This article was downloaded by: [George Washington University] On: 03 February 2015, At: 10:45 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20

Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma a

a

bc

d

b

Domenico Di Marzo , Iris Maria Forte , Paola Indovina , Elena Di Gennaro , Valeria Rizzo , b

be

ab

ad

Francesca Giorgi , Eliseo Mattioli , Carmelina Antonella Iannuzzi , Alfredo Budillon , abc

Antonio Giordano

a

& Francesca Pentimalli

a

Oncology Research Center of Mercogliano (CROM); Istituto Nazionale Per Lo Studio E La Cura Dei Tumori “Fondazione Giovanni Pascale”; IRCCS; Italy b

Department of Medicine, Surgery and Neuroscience; University of Siena; Siena, Italy

c

Click for updates

Sbarro Institute for Cancer Research and Molecular Medicine; Center for Biotechnology; College of Science and Technology; Temple University; Philadelphia, PA USA d

Experimental Pharmacology Unit; Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Giovanni Pascale” - IRCCS; Naples, Italy e

National Cancer Research Centre; Istituto Tumori “Giovanni Paolo II”; Bari, Italy Published online: 17 Dec 2013.

To cite this article: Domenico Di Marzo, Iris Maria Forte, Paola Indovina, Elena Di Gennaro, Valeria Rizzo, Francesca Giorgi, Eliseo Mattioli, Carmelina Antonella Iannuzzi, Alfredo Budillon, Antonio Giordano & Francesca Pentimalli (2014) Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma, Cell Cycle, 13:4, 652-665, DOI: 10.4161/cc.27546 To link to this article: http://dx.doi.org/10.4161/cc.27546

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

Report

Cell Cycle 13:4, 652–665; February 15, 2014; © 2014 Landes Bioscience

Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma 2

1 Oncology Research Center of Mercogliano (CROM); Istituto Nazionale Per Lo Studio E La Cura Dei Tumori “Fondazione Giovanni Pascale”; IRCCS; Italy; Department of Medicine, Surgery and Neuroscience; University of Siena; Siena, Italy; 3Sbarro Institute for Cancer Research and Molecular Medicine; Center for Biotechnology; College of Science and Technology; Temple University; Philadelphia, PA USA; 4Experimental Pharmacology Unit; Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Giovanni Pascale” - IRCCS; Naples, Italy; 5National Cancer Research Centre; Istituto Tumori “Giovanni Paolo II”; Bari, Italy

These authors contributed equally to this work.

Downloaded by [George Washington University] at 10:45 03 February 2015



Keywords: TP53 mutations, RITA, nutlin-3, p21, mesothelioma, apoptosis Abbreviations: CI, combination index; FACS, fluorescence activated cell sorting; HDAC, histone deacetylase; IAP, inhibitor of apoptosis protein; IC50, inhibitory concentration 50; MDM2, MDM2 oncogene, E3 ubiquitin protein ligase; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NBT, nitro blue tetrazolium; PFT, pifithrin; RITA: reactivation of p53 and induction of tumor cell apoptosis; SV40, simian virus 40; TrxR1, thioredoxin reductase 1; wt, wild-type

Malignant mesothelioma, a very aggressive tumor associated to asbestos exposure, is expected to increase in incidence, and unfortunately, no curative modality exists. Reactivation of p53 is a new attractive antitumoral strategy. p53 is rarely mutated in mesothelioma, but it is inactivated in most tumors by the lack of p14ARF. Here, we evaluated the feasibility of this approach in pleural mesothelioma by testing RITA and nutlin-3, two molecules able to restore p53 function through a different mechanism, on a panel of mesothelioma cell lines representing the epithelioid (NCI-H28, NCI-H2452, IST-MES 2), biphasic (MSTO-211H), and sarcomatoid (NCI-H2052) histotypes compared with the normal mesothelial HMChTERT. RITA triggered robust caspase-dependent apoptosis specifically in epithelioid and biphasic mesothelioma cell lines, both through wild-type and mutant p53, concomitant to p21 downregulation. Conversely, nutlin-3 induced a p21-dependent growth arrest, rather than apoptosis, and was slightly toxic on HMC-hTERT. Interestingly, we identified a previously undetected point mutation of p53 (p.Arg249Ser) in IST-MES 2, and showed that RITA is also able to reactivate this p53 mutant protein and its apoptotic function. RITA reduced tumor growth in a MSTO-211H-derived xenograft model of mesothelioma and synergized with cisplatin, which is the mainstay of treatment for this tumor. Our data indicate that reactivation of p53 and concomitant p21 downregulation effectively induce cell death in mesothelioma, a tumor characterized by a high intrinsic resistance to apoptosis. Altogether, our findings provide the preclinical framework supporting the use of p53-reactivating agents alone, or in combination regimens, to improve the outcome of patients with mesothelioma.

Introduction Malignant pleural mesothelioma is a highly aggressive tumor deriving from the mesothelium lining the pleural cavity. Mesothelioma is classified into 3 histological subtypes: epithelioid, sarcomatoid, and biphasic, characterized, respectively, by epithelial cells, spindle-shaped cells, or both cell types.1 The main risk factor for mesothelioma development is exposure to asbestos. Although asbestos use has been banned in many countries, its

consumption in the developing world is still high, and mesothelioma incidence is expected to increase in the next decades also owing to the long latency between exposure and tumor onset.2,3 Unfortunately, the current therapeutic options for patients with mesothelioma have only limited effects in modifying the disease natural history. The recent introduction of newer agents, such as the antifolate pemetrexed in combination with cisplatin, showed a significant improvement of the patient’s quality of life; however, the median overall survival remains 9-17 mo from

*Correspondence to: Francesca Pentimalli; Email: [email protected]; Antonio Giordano; Email: [email protected] Submitted: 07/11/2013; Revised: 08/02/2013; Accepted: 12/16/2013; Published Online: 12/17/2013 http://dx.doi.org/10.4161/cc.27546 652

Cell Cycle

Volume 13 Issue 4

©2014 Landes Bioscience. Do not distribute.

Domenico Di Marzo1,†, Iris Maria Forte1,†, Paola Indovina2,3, Elena Di Gennaro4, Valeria Rizzo2, Francesca Giorgi2, Eliseo Mattioli2,5, Carmelina Antonella Iannuzzi1,2, Alfredo Budillon1,4, Antonio Giordano1,2,3,*, and Francesca Pentimalli1,*

Downloaded by [George Washington University] at 10:45 03 February 2015

diagnosis, regardless of stage.4 Therefore, there is an urgent need to develop new strategies to improve disease management and clinical outcome. One approach to cancer therapy is based on the restoration of pathways that trigger apoptosis in cancer cells. These are usually latent in normal cells, whereas they are activated in tumor cells by a wide range of cues. Unfortunately, however, tumor cells are often resistant to apoptosis, because they inactivate key players of this process, such as the TP53 gene.5 TP53 is mutated in approximately half of human cancers and inactivated in most tumors by interaction with viral proteins, overexpression of its ubiquitin-ligase MDM2, lack of p14 ARF, which interferes with MDM2 activity, or other mechanisms.6 Therefore, p53 represents one of the most appealing targets for drug intervention. In mesothelioma TP53 mutations are described to be rare;7,8 however, homozygous deletions of the CDKN2A locus, encoding p16INK4a and p14 ARF, occur with a frequency of >70%, likely resulting in p53 functional inactivation.9-11 Indeed, a study showed that mesothelioma cells retaining wt p53 but lacking p14 ARF are susceptible to the cytolytic effect of ONYX-015, an oncolytic virus conceived to selectively replicate in tumor cells bearing a dysfunctional p53 pathway.12 The same authors reported that adenovirus-mediated p14 ARF replacement was able to induce growth arrest in several mesothelioma cell lines by inducing p53 and, consequently, p21 expression, suggesting that p53 activity can be restored in mesothelioma cells.13 Consistently, another study showed that, upon MDM2 inhibition or p14 ARF reintroduction, p53 becomes functional and able to induce apoptosis following cisplatin treatment in mesothelioma cells.14 Recently, several small molecules restoring p53 function were designed, opening the way to the development of new anticancer strategies. The well-defined interaction between MDM2 and p53 has provided the basis for the design of molecules such as nutlin-3, which, by binding MDM2, induces the release and accumulation of p53.15 More recently, another molecule, RITA (reactivation of p53 and induction of tumor cell apoptosis) was found to prevent p53–MDM2 interaction and induce p53-mediated apoptosis in tumor cell lines expressing both wt p5316 or some mutant forms.17,18 RITA, in particular, has the appealing property of inducing apoptosis rather than growth arrest in various cancer cell lines, either by promoting the proteasome-mediated degradation of the p53 transcriptional cofactor hnRNPK, which is required for the induction of growth arrest genes such as p21,19 or through other yet unidentified mechanisms.20,21 On these bases, the pharmacological targeting of p53 in mesothelioma holds the potential to be a new promising antitumor approach. Therefore, we set out to evaluate the effects of 2 small molecules able to restore p53 function with a different mode of action, RITA and nutlin-3, on human mesothelioma cell lines.

