isolates to apoptosis induced byTRAIL and docetaxel

As shown in Fig. 6A, nutlin-3 increased TRAIL-R2 onthe cell surface to varying degrees in a panel of 5 freshmelanoma isolates harboring wild-type p53. This

was associated with increases in TRAIL-R2 mRNA,as shown for Mel-MC and Mel-JC cells (Fig. 6A).Figure 6B shows that cotreatment with nutlin-3 andTRAIL enhanced apoptosis in fresh melanoma isolates,which was inhibited by a recombinant TRAIL-R2/Fcchimera.

p53

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Figure 3. Upregulation of TRAIL-R2 by nutlin-3 is dependent on p53. A, left panel, Mel-JD and MM200 cells were treated with nutlin-3 (10 mmol/L) for16 hours. The levels of expression of TRAIL-R2 in permeabilized cells were measured by flow cytometry. Right panel, whole-cell lysates from Mel-JD andMM200 cells treated with nutlin-3 (10 mmol/L) for 16 hours were subjected to Western blot analysis of TRAIL-R2. B, total RNA was extracted fromMel-JD (upper panel) and MM200 (lower panel) cells treated with nutlin-3 (10 mmol/L) for 16 hours and subjected to real-time qPCR analysis of TRAIL-R2 and-R1 mRNA. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. C, Mel-JD and MM200 cells with or withoutpretreatment with actinomycin D (Act D; 100 ng/mL) for 1 hour before the addition of nutlin-3 (10 mmol/L) for a further 16 hours. Total RNA was extracted andsubjected to real-time qPCR analysis of TRAIL-R2 and TRAIL-R1 mRNA. The relative abundance of TRAIL-R2 mRNA expression before treatment wasarbitrarily designated as 1. D, left panel, Mel-JD andMM200 cells were transfected with the control and p53 siRNA, respectively. Twenty-four hours later, cellswere treated with nutlin-3 (10 mmol/L) for a further 24 hours. Whole-cell lysates were subjected to Western blot analysis. Right panel, Mel-JD andMM200 cells were transfected with the control or p53 siRNA. Twenty-four hours later, cells were treated with nutlin-3 (10 mmol/L). Total RNA was thenextracted and subjected to real-time qPCR analysis of TRAIL-R2 mRNA. The relative abundance of TRAIL-R2 mRNA expression in cells transfected with thecontrol siRNA was arbitrarily designated as 1. Bars, SE (n ¼ 3).

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As shown in Fig. 6C, docetaxel alone inducedmoderatelevels of apoptosis in the fresh melanoma isolates Mel-MC andMel-JC, which were reduced by the combinationof docetaxel and nutlin-3, indicative of a protective effect

of nutlin-3 on fresh melanoma isolates against docetaxel-induced apoptosis. Similar to findings in cultured mel-anoma cell lines, nutlin-3 upregulated Mcl-1 in Mel-MCand Mel-JC cells (Fig. 6D).

A

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Figure 4. Upregulation of Mcl-1 is responsible for nutlin-3–mediated protection of melanoma cells against docetaxel-induced apoptosis. A, upperpanel, whole-cell lysates from Mel-JD and MM200 cells treated with nutlin-3 (10 mmol/L) for indicated periods were subjected to Western blot analysis.Lower panel, quantitation of relative intensity of Western blot bands of Mcl-1 as shown in the upper panel. B, upper panel, whole-cell lysates fromMel-RM and MM200 cells transduced with the control or Mcl-1 shRNA were subjected to Western blot analysis. Lower panel, Mel-RM and MM200 cellstransduced with the control or Mcl-1 shRNA were treated as indicated for 48 hours. Apoptosis was measured by the PI method. C, whole-cell lysatesfrom Mel-RM and MM200 cells with Mcl-1 stably knocked down by shRNA treated with nutlin-3 (10 mmol/L), docetaxel (20 nmol/L), or the combinationof both for 48 hours were subjected to Western blot analysis of caspase-9 and -3, and PARP. D, Mel-RM andMM200 cells with Mcl-1 stably knocked down byshRNA were treated as indicated for 24 hours. Apoptosis was measured by the PI method. Bars, SE (n ¼ 3).

