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Lung Cancer 82 (2013) 214– 221

Contents lists available at ScienceDirect

Lung Cancer

jou rn al h om epa g e: www.elsev ier .com/ locate / lungcan

otentiation of anticancer effect of valproic acid, an antiepilepticgent with histone deacetylase inhibitory activity, by theyclin-dependent kinase inhibitor P276-00 in humanon-small-cell lung cancer cell lines

itesh Shirsatha,1, Maggie Rathosa,1, Umesh Chaudharia,1, H. Sivaramakrishnanb,alpana Joshia,∗

Department of Pharmacology, Piramal Life Sciences, Piramal Enterprises Limited, 1 Nirlon Complex, Goregaon (East), Mumbai, Maharashtra 400 063, IndiaHead R & D, Piramal Life Sciences, Piramal Enterprises Limited, 1-Nirlon Complex, Goregaon (East), Mumbai, Maharashtra 400 063, India

r t i c l e i n f o

rticle history:eceived 29 April 2013eceived in revised form 1 August 2013ccepted 7 August 2013

eywords:DK inhibitor276-00alproic acidDACombination studieson-small-cell lung cancinoma

a b s t r a c t

Background: P276-00 is a novel cyclin-dependent kinase (CDK) inhibitor is in Phase II clinical trials.Valproic acid (VPA), an antiepileptic agent has been associated with anticancer activity, through theinhibition of histone deacetylase I. Here we investigate the effect of the combination of VPA and P276-00,in non-small-cell lung cancer (NSCLC) cell lines.Materials and methods: Cell growth inhibition was studied using the Propidium iodide (PI) assay. Cell cycleanalysis and recovery were detected by flow cytometry. The expression levels of various proteins weredetected by western blot. Inhibition of colony formation in H460 was checked in vitro. In vivo efficacywas studied in H460 xenograft model.Results: The combination of P276-00 and VPA showed synergistic effect on p53+ and p53− NSCLC cell linesin antiproliferative assay at both constant and non-constant ratio with marked decrease in colony formingpotential. Flow cytometric analysis confirmed a significant time dependent increase in apoptosis with 64%apoptotic population at 96 h compared to VPA (1%) and P276-00 (28%) alone (p < 0.0001). Incubation ofthe cells after treatment, in fresh medium without drugs, led to the recovery of cells treated with P276-00alone but not the cells treated with the combination of both the drugs. The combination treatment up-regulated tumor suppressor proteins like p53, p21 and p27 along with down-regulation of proliferation

and survival proteins viz. cyclin D1 and Bcl-2. This was also associated with the upregulation of thepro-apoptotic protein Bax and significant accumulation of hyperacetylated histones in the combinationtreatment. Interestingly, VPA in combination with P276-00 was much more effective as an antitumoragent than alone, in the H460 xenograft tumor model in SCID mice.Conclusions: This study indicates that the combination of HDAC inhibitor VPA with CDK inhibitor P276-00is promising novel molecularly targeted therapeutic approach for NSCLC treatment.

. Introduction

Lung cancer historically remains the most common malignancyorldwide and accounts for more deaths than any other cancer

n both men and women. An estimated 226,160 new cases of lung

ancer are expected in 2012, accounting for about 14% of all canceriagnoses. The 5-year survival rate for all stages has been only 15%.SCLC comprises approximately 85% of all lung cancer cases [1–3].

∗ Corresponding author: Tel.: +91 22 30818421.E-mail addresses: kalpana.joshi@piramal.com, kalpanajoshi2004@yahoo.co.in

K. Joshi).1 All the three authors have contributed equally.

169-5002/$ – see front matter © 2013 Published by Elsevier Ireland Ltd.ttp://dx.doi.org/10.1016/j.lungcan.2013.08.010

© 2013 Published by Elsevier Ireland Ltd.

Chemotherapy against NSCLC is one therapeutic approach forpatients who have unresectable tumors in an advanced stage. Buttreatment outcomes are still poor [4]. Although newer chemother-apeutic reagents (paclitaxel, erlotinib, docetaxel, gemcitabine,vinorelbine and irinotecan) are more active for lung cancer thanprior drugs and do provide a moderate therapeutic benefit in theadjuvant setting, they still have little effect on recurrent disease.The notion of combining targeted agents with additional standardchemotherapy for NSCLC is currently the focus of intense interest[5].