Results Expression and mutation status of p53 in mesothelioma cell lines Although p53 is rarely found mutated in mesothelioma specimens, it is often inactivated by alterations in its pathway. So,

www.landesbioscience.com

before testing RITA and nutlin-3 effect, we determined p53 status in a panel of mesothelioma cell lines by analyzing at first the protein levels of p53 itself and p14 ARF, which inhibits MDM2 dependent-p53 degradation and is not expressed in most mesotheliomas.22 In particular, we analyzed p53 and p14 ARF expression in a panel of mesothelioma cell lines, of epithelioid (NCI-H28, NCI-H2452, IST-MES 2), biphasic (MSTO-211H), and sarcomatoid (NCI-H2052) histotypes, and in the normal mesothelial cell line HMC-hTERT. Consistently, p14 ARF was expressed only in HMC-hTERT, whereas it could not be detected in any of the mesothelioma cell lines. Conversely, p53 was detected, although at different levels, in all the cell lines analyzed (Fig. 1A). We then assessed TP53 mutational status by cDNA sequencing (Fig. 1B). As expected, we found TP53 to be wt in HMChTERT and in NCI-H28, NCI-H2052, and MSTO-211H. In NCI-H2452, p53 is reported to be truncated,23 and, consistently, we could not amplify full-length TP53 with our strategy. Interestingly, in IST-MES 2, we identified a mutation at TP53 codon 249 (p.Arg249Ser; G > T), which was previously described in aflatoxin-induced hepatocarcinoma 24 but never in mesothelioma. p.Arg249Ser mutation drastically affects p53 trascriptional activity, which is reduced to only 5–15% for several target genes, such as MDM2 and p21 (http://p53.free.fr/ Database/p53_database.htmt). We verified that the p.Arg249Ser mutation detected in IST-MES 2 did not occur in our lab by repeating TP53 sequencing on a new cell sample, kindly provided by the ISTGE cell repository. All the cell lines in which we successfully sequenced full-length TP53 cDNA (all, including HMC-hTERT, with the exception of NCI-H2452), compared with TP53 reference sequence, carried the same polymorphism at 1odon 72 p.Pro72Arg; G > C, which occurs at high frequency in some populations.25 IST-MES 2, beyond the mutation at codon 249 of TP53, carried a polymorphism at codon 213 (Fig. 1B) (http://p53.free.fr/Database/p53_database.htmt). p53-reactivating agents affect mesothelioma cell viability We investigated the effect of 2 different agents, RITA and nutlin-3, aimed at reactivating p53 function with a different mechanism of action, on mesothelioma cell growth by MTS assay at 24 h (Fig. 1C), 48 h, and 72 h (Fig. S1; Table S1). Although RITA was synthesized aiming to restore p53 tumor suppressor activity in wt p53 cancer cells, it was recently shown to rescue the transcriptional transactivation function of some hot-spot p53 mutants.17 So, we investigated RITA effects on mesothelioma cells harboring both wt and mutated p53. Interestingly, RITA showed a cytotoxic effect in all mesothelioma cell lines, except in NCI-H2052, which represents the most aggressive tumor histotype. In particular, RITA affected cell viability at nanomolar doses in the epithelioid cell lines NCI-H28, ISTMES 2, and NCI-H2452. Also, the biphasic MSTO-211H cell line was highly susceptible to RITA, although at a higher dose (1.6 μM). RITA IC50 values are reported in the table (Fig. 1C). Remarkably, RITA did not show toxic effects in the normal mesothelial HMC-hTERT. Conversely, nutlin-3 was slightly toxic on HMC-hTERT and only moderately reduced viability of the mesothelioma cell lines at 10–20 μM doses (Fig. 1C; Fig.  S1; Table S1).

Cell Cycle 653

©2014 Landes Bioscience. Do not distribute.

Report

Report

654

Cell Cycle

Volume 13 Issue 4

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

Figure 1. Effect of p53-reactivating drugs in mesothelioma cell lines. (A) Expression levels of p53 and p14ARF assessed by western blot in a panel of mesothelioma cell lines and in HMC-hTERT derived from normal mesothelium. The blot against p14 derives from a long exposure; no bands specific for p14 were observed in mesothelioma cell lines consistent to the fact that the CDKN2A locus is reported to be homozygously deleted on the COSMIC database (http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/) in NCI-H28, NCI-H2452, MSTO-211H and NCI-H2052. The size of the p53 band detected in NCI-H2452 is consistent with a protein truncation as previously reported. 23 GAPDH was used as a loading control. Representative blots are shown out of 3 independent western blot experiments. (B) TP53 mutational status was assessed through sequencing of the full-length encoding cDNA. The table reports the polymorphisms and/or mutations found. For NCI-H2452 no PCR amplification of cDNA was achieved with our strategy. (C) Left: MTS assay was performed in the indicated cell lines treated with several doses of RITA (top) and nutlin-3 (bottom). The results are reported as the means of 3 independent experiments, each conducted in triplicate, and expressed as percentages of cell viability (calculated with respect to the control cells treated with DMSO alone). The absorbance values of treated and control samples were subjected to 1-way Anova with Dunnett post-test. Statistically significant differences between treated and control cells are indicated with: *significant (P < 0.05); **very significant (P < 0.01); and ***extremely significant (P < 0.001). Right: the table reports the IC50 values of RITA for every cell line 24 h after treatment or 48 h for MSTO-211H; IC50 values for nutlin-3 were not determined (ND). (D) Long-term RITA effects were assessed by clonogenic assay. Colonies were stained with crystal violet 2 wk after a 24 h-treatment with RITA or DMSO as control. RITA was used at the IC50, as reported in Figure 1C, or, for NCI-H2052, with the maximum dose used. A representative experiment, out of 3 independent ones, is shown.

We then assessed whether RITA was able to exert a long-term inhibition of mesothelioma cell growth through clonogenic assay. Indeed, RITA treatment dramatically reduced the number of colonies in all mesothelioma cell lines except NCI-H2052 (Fig. 1D). p53-reactivating agents affect cell cycle progression of mesothelioma cell lines To evaluate RITA and nutlin-3 effects on cell cycle progression, we analyzed by flow cytometry cell cycle profiles of mesothelioma cell lines after treatment with these agents. The effects of RITA and nutlin-3 on cell cycle distribution were remarkably different. In fact, RITA increased S-phase population in all responsive mesothelioma cell lines (both in wt and mutated TP53 cell lines) (Fig. 2A; Table S2), and its effect persisted at 48 and 72 h (data not shown). Our results are consistent with a previous study, showing that RITA stalled replication fork elongation prolonging S-phase progression.27 Moreover, in all cell lines, except HMC-hTERT and NCI-H2052, RITA dramatically increased the sub-G1 fraction, which is suggestive of cell death. Conversely, nutlin-3 induced a G1 cell cycle arrest (Fig. 2A), which persisted 72 h after treatment (data not shown), along with reduction of S-phase percentage in mesothelioma cell lines carrying wt TP53 (MSTO-211H and NCI-H2052), with the exception of NCI-H28, which showed a persistent G2 cell cycle arrest. No significant remarkable alterations in cell cycle were observed consistently following treatment with nutlin-3 in the cell lines carrying mutant TP53 (IST-MES 2 and NCI-H2452) (Fig. 2A; Table S2). To investigate at the molecular level the possible mechanisms underlying these effects on cell cycle, we assessed through western blot, following treatments with RITA and nutlin-3, the protein levels of p53 and p21, a key target of p53 with a crucial role in controlling cell cycle, the G1 checkpoint, and apoptosis.28 In all cell lines analyzed, both RITA and nutlin-3 effectively induced p53 expression in a dose-dependent manner, except in NCI-H2452, in which mutant p53 level was maximal at doses of RITA below the IC50 (Fig. 2B). Cell lines carrying mutant TP53 (IST-MES 2 and NCI-H2452) did not show p21 basal expression, as expected, whereas in cell lines harboring wt TP53 (NCI-H28, MSTO-211H, and NCIH2052), nutlin-3 strongly induced p21 expresFigure 2. RITA and nutlin-3 show different effects on cell cycle and differently modulate p53 and p21 levels. (A) Cell cycle analysis of mesothelioma cell lines (NCI-H28, NCI-H2452, sion in a dose-dependent manner. Conversely, IST-MES 2, MSTO-211H, and NCI-H2052) and HMC-hTERT treated with RITA at its IC50 values RITA reduced p21 expression, consistent with and 10 μM nutlin-3 for 24 h. For NCI-H2052 and HMC-hTERT, RITA IC50 was not determinwhat was previously reported in other cell lines19 able, therefore these cell lines were treated with RITA at the maximum dose used (1.6 μM). (Fig. 2B). The table reports the mean ± standard deviation values from 2 independent experiments. So, our data suggest that p21 expression is cruRaw data from both experiments are reported in Table S2. (B) p53, p21, and MDM2 protein levels were analyzed by western blot following cell treatment with 2 doses of RITA (IC50 and cial to determine mesothelioma cell response to 0.1 μM) and nutlin-3 (10 μM and 20 μM) for 24 h. GAPDH was used as a loading control. p53-reactivating agents. Indeed, nutlin-3-induced Representative blots are shown out of 3 independent western blot experiments.

www.landesbioscience.com

Cell Cycle 655

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

Both RITA and nutlin-3 treatments deeply affected cell morphology (Fig. S2A); cells treated with RITA showed typical features of apoptosis, such as cell rounding, tendency to float, highly fluorescent condensed chromatin and apoptotic bodies (visible through Höechst staining, Fig. S2B), whereas cells treated with nutlin-3 changed from a well-spread polygonal shape to a more elongated one, consistent to what was reported in other cell lines.26

656

Figure 3. RITA triggers caspase- and p53-dependent apoptosis in mesothelioma cell lines carrying both wild-type and mutant p53. (A) NCI-H28, NCI-H2452, IST-MES 2, MSTO-211H, and NCI-H2052 were treated for 24 h with RITA (IC50), RITA (IC50) + Z-VAD-FMK (100 μM) or nutlin-3 (10 μM) and analyzed by annexin-V assay. NCI-H2052 were treated with the highest dose of RITA used (1.6 μM). The graphs show the percentages of early (population of cells positive for annexin-V staining) and late apoptosis (population of cells positive for both annexin-V and propidium iodide staining). A representative experiment, out of 2 independent ones, is shown. (B) The same mesothelioma cell lines were analyzed by MTS assay following treatment with RITA (IC50), pifithrin-α (25 μM), and RITA (IC50) + pifithrin-α (25 μM) for 24 h. Statistically significant differences were evaluated by Anova with Tukey post-test and indicated with: *significant (P < 0.05); **very significant (P < 0.01); and ***extremely significant (P < 0.001). The results are reported as the means of 3 independent experiments, each conducted in triplicate.