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Discussion

Nutlin-3 has been reported to induce apoptosis in anumber of human cancer cells, such as lymphoma andneuroblastoma, by activating the p53-mediated apoptoticpathway (6–8). In addition, E2F1 and p73 that can alsobind to the nutlin-3–binding region of MDM2 have also

been shown to involve in nutlin-3–induced apoptosis(33). Nevertheless, our finding that nutlin-3 does notinduce apoptosis of melanoma cells is not surprising,as failure of nutlin-3 to induce apoptosis has also beenshown in various other cancer types (34, 35) and it is inline with the perception that wild-type p53 does not act asan effective tumor suppressor inmelanoma. Resistance of

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Figure 5. Upregulation of Mcl-1 by nutlin-3 involves both transcriptional and posttranslational mechanisms in melanoma cells. A, left panel, total RNAfrom Mel-JD and MM200 cells treated with nutlin-3 (10 mmol/L) for indicated periods were subjected to real-time qPCR analysis of Mcl-1 mRNA. The relativeabundance of mRNA expression before treatment was arbitrarily designated as 1. Right panel, Mel-JD and MM200 cells were treated with actinomycinD (Act D; 100 ng/mL) for 1 hour before the addition of nutlin-3 (10 mmol/L) for a further 16 hours. Total RNA was extracted and subjected to real-time qPCRanalysis of Mcl-1 mRNA expression. The relative abundance of Mcl-1 mRNA expression before treatment was arbitrarily designated as 1. B, whole-celllysates from Mel-JD and MM200 cells with or without treatment with nutlin-3 (10 mmol/L) for 16 hours followed by the addition of cycloheximide(10 mg/mL) for the indicated periodswere subjected toWestern blot analysis. C, whole-cell lysates fromMel-JD andMM200 cells with or without treatment withnutlin-3 (10 mmol/L) for 6 hours were subjected to immunoprecipitation using an antibody against Mcl-1. The resulting precipitates were subjected toSDS-PAGE and probed with an antibody against ubiquitin. D, left panel, total RNA from Mel-FH (mutant p53) and ME4405 (p53-null) cells treated withnutlin-3 (10 mmol/L) for indicated periods were subjected to real-time qPCR analysis of Mcl-1 mRNA expression. The relative abundance of Mcl-1mRNA expression before treatment was arbitrarily designated as 1. Right panel, Mel-JD and MM200 were transfected with the control or p53 siRNA.Twenty-four hours later, cells were treated with nutlin-3 (10 mmol/L) for a further 16 hours. Whole-cell lysates were subjected to Western blot. Bars,SE (n ¼ 3).

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melanoma cells to nutlin-3–induced apoptosis is not dueto dysregulation of proapoptotic transcriptional targets ofp53, in that PUMA, Noxa, Bid, Bax, and Apaf-1 were allincreased by nutlin-3 along with p53. In contrast, upre-gulation of Mcl-1 appears to play a role, as nutlin-3induced moderate levels of apoptosis in wild-type p53melanoma cells deficient in Mcl-1. Upregulation ofNotch1 has been shown to be a negative-feedback

antiapoptotic mechanism in hematologic cancer cellsafter treatment with nutlin-3, but the downstream signal-ing involved is not known (36). A number of survivalpathways such as the MEK/ERK pathway are constitu-tively activated in melanoma cells (37, 38). It is concei-vable that they may contribute to protection againstapoptosis induced by nutlin-3, as inhibition of theMEK/ERK pathway has been shown to synergize with

