Reversal of platinum drug resistance by the histone deacetylase inhibitor belinostat

Lung Cancer 103 (2017) 58–65

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Reversal of platinum drug resistance by the histone deacetylase inhibitor belinostat Kenneth Kin-Wah To a,∗ , Wing-Sum Tong a , Li-wu Fu b a b

School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou 510060, China

a r t i c l e

i n f o

Article history: Received 10 September 2016 Received in revised form 18 October 2016 Accepted 27 November 2016 Keywords: Belinostat Platinum anticancer drug Drug resistance Efflux transporters DNA repair

a b s t r a c t Objectives: To investigate and elucidate the mechanism for the potentiation of cisplatin anticancer activity by belinostat in platinum (Pt)-resistant lung cancer cells. Materials and methods: Combination of cisplatin and belinostat was investigated in two pairs of parental and cisplatin-resistant non-small cell lung cancer (NSCLC) cell lines. The Pt-resistant cell models exhibited overexpression of the efflux transporter ABCC2 and enhanced DNA repair capacity. Cellular accumulation of cisplatin and extent of DNA platination were measured by inductively coupled plasma optical emission spectrometer. Expression of Pt transporters and DNA repair gene were determined by quantitative realtime PCR. Inhibition of ABCC2 transport activity was examined by flow cytometric assay. Regulation of ABCC2 at the promoter level was studied by chromatin immunoprecipitation assay. Results and conclusion: In Pt-resistant lung cancer cells, belinostat apparently circumvent the resistance through inhibition of both ABCC2 and DNA repair-mediated mechanisms. The combination of belinostat and cisplatin were found to display synergistic cytotoxic effect in cisplatin-resistant lung cancer cell lines when the two drugs were added concomitantly or when belinostat was given before cisplatin. Upon the concomitant administration of belinostat, cellular accumulation of cisplatin and formation of DNA-Pt adducts were found to be increased whereas expression levels of the efflux transporter ABCC2 and the DNA repair gene ERCC1 were inhibited in Pt-resistant cells. Belinostat-mediated downregulation of ABCC2 was associated with an increase association of a transcriptional repressor (negative cofactor 2) but reduced association of a transcriptional activator (TFIIB) to the ABCC2 promoter. The data advocates the use of belinostat as a novel drug resistance reversal agent for use in combination cancer chemotherapeutic regimens. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide [1]. Non-small cell lung cancer (NSCLC) is a major histologic subtype constituting over 80% of all lung cancers. While surgical resection is curative for early stage lung cancer, most NSCLC patients present with locally advanced or metastatic disease at the time of diagnosis. Although significant progress has been made in the management of NSCLC in recent years

Abbreviations: ABC, ATP-binding cassette; CFDA, carboxy-2’,7’-dichlorofluorescein diacetate; MDR, multidrug resistance; NC2, negative cofactor 2; NSCLC, nonsmall cell lung cancer; Pt, platinum; SRB, sulforhodamine B. ∗ Corresponding author at: School of Pharmacy, Room 801N, Lo Kwee-Seong Integrated Biomedical Sciences Building, The Chinese University of Hong Kong, Area 39, Shatin, New Territories, Hong Kong SAR, China. E-mail address: [email protected] (K.K.-W. To). http://dx.doi.org/10.1016/j.lungcan.2016.11.019 0169-5002/© 2016 Elsevier Ireland Ltd. All rights reserved.

with the use of molecular-targeted drugs, only a subpopulation of patients carrying specific genetic abnormality respond to these new agents [2]. The mainstay treatment for patients with advanced NSCLC is the conventional platinum (Pt)-based doublet regimens [3], usually composed of cisplatin plus gemcitabine/pemetrexed/vinorelbine or carboplatin plus paclitaxel. However, resistance to Pt drugs develops rapidly, which can be caused by decreased influx/increased efflux of drug, glutathione or metallothionein conjugation, activated DNA repair, or skipping lesions during DNA replication [4]. New approaches to reverse drug resistance are urgently needed. Belinostat, a hydroxamate-type inhibitor of class I, II and IV histone deacetylases (HDACs) [5], is approved for the treatment of refractory peripheral T-cell lymphoma [6]. It has also been reported to inhibit the growth of solid cancers including lung and ovarian cancer [7,8]. HDAC inhibitors have been shown to potentiate cyto-