Cell Cycle

Volume 13 Issue 4

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

cell cycle arrest seems to depend on p21 induction by p53, because it does not occur in IST-MES 2 and NCI-H2452 carrying mutant TP53 (Fig. 2B). Conversely, RITA, by either downregulating p21 or in its absence, does not induce cell cycle arrest, but rather increases the sub-G1 cell fraction (Fig. 2A). This sub-G1 cell accumulation is suggestive of apoptosis and in accordance with the observation that p21 downregulation is a crucial event to promote apoptosis.19,28 As both RITA and nutlin-3 interfere with the p53–MDM2 interaction, we also evaluated MDM2 protein levels in mesothelioma cell lines 24 h following treatment with these agents. Consistently to what previously reported, nutlin-3 strongly induces MDM2 levels in a dosedependent manner, which seems to reflect its p53-mediated transcriptional regulation, except in IST-MES 2 and NCI-H2452 carrying mutant TP53 (Fig. 2B). This was confirmed also at the transcriptional level (data not shown). Conversely, MDM2 levels upon RITA treatment, although slightly increased at transcriptional level in NCIH28, MSTO-211H, and HMC-hTERT, carrying wt TP53 (data not shown), were either unchanged or decreased in a dose-dependent manner, indicating that also a non-trascriptional type of regulation exists, as previously suggested.29 RITA induces apoptosis in epithelioid and biphasic mesothelioma cell lines Cell cycle analysis showed that RITA, compared with nutlin-3, potently increased the sub-G1 fraction of both epithelial and biphasic mesothelioma cell lines, which is suggestive of cell death and consistent with the MTS data. To determine the drug ability to trigger apoptosis in mesothelioma cell lines, we treated them with RITA (IC50 or 1.6 μM for NCI-H2052) or nutlin-3 (10 μM) for 24 h, and performed an annexin-V assay. Indeed, RITA, but not nutlin-3, was able to induce massive apoptosis in all mesothelioma cell lines except NCI-H2052 (Fig. 3A). RITA-mediated apoptosis was completely abolished by cell treatment with the pan-caspase inhibitor, Z-VAD-FMK, indicating that, consistent with previous reports,30,31 the apoptosis induced by RITA was caspase-dependent also in mesothelioma cell lines. The analysis, through western blot, of molecular markers of apoptosis—such as poly (ADP-ribose) polymerase (PARP) and caspase 3 activation—further confirmed our findings. In particular, RITA treatment induced PARP and caspase 3 cleavage in all the responsive mesothelioma cell lines (which were evident through the appearance of a cleaved band and the concomitant downregulation of the

tested whether its ectopic expression in p53-null cells would confer susceptibility to RITA. We transfected the human colon cancer HCT116 p53–/– cells with either wt p53 or p.Arg249Ser p53 expression vectors. Western blot analysis confirmed p53 expression and showed that RITA treatment was able to induce both wt and mutant ectopic p53 levels (Fig. 5A). Then we tested RITA ability to induce apoptosis through the annexin-V assay. We found that the p.Arg249Ser p53 mutant was able to render HCT116 p53–/– cells sensitive to RITA, although to a lesser extent compared with wt p53 (Fig. 5A). No apoptosis was observed in HCT116 p53–/– following RITA treatment accordingly to what previously shown.17 This finding indicates for the first time that RITA is able to trigger apoptosis also through this mutant form of p53 and supports the fact that in IST-MES 2, RITA-induced apoptosis is at least partially dependent on p53. To further characterize the p.Arg249Ser p53 mutant and evaluate whether RITA can improve its DNA binding ability, we co-transfected in HCT116 p53–/– this p53 mutant along with the PG13-luc or MG15-luc constructs, expressing, respectively, the luciferase reporter gene downstream to p53-responsive elements or to the same elements mutated in the p53 binding sites as control.35 RITA, indeed, was able to increase specifically the transcriptional activity of p.Arg249Ser p53 on the PG13-luc but not on the MG15-luc reporter, even though, as expected, this mutant was much less active than wt p53 (Fig. 5B). RITA suppresses mesothelioma cell growth in soft-agar and in vivo To evaluate the potential of RITA to function as an anticancer drug for mesothelioma treatment, we studied its effects on mesothelioma cell ability to grow in soft agar and in a pilot in vivo experiment. First, we found that RITA, at the IC50 value, impressively reduced the growth in soft agar of MSTO-211H cells, representative of the most aggressive tumor histotype responsive to the drug (Fig. 6A). Then, we treated 2 groups of nude mice (10 each) carrying MSTO211H xenografts with daily injections of RITA at a dose of 1 mg/kg for 3 wk followed by further treatment-free 3 wk. Interestingly, compared with the control group, in which mice were treated with vehicle DMSO only, RITA treatment significantly reduced the growth rate of MSTO-211H xenografts (Fig. 6B). Remarkably, in the RITA-treated group, 4 tumors showed complete regression. Analysis of tumor weights upon resection at the end of the study period showed that the median weight value was very different between the DMSO- and RITAtreated mice, approaching statistical significance (P = 0.059), although 2 xenografts in the RITA treated group seemed not to have responded to the Figure  4. RITA and nutlin-3 effects on PARP and caspase-3 activation. NCI-H28, NCItreatment (Fig. 6C). However, overall these findH2452, IST-MES 2, MSTO-211H, NCI-H2052, and HMC-hTERT were treated for 24 h with 2 ings suggest that although further experiments doses of RITA (IC50 and 0.1 μM) and nutlin-3 (10 μM and 20 μM) or with RITA at the maxiare needed to evaluate whether RITA acts in vivo mum dose used (1.6 μM) for NCI-H2052 and HMC-hTERT. PARP and caspase-3 protein levels were analyzed by western blot with antibodies detecting both the full-length and through the same mechanisms shown in vitro and if cleaved forms. GAPDH was used as a loading control. Representative blots are shown resistance may arise, the pharmacological targeting out of 3 independent western blot experiments.

www.landesbioscience.com

Cell Cycle 657

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

full-length) but not in the sarcomatoid NCI-H2052 and in the normal HMC-hTERT (Fig. 4). Conversely, concomitant PARP cleavage and caspase 3 activation following nutlin-3 treatment were present only in NCIH2452 and MSTO-211H, especially at high doses (20 μM), although no significant increase in apoptosis was detected at 10 μM through cell cycle analysis and annexin-V assay. Pifithrin-α inhibits RITA-induced apoptosis To examine whether RITA-induced apoptosis is indeed mediated by p53 activity, we used 2 chemical inhibitors of p53, pifithrin-α and pifithrin-μ (PFTα and PFTμ), which are reported to inhibit p53 transcription-dependent or -independent apoptosis, respectively.17,32,33 First, we assessed whether PFTα and PFTμ were able to counteract RITA effect on cell viability by MTS assay. Interestingly, PFTα strongly inhibited RITA effect on all susceptible mesothelioma cell lines (Fig. 3B), whereas PFTμ was unable to prevent RITA cytotoxic effect (data not shown). Similarly, PFTα significantly reverted RITA-induced apoptosis, assessed through annexin-V/propidium iodide staining (Fig. S3). Overall, these findings suggest that p53 is capable, at least in part, of mediating RITA cytotoxic effect on mesothelioma cell lines also when its activity is partially impaired by mutation (in IST-MES 2 and NCI-H2452). This seems to indicate that, although RITA probably acts through different mechanisms,34 RITA-mediated p53 reactivation is indeed crucial for the induction of apoptosis in sensitive mesothelioma cell lines. RITA induces apoptosis in HCT116 p53–/– expressing mutant p53 p.Arg249Ser To further confirm that RITA is able to induce apoptosis through the mutant p53 that we identified in IST-MES 2, we

of p53 through RITA promises to be an effective antitumoral strategy for mesothelioma in vivo as well. RITA synergizes with cisplatin in mesothelioma cell lines Platinum-based chemotherapy plus gemcitabine or pemetrexed followed by surgery and radiotherapy represent the mainstay of mesothelioma treatment, against which other therapeutic protocols are evaluated.4 Restoring p53 function could improve the effects of cisplatin, as well as of other DNA damaging agents, by increasing the apoptotic response following the DNA damage induced by chemotherapy. Indeed, RITA was recently shown to enhance cisplatin-induced cytotoxicity in head and neck cancer.20 To assess whether RITA could potentially be used in combination with the conventional treatment in mesothelioma,

658

we investigated by MTS assay its effect along with cisplatin. First, we determined the IC50 of cisplatin at 72 h on the mesothelioma cell lines sensitive to both RITA and cisplatin (Fig. 7A). Then, based on their IC50 values, we challenged the cells for 72 h with both drugs at different concentrations in a constant ratio (Fig. 7B). We found that the 2-drug combination was effective in all mesothelioma cell lines. In particular, analysis through the Chou–Talalay method showed combination index (CI) values that were always lower than 1 (Fig. 7C). The observed synergism between RITA and cisplatin encourages further testing of this drug combination to achieve potential improvements in mesothelioma treatment and side-effect reductions.