Chromatin remodeling via histone acetylation by histone acetyl-transferases and histone deacetylases (HDACs) is considered as akey element in the dynamic regulation of many genes that playcrucial roles in cell growth and differentiation [6]. Additionally,

N. Shirsath et al. / Lung Cancer 82 (2013) 214– 221 215

Fig. 1. Cytotoxic potentiation of VPA by P276-00 in H460 and H23 cells was detected by PI assay. Cells were treated with P276-00 IC30 (0.6 �M) and IC50 (1 �M) aloneor in combination with range of VPA concentration 0.1, 0.3, 0.7, 1 mM (upper limit of antiepileptic range) at 72 h and 96 h. Controls remained untreated. These data arerepresentative of 2 independent studies. With decreasing concentration of VPA the antiproliferative activity in H460 (A) is sustained showing significant enhanced activity(p < 0.005). P276-00 has synergistic effect when combined with VPA at 1, 0.7 and 0.3 mM (clinically safe dose). Combination index (CI) was calculated using CompuSynsoftware and showed CI < 1. Similarly in H23 (B) combination was synergistic with CI < 1.

2 Cancer 82 (2013) 214– 221

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Fig. 2. Growth inhibition of NSCLC cell line H460 in colony forming assay. Combi-nation of P276-00 along with VPA inhibits colony formation of H460 cell line. NSCLCcell line H460 was grown in six-well plates (500 cells/well) in triplicates. After 24 h,the culture medium was replaced with fresh medium containing 10% FBS as control,or same medium containing 1 mM of VPA and 1 �M of P276-00 for 96 h. The culturemedium was changed once every three days post treatment for three weeks. The

16 N. Shirsath et al. / Lung

DACs also deacetylate a plethora of other proteins including p53,-Myc and others. Thus, histone deacetylases have been shown toe involved in oncogenic transformation by mediating the functionf transcription factors in the pathogenesis of solid tumors alongith certain forms of haematologic malignancies [7].

Thus, Valproic acid (VPA) which is a well-tolerated anticonvul-ant/antiepileptic agent has been identified recently as a histoneeacetylase inhibitor. Epigenetic modifications are observed uni-ersally during the tumorigenesis of lung cancer. Its veiledbility to inhibit histone deacetylases (HDACs) and to reactivateormant tumor suppressor genes has highlighted its poten-ial role in the treatment of cancer [8,9]. However utility ofDAC inhibition in other cancers such as NSCLC is less estab-

ished.Cyclin-dependent kinase inhibitors have shown the ability not

nly to block neoplastic cell proliferation, but also to induce,hrough a variety of mechanisms, programmed cell death. In addi-ion, there is abundant preclinical evidence that CDK inhibitors canotentiate, generally in a dose- and sequence-dependent manner,he anti-tumor effects of many established cytostatic and cytotoxicgents [10–13]. P276-00, one of the few CDK inhibitors to enter thelinical arena, is an inhibitor of multiple cyclin-dependent kinasesnd a transcriptional repressor. As shown earlier P276-00 morepecifically, acts as a potent inhibitor of the CDK4-D1, CDK1-B andDK9-T, disrupting the function of several other short-lived pro-eins, which are dependent upon continued transcription [14–16].lso, it inhibits phosphorylation of the C-terminal repeat domainf RNA polymerase II and disrupts initiation and elongation [17].he present study was therefore undertaken to assess the syner-ism of epigenetic-modulating activity of the HDAC inhibitor VPAnd the CDK inhibitor P276-00 as a novel therapeutic approach inhe treatment of NSCLC.

. Materials and reagents

.1. Cell lines and reagents

The human non-small-cancer cell lines H460, H23, A549nd H1299 were obtained from ATCC (Rockville, MD, USA).ll cell lines were cultured in RPMI-1640 medium contain-

ng 10% fetal bovine serum (FBS) (Hyclone, UT, USA), 2 mM-glutamine (Gibco, Grand Island, NY, USA), 100 U/mL penicillinnd 100 �g/mL streptomycin (Gibco) at 37 ◦C in a humidifiedtmosphere with 5% CO2. P276-00 and VPA were synthesized atiramal Enterprises Limited, Mumbai, India. P276-00 was dissolvedn dimethyl sulfoxide (DMSO) at a concentration of 10 mM andtored at −20 ◦C until use; required dilutions were made in cul-ure medium RPMI-1640 immediately before use. Sterile 100 mMtock solution of VPA was made in distilled water and was usedresh.