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Figure 6. Nutlin-3 differentially regulates sensitivities of fresh melanoma isolates to apoptosis induced by TRAIL and docetaxel. A, left panel, theexpression of TRAIL-R2 on the cell surface was measured by flow cytometry in fresh melanoma isolates with or without treatment with nutlin-3(10 mmol/L) for 16 hours. Right panel, total RNA from the fresh melanoma isolates Mel-MC and Mel-JC with or without treatment with nutlin-3 (10 mmol/L)for 16 hours was subjected to real-time qPCR analysis of TRAIL-R2 mRNA. The relative abundance of mRNA expression before treatment was arbitrarilydesignated as 1. B, left panel, fresh melanoma isolates treated with nutlin-3 (10 mmol/L), TRAIL (200 ng/mL), or the combination of both for 24 hours beforeapoptosis was measured by the PI method in flow cytometry. Right panel, Mel-MC and Mel-JC cells with or without pretreatment with a recombinantTRAIL-R2/Fc chimera (10 mg/mL) for 1 hour were treated with TRAIL (200 ng/mL) or the combination of nutlin-3 (10 mmol/L) and TRAIL for 24 hoursbefore apoptosis wasmeasured by the PI method in flow cytometry. C, Mel-MC andMel-JC cells were treated with nutlin-3 (10 mmol/L), docetaxel (20 nmol/L),or the combination of both for 48 hours before apoptosis was measured by the PI method in flow cytometry. D, whole-cell lysates from Mel-MC andMel-JC cells treated with nutlin-3 (10 mmol/L) for 16 hours were subjected to Western blot analysis of Mcl-1. Bars, SE (n ¼ 3).

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nutlin-3 in the induction of apoptosis in leukemia cells(39).

Despite the failure of nutlin-3 to induce apoptosis, itenhanced TRAIL-induced apoptotic cell death in mela-noma cells harboring wild-type p53. This is of particularinterest in that past studies have indicated that melanomacells in vivo expressed in general low levels of TRAILdeath receptors, in particular TRAIL-R2 (16, 17). Sensiti-zation of melanoma cells to TRAIL-induced apoptosis bynutlin-3 was associated with increased activation of cas-pase-8, consistent with upregulation of TRAIL-2 on themelanoma cell surface. siRNA knockdown of TRAIL-R2blocked the sensitization, substantiating the essentialrole of the increased interactions between TRAIL andTRAIL-R2.

Nutlin-3–mediated upregulation of TRAIL-R2 wasdue to increased TRAIL-R2 gene transcription, whichappeared to be mediated by p53. Intriguingly, we foundin separate studies that the DNA-damaging drug cis-platin did not upregulate TRAIL-R2 at the protein level,even though it increased TRAIL-R2 mRNA along withp53 (18, 19; data not shown). Together, these findingsindicate that the ability of p53 to activate TRAIL-R2transcription per se is intact in melanoma cells andsuggest that p53-mediated upregulation of TRAIL-R2requires cooperation of posttranscriptional mechanismsthat may be impaired in melanoma cells under geno-toxic stress.

An unexpected, yet important finding of this study isthe inhibition of docetaxel-induced apoptosis by nutlin-3.Blockage of apoptotic signaling induced by docetaxelappeared to occur upstream of mitochondria, as nutlin-3 inhibited reduction in DYm andmitochondrial release ofcytochrome c. These observations along with the findingsthat multiple BH3-only proteins were increased bynutlin-3 suggest that nutlin-3 may also upregulate 1 ormore antiapoptotic Bcl-2 family members to neutralizethe proapoptotic proteins. Indeed, nutlin-3 markedlyupregulated Mcl-1, which is known to be of particularimportance in melanoma cells (27, 40). Inhibition of Mcl-1reversed the inhibitory effect of nutlin-3 on docetaxel-induced apoptosis, indicating that upregulation of Mcl-1is responsible for the protection ofmelanoma cells againstdocetaxel by nutlin-3. Notably, upregulation of Mcl-1attenuated TRAIL-induced apoptotic signaling, but thisinhibitory effect was largely overridden by strong apop-totic signaling resulting from the increased expression ofTRAIL-R2 in the presence of nutlin-3.