Cell Cycle

Volume 13 Issue 4

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

Figure 5. RITA is able to trigger apoptosis through mutant p.Arg249Ser p53. (A) HCT116 p53–/– were transfected with the pCEFL-HA vector expressing wt p53 cDNA (cloned from NCI-H28) or p.Arg249Ser p53 cDNA (cloned from IST-MES 2). Following 18 h from transfection, cells were treated with 1.6 μM RITA for 24 h then harvested and analyzed through the annexin-V assay; p53 expression levels in transfected cells were analyzed in parallel through western blot. GAPDH was used as a loading control. (B) HCT116 p53–/– were co-transfected with pCEFL-HA expressing wt p53 or p.Arg249Ser p53 and the PG13-luc or MG15-luc vectors carrying either wt or mutated p53 binding sequences upstream the luciferase reporter gene. The results represent the means of 3 independent experiments. Statistically significant differences were evaluated by Student t test and indicated with *(P < 0.05).

Downloaded by [George Washington University] at 10:45 03 February 2015

Malignant mesothelioma is a very aggressive cancer correlated with asbestos exposure. Although asbestos has been banned in many countries, it is still widely used in the world and often it is not properly dismissed, which will probably cause a shift from occupational to environmental exposure. So, while many studies predict an increase in the incidence of mesothelioma, its prognosis is very poor, and no current curative modality is available. Therefore there is an urgent need to identify new therapeutic approaches for patients with mesothelioma. Although mesothelioma is usually wt for the TP53 gene, most tumors carry homozygous deletions of the INK4A/ARF locus

Figure 6. RITA suppresses mesothelioma cell growth in soft agar and in vivo. (A) MSTO-211H were seeded in 24-well plates containing semisolid medium (soft agar) and cultured with vehicle DMSO (control) or RITA IC50 (1.6 μM) for 2 wk. Colony formation was assessed by staining with NBT. Statistically significant differences were evaluated by Student t test and indicated with **(P < 0.01). (B) Growth curves of MSTO-211H xenografts in 2 groups of nude mice treated with vehicle DMSO or RITA, respectively. The graph represents the median values of the tumor volumes at each measurement. The length of drug treatments is indicated through the syringes. Statistically significant differences were evaluated by Mann–Whitney test and indicated with *(P < 0.05). + indicates P = 0.05. (C) Dot plots showing the median and interquartile range of the weights of tumors excised from the 2 groups of mice treated with DMSO or RITA. The P value obtained by the Mann–Whitney test is reported (P = 0.059). www.landesbioscience.com

encoding p14 ARF, which normally inhibits MDM2-mediated p53 degradation, thereby resulting in p53 inactivation. Moreover, some studies indicate a possible role of the simian virus (SV)40, which could act as a co-carcinogen of asbestos in mesothelioma development.36 This could represent yet another mechanism of p53 inactivation through the interaction with the SV40 large T antigen.37 Therefore, restoring p53 activity in mesothelioma, through direct pharmacological targeting, could be an effective antitumoral approach. Consistent with this idea, previous studies showed that in mesothelioma cells lacking p14 ARF, p53 is functional and can be activated by p14 ARF adenoviral expression or MDM2 inhibition.12,14 Many research groups in the past few years took up the challenge to search for drugs able to reactivate p53. Here, we investigated the effect of 2 of these compounds, RITA and nutlin-3, which have a different mechanism of action, on a panel of mesothelioma cell lines of different histotypes. We first analyzed the mutational status of TP53 in these cell lines and identified in IST-MES 2 a p.Arg249Ser p53 mutation, which had never been described in mesothelioma. This mutation, lying in the DNAbinding domain, is considered an aflatoxin-associated mutational hotspot;38 however, it remains to be established whether it could have a causal role in mesothelioma development. Then, we showed that both RITA and nutlin-3 are indeed able to increase p53 levels in mesothelioma cell lines. Although in most mesothelioma cell lines analyzed, p53 indeed seems to be stabilized by RITA in a dose-dependent manner, in NCI-H2452, p53 level is maximal at doses below the IC50, whereas, paradoxically, it is consistently reduced at the IC50. We hypothesize that such reduction depends, at least in part, on the fact that p53 is mutated, because at higher doses (1 μM) it occurs also in IST-MES 2 (data not shown), which also carry mutated p53. Since NCI-H2452 undergo massive apoptosis when challenged with RITA, we also speculated that p53 reduction could depend on massive caspase activation, consistent with the fact that pretreatment with the Z-VAD-FMK caspase inhibitor at least partially abrogated this degradation (data not shown). Although both RITA and nutlin-3 stabilized p53 levels in mesothelioma cell lines, the 2 treatments had different consequences. RITA dramatically affected cell viability of epithelioid and biphasic mesothelioma cells, irrespectively of TP53 mutational status, without showing cytotoxic effects on the normal mesothelial cell line HMC-hTERT. Conversely, nutlin-3 had only a mild effect on mesothelioma cell viability and was quite toxic in HMC-hTERT. Cell cycle analysis showed that RITA, beyond causing an S-phase increase, which is consistent with a previously described role in stalling replication fork elongation,27 induced apoptosis in mesothelioma cell lines but not in HMC-hTERT nor in NCI-H2052, a cell line with a well-known intrinsic resistance to apoptosis, which characterizes the very aggressive sarcomatoid histotype.39,40 Treatment with nutlin-3, instead, caused a cell cycle arrest in mesothelioma cell lines carrying wt p53, whereas no arrest was achieved in cell lines carrying mutant p53. As described for other tumor types,19 nutlin-3-induced arrest seems to rely on the upregulation of the p53 target

Cell Cycle 659

©2014 Landes Bioscience. Do not distribute.

Discussion

ues below 1 indicate strong synergism.

660

Cell Cycle

Volume 13 Issue 4

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

p21. Consistently, we found that nutlin-3 was able to cause cell experiments with the PFT-α p53 inhibitor and with the ISTcycle arrest only in p53 wt cells concomitantly to p21 induction, MES 2 mutant in HCT116 argue for a direct involvement of p53. whereas IST-MES 2 and NCI-H2452, owing to a mutated p53, RITA dramatically reduced the colony-forming ability of did not express p21 following nutlin-3 treatment and did not mesothelioma cell lines, except for the resistant NCI-H2052, show cell cycle arrest. and inhibited the growth in soft agar of MSTO-211H, repreConversely, RITA treatment downregulated p21, consistent sentative of the biphasic histotype. Moreover, RITA treatment to what was previously reported,19 which probably favored apoptosis rather than growth arrest in mesothelioma cell lines. Consistently, IST-MES 2 and NCI-H2452, which are unable to express p21, show the highest sensitivity to RITA, further supporting p21 relevance in determining the cellular response to these antitumoral agents. Interestingly, we observed that whereas MDM2 protein levels were strongly induced by nutlin-3, they remained at low levels or even decreased in a dose-dependent manner in the more sensitive cell lines upon 24 h of RITA treatment. The kinetics of MDM2 regulation, upon RITA treatment, seems very complex,29 and in mesothelioma it is also possible to hypothesize that a non-transcriptional regulation also exists, which warrants further investigation. In particular, although the reduction of the MDM2 oncogene at 24 h obviously favors the apoptotic response in this context, it will be important to better define the specific contribution of MDM2 and other MDM2 possible targets to RITA-mediated apoptosis in mesothelioma. RITA-induced apoptosis in mesothelioma cell lines was caspase-dependent and seemed, indeed, triggered by p53 induction, because it was hindered by its inhibitor PFT-α. Interestingly, PFT-α was able to block RITA-induced apoptosis also in mesothelioma cell lines carrying mutant p53, suggesting that these mutants retain at least some apoptotic functions. This was further confirmed by transfecting the mutant p.Arg249Ser p53 that we identified in ISTMES 2 into HCT116 cells devoid of p53. RITA treatment was able to induce apoptosis in HCT116 transfected with this mutant p53, although to a lesser extent compared with the same cell line transfected with wt p53. Moreover, the p.Arg249Ser p53 mutant was able to transactivate a p53-responsive reporter gene, suggesting that its apoptotic function could be driven by its residual transcriptional activity. A p53 knockout mesothelioma model would be necessary to provide the definitive clue that Figure 7. Synergistic effect of RITA–cisplatin combination. (A) The table reports the IC50 values at 72 h of RITA and cisplatin on mesothelioma cell lines, as determined through RITA-induced apoptosis is indeed mediated by p53 MTS analysis of cell viability. The results represent the means of 3 independent experiin mesothelioma cell lines. The strategies that we ments, each conducted in triplicate. (B) Dose-response curves for RITA alone, cisplatin attempted to knockdown p53 in mesothelioma failed alone, and RITA–cisplatin combination in NCI-H28, NCI-H2452, IST-MES 2, and MSTOto achieve this demonstration, because RITA treat211H. The results represent the means of 3 independent experiments, each conducted ment was able to “reactivate” the expression of p53 in triplicate. (C) The table reports the mean Combination Index (CI) values of the drug combination at 50% and 75% of cell killing (CI50 and CI75) following 72 h of treatment, that we silenced both through siRNA and shRNA calculated by the CalcuSyn software for each of the 3 independent experiment. CI valin MSTO-211H (data not shown). However, the