.2. Propidium iodide assay

Cells were plated in 96-well cell culture plates3 × 106 cells/plate) and were treated with P276-00 and/orPA at the indicated concentrations. A modified propidium iodide

PI) assay was used to assess the effects on the cell growthnd plates processed as described earlier [18,19]. IC50 valuesere determined by plotting compound concentration versus

ell viability. The combination index (CI) was calculated by thehou-Talalay equation using the Compusyn software (Com-

oSyn, Inc., NY, USA) [20]. The combination index is used forhe quantification of synergism or antagonism for two drugshere CI < 1, =1, and >1 indicate synergism, additive effect, and

ntagonism respectively.

pictures of six-well plates with colonies were taken by a digital camera on day 15.

2.3. Colony forming assay

Cells were seeded into 6-well plates in triplicates at a densityof 500 cells/well in 2 ml of medium containing 10% FBS. After 24 h,fresh medium with or without drugs were added as described inthe legend of the Fig. 2 and plates processed as described earlier[21].

2.4. Analysis of cell cycle distribution

H460 cells were seeded at a density of 1.0 × 106 mL–1 and after24 h treated with the drugs of interest at the indicated concentra-tions and time period as indicated in the legends of the Fig. 3 andcompared with control. Samples were processed and analyzed asdescribed earlier [14,17].

2.5. Cell lysate preparation and western blotting

H460 cells were seeded at 1.5 × 106 cells/mL and treatedwith P276-00 or VPA or combination of both for various timepoints as mentioned in the legend of the Figs. 4 and 5.Lysates were prepared and western blotting was carried out asdescribed previously [17]. Following antibodies were used: �-actin (Sigma, MO, USA), p21, p27, p53, Bcl-2, cyclin D1, survivin,Bax, anti-rabbit-HRP and anti-mouse-HRP secondary antibodies(Santa Cruz Biotechnology, CA, USA) For detection of acety-lated histones, acidic cell lysates were prepared as describedin the website of the abcam using antibodies against acety-

lated H3 (Cell Signaling Technology, USA) [http://www.abcam.com/ps/pdf/protocols/Histone/extractioprotocol.pdf].

N. Shirsath et al. / Lung Cancer 82 (2013) 214– 221 217

Fig. 3. Effect of P276-00 and VPA on cell cycle distribution, apoptosis and cell recovery: (A) Profound enhancement of apoptosis was observed in H460 cells by flowcytometry. Cells were cultured with VPA (1 mM), P276-00 (1 �M) and combination of both for 24, 48, 72 and 96 h, harvested, and stained with PI. Compiled bar graph showstime dependant increase in apoptotic cells in H460 cells (p < 0.0001). Histogram shows shift of cells from G1 phase (M1) to apoptotic phase (M4). (B) After 96 h of treatmentcells were further incubated in fresh medium without drug for recovery for 0, 24 and 48 h. Cells were processed and analyzed by flow cytometry as described in Materialsand Methods. (C) Western blot analysis of pro- and anti-apoptotic proteins and the densitometric plots of the respective protein bands was done using the software Image J.Reduction of Bcl-2 and survivin protein levels with increased expression of Bax protein in H460 cells treated with 1 mM of VPA, 1 �M of P276-00 or combination of both for96 h was observed. Equal loading of protein was confirmed by �-actin expression.

218 N. Shirsath et al. / Lung Cancer 82 (2013) 214– 221

Fig. 4. Western blot analysis of cell cycle regulatory proteins in VPA and P276-00-treated cells. H460 cells were treated with 1 mM VPA or 1 �M P276-00 or both for 12 h.Combination of both showed significant down-regulation of cyclin D1 levels compared to drug alone. p53, p21 and p27 on the other hand, showed up-regulation after thet are sha

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reatment. Representative data of two independent experiments with similar resultsnalysis of the bands was done using the software Image J.