Althougha transcriptional increase is involved innutlin-3–induced upregulation ofMcl-1, the extent of the increasein Mcl-1 mRNA is not sufficient to account for the greaterincrease in its protein levels. Treatment with nutlin-3caused an increase in the half-life time of Mcl-1, which isassociated with reduced polyubiquitination of the protein,suggesting that nutlin-3 may affect the mechanism thatregulates Mcl-1 ubiquitination and its proteasomal degra-dation. Interestingly, transcriptional upregulation ofMcl-1by nutlin-3 appeared independent of p53, but whether

other transcription factors such as p73 and E2F1 areinvolved remains to be studied (33). E2F1 is known to bea transcriptional repressor of Mcl-1 (41). Significantly, themechanism by which nutlin-3 mediates posttranslationalregulation of Mcl-1 appeared dependent on p53. Ubiqui-tination and proteasomal degradation of Mcl-1 are regu-lated by the E3 ligase Mule and the deubiquitinase USP9X(32, 42), but whether p53 upregulated by nutlin-3 mayimpinge on these mechanisms is to be examined. Never-theless, the present study appears to be the first to revealthe regulatory effect of p53 on the expression of Mcl-1 inmelanoma cells after exposure to nutlin-3. It is of note thatp53doesnot appear to regulateMcl-1 in every scenario. Forexample, DNA-damaging agents cause accumulation ofp53 but do not increase the levels of Mcl-1 (25, 43).

Besides transcriptional regulation and posttransla-tional degradation by the ubiquitin–proteasome system(32, 42), Mcl-1 expression can also be regulated both atthe translation initiation level and by caspase-mediateddegradation during apoptosis (44, 45). We observed thatwhile treatment with TRAIL alone resulted in a mod-erate decrease in Mcl-1, the combination of nutlin-3 andTRAIL caused further downregulation of the Mcl-1levels, which could be inhibited by pretreatment withz-VAD-fmk (Supplementary Fig. 8), indicating thatincreased caspase activation caused by the combinator-ial treatment resulted in enhanced caspase-mediatedcleavage of Mcl-1 (45). Increased Mcl-1 in some malig-nancies has been shown to be related to downregulationof microRNA (miR)-29b (46). However, we have foundby qPCR-based screening that there was no significantdifference in the expression levels of miR-29b in mela-noma cells before and after treatment with nutlin-3(data not shown), suggesting that nutlin-3–mediatedupregulation of Mcl-1 may not be associated withdownregulation of miR-29b. Similarly, although trans-lation initiation regulators such as eIF4E, eIF4G, and4EBP1 have been shown to mediate translational reg-ulation of Mcl-1 (44, 47), they may not play a major rolein nutlin-3–triggered upregulation of Mcl-1, as ourresults clearly indicated that the increase in Mcl-1induced by nutlin-3 is mediated by transcriptionaland posttranslational mechanisms.

Our finding that nutlin-3 could sensitize fresh mela-noma isolates to TRAIL-induced apoptosis by upregula-tion of TRAIL-R2 is of particular importance, as they mayreflect more closely the in vivo status of TRAIL deathreceptor expression in melanoma cells and their suscept-ibility to TRAIL-induced apoptosis (15, 16). The combi-nation of nutlin-3 and TRAIL may therefore be a usefulstrategy in improving the therapeutic efficacy of TRAILin melanoma. However, the finding that nutlin-3 alsoupregulatesMcl-1 in freshly isolatedmelanoma cells callsfor attention to possibly unexpected adverse effects of theadministration of nutlin-3 in vivo. High levels of Mcl-1may antagonize other chemotherapeutic drugs inaddition to docetaxel. The role of p53 in the regulationof Mcl-1 in melanoma cells treated with nutlin-3 may

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have broad implications in understanding melanoma cellsurvival and resistance to treatment and warrants furtherinvestigation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by the NSW State Cancer Council (RG 09-23), andNational Health and Medical Research Council (569232), Australia.

Received 07/13/2010; revised 09/07/2010; accepted 09/28/2010;published 12/14/2010.

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2010;9:3363-3374. Mol Cancer Ther   Hsin-Yi Tseng, Chen Chen Jiang, Amanda Croft, et al.   Melanoma CellsApoptosis Due to Upregulation of TRAIL-R2 and Mcl-1 in Human Contrasting Effects of Nutlin-3 on TRAIL- and Docetaxel-Induced

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