www.landesbioscience.com

side effects. Remarkably, limited side effects should be expected with RITA treatment itself, because it did not show toxic effects on the normal mesothelial cell line, similarly to other studies reporting RITA to be preferentially cytotoxic to malignant cells.30 Consistent with the fact that p53 itself regulates a myriad of targets, RITA acts through multiple pathways. Moreover, RITA might even have p53-independent effects.34 Noteworthy, RITA was proved to inhibit several oncogenes and survival genes, including MYC and AKT, specifically in cancer cells,53 which is tantalizing considering the heterogeneity of genetic alterations in tumors. More recently, RITA was shown to sensitize tumor cells to agents that induce oxidative stress through a p53-dependent inhibition of thioredoxin reductase 1 (TrxR1), a key enzyme of the thioredoxin system, which protects cells from oxidative damage.54 Interestingly, TrxR1 is expressed at exceptionally high levels in mesothelioma cell lines, to which it is thought to confer a survival advantage by maintaining the redox status following the deleterious generation of reactive oxygen species induced by asbestos.55 This seems to further support RITA potential application for effective treatment of mesothelioma. In conclusion, although the molecular mechanisms underlying RITA effects in mesothelioma require further investigation, our study provides the preclinical framework for the possible use of this p53-reactivating agent in the most common forms of mesothelioma, either as a single agent or in combination with other therapies.

Materials and Methods Cell cultures NCI-H28, NCI-H2452, MSTO-211H, and NCIH2052 mesothelioma cell lines were purchased from ATCC (LGC Standards S.r.l.; http://www.lgcstandards-atcc.org/ Products/All/CRL-5820.aspx; http://www.lgcstandardsatcc.org/Products/All/CRL-5946.aspx; http://www.lgcstandards-atcc.org/Products/All/CRL-2081.aspx; http:// www.lgcstandards-atcc.org/Products/All/CRL-5915.aspx), whereas IST-MES 2 were purchased from the ISTGE Cell Repository (http://www.iclc.it; http://wwwsql.iclc.it/test/ iclc/det_list.php?line_id=5544&x=18&y=12). The normal human mesothelial cell line HMC-hTERT (immortalized with the human telomerase catalytic subunit, hTERT) was kindly provided by Dr G Gaudino.56 Mesothelioma cell lines were cultured according to the provider recommendations. HMC-hTERT were cultured in Medium199 (https://products.invitrogen.com/ivgn/product/31150030?ICID =searchproduct), 10% FBS (https://products.invitrogen.com/ivgn/ product/10099141?ICID=search-product), supplemented with 3.3 nM epidermal growth factor (http://products.invitrogen. com/ivgn/product/PHG0311?ICID=search-phg0311), 400 nM hydrocortisone (DBA Italia s.r.l.; http://www.mpbio.com/ product.php?pid=02101996), 870 nM insulin (https://products.invitrogen.com/ivgn/product/12585014?ICID =searchproduct). The human colon cancer HCT116 cell lines, a kind gift of Dr B Vogelstein to AB, were cultured in RPMI-1640 (https://products.invitrogen.com/ivgn/

Cell Cycle 661

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

reduced tumor growth of MSTO-211H xenografts, achieving tumor regression in 40% of mice, suggesting that pharmacological targeting of p53 could be a feasible antitumoral strategy to tackle mesothelioma in vivo. Further experiments, however, have to be performed to determine whether RITA acts in the in vivo setting through the same mechanisms herein shown in vitro and to explore whether resistance may arise. RITA ability to induce apoptosis in mesothelioma is noteworthy, because this tumor is very refractory to apoptosis owing to high expression of antiapoptotic proteins such as survivin,41,42 livin, and inhibitors of apoptosis proteins (IAPs).43,44 These proteins significantly contribute to mesothelioma progression promoting cell survival and correlate with mesothelioma prognosis.40 Active p53, beyond directly upregulating proapoptotic proteins, also inhibits antiapoptotic proteins like IAP and survivin.45,46 p53 functions as a safeguard mechanism against tumor development protecting cells from stresses or damage by inducing growth arrest or apoptosis. Understanding how p53 determines such different cell fates is crucial for the application of therapies based on its reactivation. In the clinical setting, it would be detrimental inducing cell cycle arrest in cancer cells, because it could favor DNA repair and interfere with the action of genotoxic chemotherapeutic drugs.19 A recent study suggests that, upon increase of p53 levels, p53 proarrest, and proapoptotic targets increase proportionally. What determines the cell fate decision between arrest and apoptosis is the balance between pro- and antiapoptotic signals, which is a dynamic equilibrium and varies within and among cells. Apoptosis is thought to have a higher execution threshold, defined as the minimum proapoptotic/antiapoptotic ratio.47 In our study RITA, but not nutlin-3, is able to overcome this threshold, probably through its action on p21. Interestingly, inhibition of antiapoptotic proteins can change this equilibrium and lower the apoptotic threshold.47 According to this view, it is likely that coupling to RITA treatment a strategy to inhibit antiapoptotic factors would lower the apoptotic threshold also in mesothelioma cells with a highly antiapoptotic milieu, such as that of the sarcomatoid histotype. Intrinsic resistance to apoptosis is also a key factor causing mesothelioma resistance to the cytotoxic effects of chemotherapy. For example, IAP-1 is responsible for a large degree of mesothelioma cell resistance to cisplatin.48 Moreover, a stable p21-dependent senescence-like growth arrest contributes to protect mesothelioma cells from drug-induced apoptosis. Consistently, inhibition of p21 expression enhanced chemosensitivity of mesothelioma cells to several antineoplastic DNA-damaging agents, suggesting that p21 activation is crucial to avoid apoptosis in response to chemotherapy.49 Similarly, p21 abrogation enhanced sensitivity of tumor cells to the apoptotic effects of HDAC inhibitors50,51 including mesothelioma cells.52 Therefore, whereas nutlin-3 treatment could be inefficient and counterproductive in mesothelioma (through p21 induction), RITA treatment could improve outcome following chemotherapy or exposure to other drugs. Accordingly, here we showed that RITA synergizes with cisplatin, which is the current mainstay of mesothelioma treatment, suggesting that the use of this drug combination could not only increase treatment efficacy but also reduce chemotherapy

662

used. Nutlin-3 was used at a range of 1.25–20 μM. RITA and nutlin-3 were dissolved in DMSO as a stock and diluted in culture medium. As a control, cells were treated with the maximum amount of DMSO used to vehicle the higher drug concentration. DMSO showed no toxic effects, consistently to what previously reported.57 Cell viability was measured by MTS assay through the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit (Promega; http://ita.promega.com/resources/protocols/ technical-bulletins/0/celltiter-96-aqueous-nonradioactive-cellproliferation-assay-protocol/). Each experiment was performed in triplicate. IC50 values were calculated using GraphPad Prism (GraphPad Software Inc; http://www.graphpad.com/scientificsoftware/prism/). For clonogenic assays, 1 × 103 - 2.5 × 103 cells were seeded in 60 mm plates and treated with RITA for 24 h, at the IC50 or, for NCI-H2052, with the maximum dose used. Two weeks after, colonies were stained with crystal violet. Cell cycle and apoptosis analysis Cell cycle was analyzed by FACS at 24, 48, and 72 h upon RITA or nutlin-3 treatment. Total cell populations were fixed in ice-cold 70% ethanol and stained with a 50 μg/mL propidium iodide solution containing 20 μg/mL RNase. DNA content was determined using a FACStar Canto (Becton Dickinson). Apoptosis was identified by analyzing the subdiploid region of the DNA content and evaluated through the Annexin V-FITC kit (Miltenyi Biotec GmbH; https://www.miltenyibiotec. com/~/media/Images/Products/Import/0001300/IM0001386. ashx) according to the manufacturer instructions, by FACS. Z-VAD-FMK (R&D Systems; http://www.rndsystems.com/ pdf/FMK001.pdf) and pifithrin-α (Sigma-Aldrich S.r.l.; http:// w w w.sigmaaldrich.com/content/dam/sigmaaldrich/docs / Sigma/Product_Information_Sheet/1/p4236pis.pdf) were dissolved in DMSO and diluted in fresh culture medium before use. Plasmids, transfections, and luciferase assay Full-length wt p53 or p53 p.Arg249Ser cDNAs were amplified from NCI-H28 and IST-MES 2, respectively, with EcoRI and NotI-containing primers for cloning into pCEFL-HA (a kind gift of Dr M Chiariello): 5′-CAACCCGAAT TCATGGAGGA GCCGCAGTCA GATCCTAG-3 and 5′-TAATTGCGGC CGCTTATCAG TCTGAGTCAG GCCCTTCTG-3′. For annexin-V assay, 1 × 106 HCT116 p53–/– cells were transfected with pCEFL-HA plasmids using Lipofectamine 2000 (Life Technologies Italia; http://products.invitrogen.com/ivgn/ product/11668019). Eighteen hours after transfection, cells were treated with DMSO or RITA (1.6 μM) for 24 h and harvested to analyze apoptosis with the annexin V-FITC kit. For Luciferase assay, HCT116 p53–/– cells were seeded in 6-well plates and co-transfected with the PG13 or MG15 constructs, expressing, respectively, the luciferase reporter gene downstream to p53 binding sequences or the same sequences mutated in the p53 binding sites (Addgene plasmid 16442 and 16443);35,58 pCEFL-HA (expressing either wt p53 or p53 p.Arg249Ser); and 0.5 μg of pRL-CMV encoding Renilla luciferase (Promega; http://ita.promega.com/resources/protocols/ technical-manuals/0/renilla-luciferase-assay-system-protocol/). Each transfection was performed in triplicate. Eighteen hours after transfection, cells were treated with DMSO or RITA for