.6. Tumor xenograft model

To examine the in vivo antitumor efficacy 0.2 mL of H460 cells5 × 106 cells/mL) were injected on the right flank s.c. with matrigeln ratio of 1:1 in 6 weeks old female severe combined immun-deficient (SCID) mice and observed daily for tumor appearance.

hen the tumors attained a diameter of 10 mm, they were ran-

omized into six groups. Control (group I), water was administered.p. as vehicle control, P276-00 (group II) 35 mg/kg in water wasdministered i.p. VPA 100 mg/kg p.o. (group III), VPA acid 200 mg/kg

own. Equal loading of protein was confirmed by �-actin expression. Densitometric

p.o. (group IV), combination of P276-00 35 mg/kg and VPA100 mg/kg (group V) and combination of P276-00 35 mg/kg andVPA 200 mg/kg (group VI) was administered everyday for 15 days.Body weight was recorded everyday. Tumor volume and other signsof toxicity were recorded on every alternate day without sacrificingthe mice and tumor growth inhibition was calculated as described

earlier [19]. Animals were maintained and experiments were car-ried out as per the institutional animal ethical committee in compli-ance with the guidelines of the Committee for the Purpose of Con-trol and Supervision on Experiments on Animals (CPCSEA), India.

N. Shirsath et al. / Lung Canc

Fig. 5. Effect of P276-00 and VPA combination on histone H3To demonstrate that VPA treatment induces the expression of H3 acethylation inlung cancer, total cellular lystates prepared from H460 cells treated with VPA (1 mM)and P276-00 (1 �M) for 24, 48 and 96 h and were subjected to western blot anal-ysis for histone H3 acetylation. Significant increases in acetylated histone H3 wereolu

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bserved compared to the untreated ones. �-actin was included to account for equaloading of the protein sample across the lanes. Quantification of each protein is donesing densitometry Image J software. acetylation.

.7. Statistical analysis

Statistical comparison was made using GraphPad PRISM® (ver-ions 5.0, GraphPad Software, Inc., USA) software where one-waynalysis of variance (ANOVA) and Tukey’s multiple comparisonost-tests were used to determine significant differences betweeneveral treatment groups. Student’s unpaired t-test was employedhen only two groups were compared. Data are presented asean ± SEM of at least three independent experiments with tripli-

ates. Statistical significance was evaluated by calculating p values.ifferences where p < 0.05 were considered statistically significant

*p < 0.05; **p < 0.01; ***p < 0.001).

. Results

.1. Cytotoxic potential of VPA in combination with P276-00 inarious non-small-cell lung carcinoma cell lines

To determine if P276-00 enhances the sensitivity of lung can-

er cells to the growth inhibitory effect of VPA, cytotoxicity assaysere done. The combination studies were conducted using NSCLC

ells with different p53 status viz. H460 p53+, H23 p53−, A549 p53+

nd H1299 p53mut. IC50 was determined for VPA and P276-00 in all

able 1ombination indices for synergy experiments with inhibitory concentration of P276-00 a

Cell lines p53 status P276-00 (IC50 �M) Valproic

A549 Positive 1 4

H23 Negative 1 3.5

H1299 Mutated 0.8 6

H460 Positive 1 3

hese data are representative of 2 independent studies.

er 82 (2013) 214– 221 219

four cell lines using range of concentrations for both the drugs andthe graphs were plotted. The IC50 values for both the compoundsand in all the four cell lines are mentioned in Table 1. Combinationpotential was evaluated using constant ratio i.e. IC50 concentra-tion of both compounds in all four cell lines (Table 1) and usingnon-constant ratio using range of concentrations of VPA with IC30and IC50 of P276-00 in two cell lines viz. H460 and H23 (Fig. 1). Toevaluate the constant ratio potential both the drugs were addedtogether for 48, 72 and 96 h and the combination was found to besynergistic in all the four cell lines with CI < 1. For non-constantratio studies were done by titrating out various concentration ofVPA i.e. 0.1, 0.3, 0.7 and 1.0 mM (upper limit of antiepileptic range)with IC30 (0.6 �M) and IC50 (1 �M) of P276-00 for A549 and H23for 72 and 96 h. As shown in Fig. 1, the combination was synergisticin H460 and H23 cell lines with CI values in the range of 0.6–0.97and 0.6–0.92, respectively.

3.2. Effect of combination of P276-00 and VPA treatment oncolony formation using H460 cell line

Next, we performed in vitro colony forming assay which havebeen used widely for assessing the long-term anticancer drugeffects and for preclinical screening of new anticancer agents [22].Our results indicated that combination of VPA at 1 mM dose (<theIC50) and IC50 of P276-00 reduced colony growth of H460 cells.As seen from Fig. 2, the combined treatment was very effective ascompared to control or drugs alone.