Cell Cycle

Volume 13 Issue 4

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

product/61870044?ICID=search-product), 10% FBS. All cell culture reagents were purchased from Life Technologies Italia, unless otherwise specified. TP53 sequencing One microgram of Trizol-isolated RNA was retrotranscribed with SuperScript reverse transcriptase III (Life Technologies Italia; http://products.invitrogen.com/ivgn/ product/15596026; https://products.invitrogen.com/ivgn/ product/18080085?ICID=search-product). The primers used for PCR amplification of full-length TP53 were: 5′-CGTCCAGGGA GCAGGTAG-3′ and 5′-CAAGCAAGGG TTCAAAGAC-3′. PCRs were performed using Pfu DNA polymerase (Agilent Technologies; http://www.chem.agilent.com/ Library/usermanuals/Public/600670.pdf). The reaction mix was denatured at 94 °C for 2 min and subjected to 40 amplification cycles, of 20 s at 94 °C, 20 s at 61 °C, 40 s at 72 °C each, followed by a 3 min extension at 72 °C. PCR products were purified using the QIAquick gel extraction kit (Qiagen S.r.l.; http://www.qiagen.com/Products/Catalog/Sample-Technologies/DNA-SampleTechnologies/DNA-Cleanup/QIAquick-Gel-Extraction-Kit). Sequencing reactions were performed by PRIMM srl, and analyzed through the Sequencher Software (Gene Codes Corporation; http://genecodes.com/). The human TP53 wt sequence used as reference was NM_000546.4 (National Center for Biotechnology Information). Western blot For total protein extraction, cells were lysed on ice for 30 min in lysis buffer containing 1 mM EDTA, 150 mM NaCl, 1%NP-40, 50 mM TRIS-HCL pH 7.5, and 10 mg/ml each of aprotinin, leupeptin, and pepstatin. Cell lysates were subjected to SDS-PAGE. Western blots were performed with antibodies against p53 (mouse monoclonal DO-1; http://www.scbt.it/datasheet-126-p53-do-1-antibody.html), p14 ARF (rabbit polyclonal sc-8340; http://www.scbt.it/datasheet-8340-p14-arf-h-132-antibody.html), p21 (mouse monoclonal sc-817; http://www.scbt. it/datasheet-817-p21-187-antibody.html), MDM2 (mouse monoclonal sc-965; http://datasheets.scbt.com/sc-965.pdf), caspase 3: (rabbit polyclonal #9662; Cell Signaling; http://www.cellsignal. com/products/9662.html), PARP: (mouse monoclonal #556494, BD PharMingen; http://www.bdbiosciences.com/external_files/ pm/doc/tds/cell_bio/live/web_enabled/66401A_556494.pdf ), and GAPDH (rabbit polyclonal sc-25778; http://www.scbt. it/datasheet-25778-gapdh-fl-335-antibody.html). Signals were detected through ECL (Amersham Biosciences; http://www. gelifesciences.com/webapp/wcs/stores/servlet/productById/it/ GELifeSciences/28980926). MTS and clonogenic assay Cells were seeded in 96-well plates at 1 × 103 – 2.5 × 103 cell/100 μL/well density and treated with increasing doses of RITA (Vincibiochem S.r.l; http://www.vincibiochem.it/ prodotti/BX1PksnibRU=/d8P8fr1qPhE ) or increasing doses of nutlin-3 (Sigma-Aldrich S.r.l; http://www.sigmaaldrich.com/ catalog/product/sigma/n6287?lang=it®ion=IT) for 24, 48, or 72 h. In particular, RITA was used at a range of 0.01–1 μM for all cell lines except for MSTO-211H and NCI-H2052, of the most aggressive histotypes, for which a range of 0.01–5 μM was

References 1.

William D. Travis, Elisabeth Brambilla, H. Konrad Müller-Hermelink, Curtis C. Harris, eds. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. Lyon, France: IARCPress International Agency for Research on Cancer (IARC) 2004. 2. LaDou J, Castleman B, Frank A, Gochfeld M, Greenberg M, Huff J, Joshi TK, Landrigan PJ, Lemen R, Myers J, et al. The case for a global ban on asbestos. Environ Health Perspect 2010; 118:897901; PMID:20601329; http://dx.doi.org/10.1289/ ehp.1002285

www.landesbioscience.com

additivity or antagonism were determined calculating the combination index (CI) according to the Chou–Talalay equation, through the Calcusyn Software (BioSoft; http://www.biosoft. com/w/calcusyn.htm).59 CI < 1 indicates synergism, CI = 1 additive effect, and CI > 1 antagonism. The r value represents the linear correlation coefficient of the median-effect plot, which indicates the conformity of the data to the mass-action law. Statistical analyses Statistical analyses were performed using the GraphPad Software. Statistically significant differences between the means of multiple matched groups were evaluated by 1-way repeated measures Anova with either Dunnett post-test, to compare all data vs. control (MTS assays), or Tukey post-test, to compare all pairs of data (pifithrin-α analyses). To compare the means of 2 matched groups, as for luciferase assay and soft agar analyses, we used paired Student t test. The nonparametric Mann–Whitney test was used for median comparison of tumor volumes, which failed the normality test, between DMSO- and RITA-treated xenotransplanted mice at each evaluation time. The Mann– Whitney test was also used for median comparison of the weights of tumors excised from DMSO- and RITA-treated mice at the end of treatment. P < 0.05 was considered statistically significant. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

We are thankful to the Sbarro Health Research Organization (http://www.shro.org), the Human Health Foundation (http:// www.hhfonlus.org) and the Commonwealth of Pennsylvania for their support. We also wish to thank Bert Vogelstein for the PG13luc and MG15-luc constructs and Caterina Missero (CEINGE Biotecnologie Avanzate-Center for Genetic Engineering, Napoli, Italy) for helpful discussions. We are very grateful to Pasquale Barba and Laura Pisapia (Consiglio Nazionale delle RicercheInstitute of Genetics and Biophysics, Naples, Italy) for technical help with the FACS analyses and to Vincenza Rubini and Rossana Daprile of the National Cancer Research Centre, Istituto Tumori “Giovanni Paolo II” of Bari for technical help with xenograft analyses. This work is dedicated to the memory of Delia Pepe. Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/cc/article/27546

3. Stayner L, Welch LS, Lemen R. The worldwide pandemic of asbestos-related diseases. Annu Rev Public Health 2013; 34:205-16; PMID:23297667; http://dx.doi.org/10.1146/ annurev-publhealth-031811-124704 4. Tsao AS, Wistuba I, Roth JA, Kindler HL. Malignant pleural mesothelioma. J Clin Oncol 2009; 27:208190; PMID:19255316; http://dx.doi.org/10.1200/ JCO.2008.19.8523 5. Bullock AN, Fersht AR. Rescuing the function of mutant p53. Nat Rev Cancer 2001; 1:68-76; PMID:11900253; http://dx.doi. org/10.1038/35094077

6.

7.

8.

Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408:307-10; PMID:11099028; http://dx.doi.org/10.1038/35042675 Metcalf RA, Welsh JA, Bennett WP, Seddon MB, Lehman TA, Pelin K, Linnainmaa K, Tammilehto L, Mattson K, Gerwin BI, et al. p53 and Kirsten-ras mutations in human mesothelioma cell lines. Cancer Res 1992; 52:2610-5; PMID:1568228 Mor O, Yaron P, Huszar M, Yellin A, Jakobovitz O, Brok-Simoni F, Rechavi G, Reichert N. Absence of p53 mutations in malignant mesotheliomas. Am J Respir Cell Mol Biol 1997; 16:9-13; PMID:8998073; http://dx.doi.org/10.1165/ajrcmb.16.1.8998073

Cell Cycle 663

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

24 h, then harvested and analyzed for luciferase activity through the Dual-Glo™ Luciferase Assay System (Promega; http://ita. promega.com/resources/protocols/technical-manuals/0/dualglo-luciferase-assay-system-protocol/). Luciferase activity was normalized against the Renilla internal control. Soft agar assay and in vivo experiments MSTO-211H cells (2 × 103) were resuspended in a top-agar solution (0.3% agar, 10% FBS, RPMI and either DMSO or 1.6 μM RITA) and seeded on a 0.8% agar base, (Bacto™ Agar; Conda; http://www.condalab.com/pdf/1433.pdf). Two weeks after, colonies were stained with nitro blue tetrazolium (NBT) dye (Life Technologies; http://products.invitrogen.com/ivgn/ product/N6495?ICID=search-product). Xenograft experiments were performed at the Animal Model Facility of the Biogem Institute of Genetics Research (Ariano Irpino). Animals were maintained in compliance with the current normative (Italian DL.vo N° 116–27/01/1992 and EU 86/609/ CEE-24/11/1986). All animal experiments were approved by the local animal experiment approval committee (project number G08/75). MSTO-211H cells (1.2 × 106) were injected on the right flank of 20 female nude mice, 6–7-wk-old. Treatment started 13 d after inoculation when xenografts appeared consistently palpable. Mice were daily treated with intratumoral administration of either 150 μL of 5% DMSO (10 mice) or 150 μL of RITA at a 1 mg/kg dose (10 mice) for 5 consecutive days, followed by 2 treatment-free days for a total of 3 wk. Xenograft volumes were measured twice weekly through a caliper during the whole study period. Tumor volumes were calculated through the formula: 1/2(length × width2).60 At the end of the study period mice were sacrificed and tumors were weighted upon resection. Drug combination studies For drug combination studies, we first determined cisplatin (Calbiochem; http://www.merckm i l l i p o r e . c o m / i s - b i n / I N T E R S H O P. e n f i n it y / W F S / Merck-IT-Site/en_US/-/EUR/ViewProductDetail-UserPro tocolDataSheet?ProductUUID =2ZWb.s1OkKQA A AEaI_ pqKZH5&PortalCatalogUUID =ywGb.s1LAyMA A AEWzd Uf VhTl&SelectedDocumentType=UserProtocolDataSheet) cytotoxicity, as for RITA, by MTS assay as described above and elsewhere.61 Serial dilutions of RITA and cisplatin, combined in various doses at constant ratio, were tested by MTS. Concentration-effect curves were generated as a plot of the percentage of viable cells vs. drug concentration and synergism,

Cheng JQ, Jhanwar SC, Klein WM, Bell DW, Lee WC, Altomare DA, Nobori T, Olopade OI, Buckler AJ, Testa JR. p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma. Cancer Res 1994; 54:5547-51; PMID:7923195 10. Prins JB, Williamson KA, Kamp MM, Van Hezik EJ, Van der Kwast TH, Hagemeijer A, Versnel MA. The gene for the cyclin-dependent-kinase-4 inhibitor, CDKN2A, is preferentially deleted in malignant mesothelioma. Int J Cancer 1998; 75:649-53; PMID:9466670; http://dx.doi.org/10.1002/ ( SICI )1097- 0215 (19980209 ) 75 : 4 < 6 49 : : A ID IJC25>3.0.CO;2-2 11. Lecomte C, Andujar P, Renier A, Kheuang L, Abramowski V, Mellottee L, Fleury-Feith J, ZucmanRossi J, Giovannini M, Jaurand MC. Similar tumor suppressor gene alteration profiles in asbestosinduced murine and human mesothelioma. Cell Cycle 2005; 4:1862-9; PMID:16319530; http:// dx.doi.org/10.4161/cc.4.12.2300 12. Yang CT, You L, Uematsu K, Yeh CC, McCormick F, Jablons DM. p14(ARF) modulates the cytolytic effect of ONYX-015 in mesothelioma cells with wild-type p53. Cancer Res 2001; 61:5959-63; PMID:11507034 13. Yang CT, You L, Yeh CC, Chang JW, Zhang F, McCormick F, Jablons DM. Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells. J Natl Cancer Inst 2000; 92:636-41; PMID:10772681; http://dx.doi.org/10.1093/ jnci/92.8.636 14. Hopkins-Donaldson S, Belyanskaya LL, SimõesWüst AP, Sigrist B, Kurtz S, Zangemeister-Wittke U, Stahel R. p53-induced apoptosis occurs in the absence of p14(ARF) in malignant pleural mesothelioma. Neoplasia 2006; 8:551-9; PMID:16867217; http:// dx.doi.org/10.1593/neo.06148 15. Di Cintio A, Di Gennaro E, Budillon A. Restoring p53 function in cancer: novel therapeutic approaches for applying the brakes to tumorigenesis. Recent Pat Anticancer Drug Discov 2010; 5:1-13; PMID:19663772; http://dx.doi. org/10.2174/157489210789702172 16. Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, Pramanik A, Selivanova G. Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat Med 2004; 10:1321-8; PMID:15558054; http:// dx.doi.org/10.1038/nm1146 17. Zhao CY, Grinkevich VV, Nikulenkov F, Bao W, Selivanova G. Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA. Cell Cycle 2010; 9:1847-55; PMID:20436301; http:// dx.doi.org/10.4161/cc.9.9.11545 18. Girardini JE, Del Sal G. Improving pharmacological rescue of p53 function: RITA targets mutant p53. Cell Cycle 2010; 9:2062; PMID:20559030; http:// dx.doi.org/10.4161/cc.9.11.11859 19. Enge M, Bao W, Hedström E, Jackson SP, Moumen A, Selivanova G. MDM2-dependent downregulation of p21 and hnRNP K provides a switch between apoptosis and growth arrest induced by pharmacologically activated p53. Cancer Cell 2009; 15:17183; PMID:19249676; http://dx.doi.org/10.1016/j. ccr.2009.01.019 20. Roh JL, Ko JH, Moon SJ, Ryu CH, Choi JY, Koch WM. The p53-reactivating small-molecule RITA enhances cisplatin-induced cytotoxicity and apoptosis in head and neck cancer. Cancer Lett 2012; 325:3541; PMID:22634494; http://dx.doi.org/10.1016/j. canlet.2012.05.020 21. Henze J, Mühlenberg T, Simon S, Grabellus F, Rubin B, Taeger G, Schuler M, Treckmann J, DebiecRychter M, Taguchi T, et al. p53 modulation as a therapeutic strategy in gastrointestinal stromal tumors. PLoS One 2012; 7:e37776; PMID:22662219; http:// dx.doi.org/10.1371/journal.pone.0037776

664

22. Musti M, Kettunen E, Dragonieri S, Lindholm P, Cavone D, Serio G, Knuutila S. Cytogenetic and molecular genetic changes in malignant mesothelioma. Cancer Genet Cytogenet 2006; 170:9-15; PMID:16965949; http://dx.doi.org/10.1016/j. cancergencyto.2006.04.011 23. Romagnoli S, Fasoli E, Vaira V, Falleni M, Pellegrini C, Catania A, Roncalli M, Marchetti A, Santambrogio L, Coggi G, et al. Identification of potential therapeutic targets in malignant mesothelioma using cell cycle gene expression analysis. Am J Pathol 2009; 174:762-70; PMID:19218339; http:// dx.doi.org/10.2353/ajpath.2009.080721 24. Lunn RM, Zhang YJ, Wang LY, Chen CJ, Lee PH, Lee CS, Tsai WY, Santella RM. p53 mutations, chronic hepatitis B virus infection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan. Cancer Res 1997; 57:3471-7; PMID:9270015 25. Whibley C, Pharoah PD, Hollstein M. p53 polymorphisms: cancer implications. Nat Rev Cancer 2009; 9:95-107; PMID:19165225; http://dx.doi. org/10.1038/nrc2584 26. Moran DM, Maki CG. Nutlin-3a induces cytoskeletal rearrangement and inhibits the migration and invasion capacity of p53 wild-type cancer cells. Mol Cancer Ther 2010; 9:895-905; PMID:20371712; http://dx.doi.org/10.1158/1535-7163.MCT-09-1220 27. Ahmed A, Yang J, Maya-Mendoza A, Jackson DA, Ashcroft M. Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK1. Cell Death Dis 2011; 2:e160; PMID:21593792; http://dx.doi.org/10.1038/cddis.2011.42 28. Abbas T, Dutta A. p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 2009; 9:40014; PMID:19440234; http://dx.doi.org/10.1038/ nrc2657 29. Rinaldo C, Prodosmo A, Siepi F, Moncada A, Sacchi A, Selivanova G, Soddu S. HIPK2 regulation by MDM2 determines tumor cell response to the p53-reactivating drugs nutlin-3 and RITA. Cancer Res 2009; 69:6241-8; PMID:19638586; http:// dx.doi.org/10.1158/0008-5472.CAN-09-0337 30. Saha MN, Jiang H, Mukai A, Chang H. RITA inhibits multiple myeloma cell growth through induction of p53-mediated caspase-dependent apoptosis and synergistically enhances nutlin-induced cytotoxic responses. Mol Cancer Ther 2010; 9:3041-51; PMID:21062913; http://dx.doi.org/10.1158/15357163.MCT-10-0471 31. Koster R, Timmer-Bosscha H, Bischoff R, Gietema JA, de Jong S. Disruption of the MDM2-p53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway. Cell Death Dis 2011; 2:e148; PMID:21509038; http://dx.doi. org/10.1038/cddis.2011.33 32. Komarov PG, Komarova EA, Kondratov RV, Christov-Tselkov K, Coon JS, Chernov MV, Gudkov AV. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 1999; 285:1733-7; PMID:10481009; http://dx.doi. org/10.1126/science.285.5434.1733 33. Strom E, Sathe S, Komarov PG, Chernova OB, Pavlovska I, Shyshynova I, Bosykh DA, Burdelya LG, Macklis RM, Skaliter R, et al. Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat Chem Biol 2006; 2:4749; PMID:16862141; http://dx.doi.org/10.1038/ nchembio809 34. Di Conza G, Buttarelli M, Monti O, Pellegrino M, Mancini F, Pontecorvi A, Scotlandi K, Moretti F. IGF-1R/MDM2 relationship confers enhanced sensitivity to RITA in Ewing sarcoma cells. Mol Cancer Ther 2012; 11:1247-56; PMID:22461661; http:// dx.doi.org/10.1158/1535-7163.MCT-11-0913