3.3. Combination of P276-00 and VPA induces apoptosisconcomitant with decrease in antiapoptotic proteins

The above results were also confirmed by cell cycle analysisusing flow cytometry wherein induction of apoptosis in H460 wasobserved post combination treatment. As shown in Fig. 3(A), thecombined treatment of both drugs significantly increased cell apo-ptosis compared to drug alone in a time dependent manner. Thecombination of P276-00 and VPA showed highly significant apo-ptosis of 24, 42, 50 and 62% at 24, 48, 72 and 96 h, respectively, ascompared to either drug alone (p < 0.0001). The percent distributionof the cells in various phases of the cell cycle is also depicted in thebar graph as shown in Fig. 3(A). Simultaneously, we checked theexpression of the antiapoptotic proteins Bcl-2 and survivin aftertreatment with P276-00 or VPA or both by western blotting. Asshown in the Fig. 3(C) and the corresponding densitometry plot ofthe western bands, the combination of P276-00 and VPA decreasedthe expression of both antiapoptotic proteins Bcl-2 and survivinsignificantly (p < 0.0001) which may account for the drug synergy.However, expression of Bax showed 75% enhancement in expres-sion compared to untreated and VPA alone after 96 h.

3.4. P276-00 and VPA combination in cell recovery assay

Synchronized non-small-cell lung cancer cell line H460 was firsttreated with 1 �M P276-00 and 1 mM VPA, alone or in combinationfor 96 h and then the cells were allowed to grow in medium withoutthe compound for 0, 24 and 48 h for recovery. After recovery at

nd Valproic acid in NSCLC cell lines at constant ration.

acid (IC50 mM) CI (48 h) CI (72 h) CI (96 h)

0.99 0.52 0.650.75 0.63 0.740.54 0.72 0.610.8 0.72 0.72

2 Cancer 82 (2013) 214– 221

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20 N. Shirsath et al. / Lung

ifferent time points, including 0 h, the cells were harvested and cellycle analysis was done. The combination showed persistent effectn cell recovery post treatment with high apoptotic population of2, 40 and 42% at 0, 24 and 48 h, respectively. In P276-00 alonereated cells the apoptotic population declined gradually viz. 42,0 and 8% at 0, 24 and 48 h post recovery, respectively, while VPAas consistently around 5% at all three time points. This indicates

hat after removal of the compounds the cells in the combinationontinue to undergo apoptosis signifying that P276-00 potentiatesPA anticancer activity, persistently (Fig. 3(B)).

.5. P276-00 regulates the positive and negative cell cycleegulatory proteins

It has been shown previously that increase in p53 levels leadso increase in p21; an increase in cellular levels of p21 and p27ave been shown to correlate with increased inactivation of cyclin-ependent kinases, leading to an inability of the cell to proceed intohe S-phase of the cell cycle [23,24]. This phenomenon of accumu-ation of both leads to concomitant G1 arrest and enhanced sub1 population, which was observed in the combination of P276-0 and VPA. Combination treatment markedly induced p53 levelsnd upregulated the level of p21 by 40% along with increase in27 levels by 25%, compared to untreated control. Conversely, welso analyzed the effect on cyclin D1, which is necessary for cellycle progression, and whether down regulation of this protein by276-00 a known CDK inhibitor could be responsible for the moreronounced VPA induced apoptosis. Our results showed that rela-ive to untreated control, VPA treatment increased cyclin D1 levelsy 30% as compared to control. Furthermore, the addition of P276-0 for 96 h to the cells decreased cyclin D1 protein levels by 70%Fig. 4).

.6. Effect of P276-00 and VPA combination on the acetylation ofistones

VPA like other short-chain fatty acids is reported to inhibit HDACn several cell types. In addition VPA not only inhibits class I HDACs,ut also induces proteosomal degradation of class II HDACs [9,25].o determine whether VPA elicited a similar effect in H460, wessessed the levels of acetylated histone H3 in the absence andresence of VPA in combination with P276-00 at clinically safeose. Weak histone H3 acetylation was evident in untreated cells,hereas the addition of VPA (1 mM) along with P276-00 strikingly

nhanced acetylated histone H3 levels within 24 h (Fig. 5).

.7. P276-00 potentiates anti-tumor effect of VPA in xenograftodel of H460

The tumor growth inhibition was significant in the combinationreatment with tumor growth inhibition of 74% and 82% comparedo control (p < 0.001). While for P276-00, VPA 100 mg/kg and VPA00 mg/kg, the tumor growth inhibition was 46%, 54% and 60%,espectively (Fig. 6). The doses and schedule were well toleratedn all the groups.

. Discussion

It has been suggested that HDAC inhibitors may be able to workn tandem with chemotherapy and radiation to improve tumoresponsiveness [26,27]. This is hypothesized to occur becauseDAC inhibitor effectively relaxes chromatin structure, thereby

aking the DNA more susceptible to damage induced by the

hemotherapy. Epigenetic mechanisms such as DNA methylationnd histone deacetylation play a fundamental role in the prolifera-ion of human cancer cells. This leads to activate tumor suppressive

and P276-00 + VPA (200 mg/kg) on tumors were significant (p < 0.001) with tumorgrowth inhibition of 74% and 82%, respectively, compared to vehicle.

genes, including p21 and p53, while suppressing oncogene expres-sion by increasing histone acetylation [28].

In the present study, the concentrations selected for VPA wereclinically proven, safe and achievable dose i.e. 0.6 mM, thera-peutic serum concentration, and 1 mM, which is upper limit ofantiepileptic range. It is reported that concentration above 1 mMi.e. 2.7 mM is associated with limited toxicity, such as lethargywith benign outcome while 3.6 and 5.1 mM is associated withlife-threatening toxicities, such as coma [29]. Advantageously, asynergistic interaction may allow for lower doses of each compo-nent to be administered to a patient, thereby decreasing the toxicityof chemotherapy, whilst producing and/or maintaining the sametherapeutic effect. Here, we have demonstrated that P276-00 syn-ergistically and significantly potentiates anticancer activity of VPAat clinically achievable concentration, which is much below the IC50value in human NSCLC cell lines. Preferably, P276-00 and the VPAinteract in a synergistic manner, as used herein, the term “synergis-tic” means that VPA produces a greater effect at 1 mM when usedin combination with P276-00 than would be expected from addingit individually.

The endogenous CDK inhibitors p21 and p27 are cyclin-dependent kinase inhibitors that have important roles in blockingthe cell cycle in the G1 phase [16,17]. In this study, we observedthat there is prominent arrest of cancer cells in the G0/G1 phase ofthe cell cycle. This is likely due to the induction of p53 which in turnleads to increase in p21 levels and concomitant increase in p27 fol-lowing treatment of H460 cancer cells with VPA and P276-00, sup-porting their contribution as a possible mechanism by which theseagents inhibit its growth. P276-00 alone and in combination withVPA decreased levels of cyclin D1 which modulate the activity of thedownstream pRb/E2F axis, triggering cell cycle arrest and apoptosis[14,15]. Recently this has been demonstrated by us in head and neckcancer cells [30]. We show that treatment with combination thanalone, dramatically and significantly increased the number of apop-totic cells in H460 cancer cell line. This effect was associated with adecrease in levels of the anti-apoptotic proteins Bcl-2 and survivin.Many anticancer agents increase Bax protein and/or decrease Bcl-2protein during the apoptotic process. Similarly, VPA and P276-00-induced apoptosis in H460 cells was accompanied by an elevationof the Bax to Bcl-2 ratio due to the down regulation of Bcl-2. p53induces apoptosis in response to DNA damage and regulates Baxand Bcl-2 protein expression [31]. Similar response was observed

in the combination group where p53 levels were significantly upregulated and which could have led to modulation of Bax and Bcl-2expression. Thus it was clearly demonstrated that the combina-tion effect on the phenotypic expression of Bax, Bcl-2, and survivin

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N. Shirsath et al. / Lung

ed to a net increase in the ratio of pro- versus anti-apoptoticroteins.

In conclusion, this is the first report that shows a CDK inhibitoruch as P276-00 potentiates VPA activity that is effective at clini-ally achievable dose of 1 mM (<IC50) on NSCLC cells. Thus, HDACnhibitors and cyclin-dependent kinase inhibitors both target cellycle progression at different levels. Taken together, this study indi-ates an exciting potential for the use of CDK inhibitor P276-00 withhe HDAC inhibitor VPA as a future therapy and novel regime forSCLC.

onflict of interest statement

None declared.

cknowledgements

The work has been supported and carried out at Piramal Enter-rises Limited, Goregaon, Mumbai, India. We extend our thanks forhe support.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.lungcan.013.08.010.

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