Cell Cycle

35. Kern SE, Pietenpol JA, Thiagalingam S, Seymour A, Kinzler KW, Vogelstein B. Oncogenic forms of p53 inhibit p53-regulated gene expression. Science 1992; 256:827-30; PMID:1589764; http://dx.doi. org/10.1126/science.1589764 36. Mazzoni E, Corallini A, Cristaudo A, Taronna A, Tassi G, Manfrini M, Comar M, Bovenzi M, Guaschino R, Vaniglia F, et al. High prevalence of serum antibodies reacting with simian virus 40 capsid protein mimotopes in patients affected by malignant pleural mesothelioma. Proc Natl Acad Sci U S A 2012; 109:18066-71; PMID:23071320; http:// dx.doi.org/10.1073/pnas.1213238109 37. Carbone M, Rizzo P, Grimley PM, Procopio A, Mew DJ, Shridhar V, de Bartolomeis A, Esposito V, Giuliano MT, Steinberg SM, et al. Simian virus-40 large-T antigen binds p53 in human mesotheliomas. Nat Med 1997; 3:908-12; PMID:9256284; http:// dx.doi.org/10.1038/nm0897-908 38. Soini Y, Chia SC, Bennett WP, Groopman JD, Wang JS, DeBenedetti VM, Cawley H, Welsh JA, Hansen C, Bergasa NV, et al. An aflatoxin-associated mutational hotspot at codon 249 in the p53 tumor suppressor gene occurs in hepatocellular carcinomas from Mexico. Carcinogenesis 1996; 17:100712; PMID:8640905; http://dx.doi.org/10.1093/ carcin/17.5.1007 39. Aung W, Hasegawa S, Furukawa T, Saga T. Potential role of ferritin heavy chain in oxidative stress and apoptosis in human mesothelial and mesothelioma cells: implications for asbestos-induced oncogenesis. Carcinogenesis 2007; 28:2047-52; PMID:17434931; http://dx.doi.org/10.1093/carcin/bgm090 40. Villanova F, Procopio A, Rippo MR. Malignant mesothelioma resistance to apoptosis: recent discoveries and their implication for effective therapeutic strategies. Curr Med Chem 2008; 15:631-41; PMID:18336278; http://dx.doi.org/10.2174/092986708783885273 41. Xia C, Xu Z, Yuan X, Uematsu K, You L, Li K, Li L, McCormick F, Jablons DM. Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides. Mol Cancer Ther 2002; 1:687-94; PMID:12479365 42. Falleni M, Pellegrini C, Marchetti A, Roncalli M, Nosotti M, Palleschi A, Santambrogio L, Coggi G, Bosari S. Quantitative evaluation of the apoptosis regulating genes Survivin, Bcl-2 and Bax in inflammatory and malignant pleural lesions. Lung Cancer 2005; 48:211-6; PMID:15829320; http://dx.doi. org/10.1016/j.lungcan.2004.10.003 43. Zaffaroni N, Costa A, Pennati M, De Marco C, Affini E, Madeo M, Erdas R, Cabras A, Kusamura S, Baratti D, et al. Survivin is highly expressed and promotes cell survival in malignant peritoneal mesothelioma. Cell Oncol 2007; 29:453-66; PMID:18032822 44. Gordon GJ, Mani M, Mukhopadhyay L, Dong L, Edenfield HR, Glickman JN, Yeap BY, Sugarbaker DJ, Bueno R. Expression patterns of inhibitor of apoptosis proteins in malignant pleural mesothelioma. J Pathol 2007; 211:447-54; PMID:17253596; http://dx.doi.org/10.1002/path.2121 45. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002; 277:3247-57; PMID:11714700; http://dx.doi. org/10.1074/jbc.M106643200 46. Mirza A, McGuirk M, Hockenberry TN, Wu Q, Ashar H, Black S, Wen SF, Wang L, Kirschmeier P, Bishop WR, et al. Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene 2002; 21:2613-22; PMID:11965534; http://dx.doi. org/10.1038/sj.onc.1205353 47. Kracikova M, Akiri G, George A, Sachidanandam R, Aaronson SA. A threshold mechanism mediates p53 cell fate decision between growth arrest and apoptosis. Cell Death Differ 2013; 20:57688; PMID:23306555; http://dx.doi.org/10.1038/ cdd.2012.155

Volume 13 Issue 4

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

9.

www.landesbioscience.com

53. Grinkevich VV, Nikulenkov F, Shi Y, Enge M, Bao W, Maljukova A, Gluch A, Kel A, Sangfelt O, Selivanova G. Ablation of key oncogenic pathways by RITA-reactivated p53 is required for efficient apoptosis. Cancer Cell 2009; 15:441-53; PMID:19411072; http://dx.doi.org/10.1016/j.ccr.2009.03.021 54. Hedström E, Eriksson S, Zawacka-Pankau J, Arnér ES, Selivanova G. p53-dependent inhibition of TrxR1 contributes to the tumor-specific induction of apoptosis by RITA. Cell Cycle 2009; 8:357683; PMID:19838062; http://dx.doi.org/10.4161/ cc.8.21.9977 55. Nilsonne G, Sun X, Nyström C, Rundlöf AK, Potamitou Fernandes A, Björnstedt M, Dobra K. Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress. Free Radic Biol Med 2006; 41:874-85; PMID:16934670; http://dx.doi.org/10.1016/j. freeradbiomed.2006.04.031 56. Busacca S, Germano S, De Cecco L, Rinaldi M, Comoglio F, Favero F, Murer B, Mutti L, Pierotti M, Gaudino G. MicroRNA signature of malignant mesothelioma with potential diagnostic and prognostic implications. Am J Respir Cell Mol Biol 2010; 42:312-9; PMID:19502386; http://dx.doi. org/10.1165/rcmb.2009-0060OC

57. Indovina P, Giorgi F, Rizzo V, Khadang B, Schenone S, Di Marzo D, Forte IM, Tomei V, Mattioli E, D’Urso V, et al. New pyrazolo[3,4-d]pyrimidine SRC inhibitors induce apoptosis in mesothelioma cell lines through p27 nuclear stabilization. Oncogene 2012; 31:929-38; PMID:21785466; http://dx.doi. org/10.1038/onc.2011.286 58. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993; 75:817-25; PMID:8242752; http://dx.doi.org/10.1016/0092-8674(93)90500-P 59. Chou TC, Talalay P. Quantitative analysis of doseeffect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22:27-55; PMID:6382953; http://dx.doi. org/10.1016/0065-2571(84)90007-4 60. Euhus DM, Hudd C, LaRegina MC, Johnson FE. Tumor measurement in the nude mouse. J Surg Oncol 1986; 31:229-34; PMID:3724177; http://dx.doi. org/10.1002/jso.2930310402 61. Indovina P, Marcelli E, Di Marzo D, Casini N, Forte IM, Giorgi F, Alfano L, Pentimalli F, Giordano A. Abrogating G 2/M checkpoint through WEE1 inhibition in combination with chemotherapy as a promising therapeutic approach for mesothelioma. Cancer Biol Ther 2013; 15; PMID:24365782

Cell Cycle 665

©2014 Landes Bioscience. Do not distribute.

Downloaded by [George Washington University] at 10:45 03 February 2015

48. Gordon GJ, Appasani K, Parcells JP, Mukhopadhyay NK, Jaklitsch MT, Richards WG, Sugarbaker DJ, Bueno R. Inhibitor of apoptosis protein-1 promotes tumor cell survival in mesothelioma. Carcinogenesis 2002; 23:1017-24; PMID:12082024; http://dx.doi. org/10.1093/carcin/23.6.1017 49. Lazzarini R, Moretti S, Orecchia S, Betta PG, Procopio A, Catalano A. Enhanced antitumor therapy by inhibition of p21waf1 in human malignant mesothelioma. Clin Cancer Res 2008; 14:5099-107; PMID:18698027; http://dx.doi.org/10.1158/10780432.CCR-08-0255 50. Sandor V, Senderowicz A, Mertins S, Sackett D, Sausville E, Blagosklonny MV, Bates SE. P21dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228. Br J Cancer 2000; 83:817-25; PMID:10952788; http://dx.doi. org/10.1054/bjoc.2000.1327 51. Almenara J, Rosato R, Grant S. Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Leukemia 2002; 16:1331-43; PMID:12094258; http://dx.doi.org/10.1038/sj.leu.2402535 52. Nguyen DM, Schrump WD, Chen GA, Tsai W, Nguyen P, Trepel JB, Schrump DS. Abrogation of p21 expression by flavopiridol enhances depsipeptide-mediated apoptosis in malignant pleural mesothelioma cells. Clin Cancer Res 2004; 10:1813-25; PMID:15014036; http://dx.doi.org/10.1158/10780432.CCR-0901-3

Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma.

Malignant mesothelioma, a very aggressive tumor associated to asbestos exposure, is expected to increase in incidence, and unfortunately, no curative ...
3MB Sizes 0 Downloads 0 Views