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toxic therapy and radiation but the precise mechanisms have not been fully elucidated [9,10]. The aim of this study was to investigate the potentiation effect of belinostat on the anticancer activity of cisplatin in Pt-resistant NSCLC. 2. Materials and methods 2.1. Chemicals and reagents Belinostat was purchased from Selleckchem (Houston, TX, USA). Benzbromarone and carboxy-2 ,7 -dichlorofluorescein diacetate (CFDA) were obtained from Sigma Chemical (St Louis, MO, USA). Cisplatin was purchased from Acros Organics (Thermo Fisher Scientific, New Jersey, USA). 2.2. Cell culture Human NSCLC cell lines H460 and A549 are generous gift from Dr. Susan Bates (National Cancer Institute, USA). Cisplatin resistance was induced in H460 and A549 cells by prolonged incubation in increasing concentration of cisplatin to generate cisplatinresistant sublines, H460 cisR and A549 cisR, respectively. Human NSCLC H1299 cells were purchased from American Type Culture Collection (Manassas, VA, USA). The human embryonic kidney cell line HEK293 and its stable pcDNA3-, or ABCC2-transfected sublines were used to demonstrate the effect of belinostat on ABCC2. A549 and A549 cisR were maintained in DMEM medium whereas H460 and H460 cisR were grown in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 units/mL streptomycin sulfate, and 100 units/mL penicillin G sulfate, and incubated at 37 ◦ C in 5% CO2 . The transfected HEK293 cells were cultured in DMEM medium supplemented with 2 mg/mL G418. 2.3. Reverse transcription and quantitative real-time PCR Quantitative real-time PCR was performed as described previously [11] to evaluate the expression of ABCC2, ATP7A, ATP7B, CTR1 and ERCC1 in cells treated with belinostat. The specific primer sequences are shown in Supplementary Table 1. 2.4. Growth inhibition assay and analysis of drug combination The growth inhibitory effect of individual anticancer drugs was evaluated by the sulforhodamine B (SRB) assay as described previously [12]. The median-drug effect analysis method was used to evaluate the nature of the drug combination [13]. Cells grown in 96-well plates were treated with either cisplatin or belinostat alone or their combination in a fixed ratio of each drug in increasing concentrations. Combination index (CI) was then calculated to assess the outcome of the drug combination at different fraction of cells affected (Fa) as described previously [13].

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2.6. Analysis of ABCC2 inhibition kinetics The inhibition kinetic of ABCC2-mediated efflux of CFDA by belinostat was evaluated as described previously in ABCC2-stably transfected HEK293 cells [11]. The quantity of ABCC2-mediated CFDA efflux was measured by flow cytometric assay as above and calculated by subtracting values obtained at 37 ◦ C from that at 0 ◦ C. The inhibitory effect of belinostat on ABCC2 was analyzed by the Dixon plot. 2.7. Chromatin immunoprecipitation (ChIP) assay ChIP assay was performed as described previously [14]. ChIP was carried out overnight at 4 ◦ C with monoclonal antibody against TFIIB, NC2, or normal IgG. The amount of immunoprecipitated DNA was assessed by quantitative real-time PCR, using primers spanning the ABCC2 promoter (Supplementary Table 1), and compared with the amount of input DNA before immunoprecipitation. Fold enrichment in each immunoprecipitation was determined by comparing the Ct value for the immunoprecipitated DNA versus the input DNA from real-time PCR. Only 10% of the total input was used in PCR reactions. 2.8. Cellular Pt accumulation and DNA platination Cellular Pt accumulation and DNA platination were measured as described previously [15]. Pt content in the samples was analyzed by inductively coupled plasma optical emission spectrometer (ICPOES) (Optima 4300DV, PerkinElmer, MA, USA). 2.9. Transporter ATPase assay The effect of belinostat on the vanadate-sensitive ATPase activity of ABCC2 was measured by using the BD Gentest ATPase assay kit (BD Biosciences) according to the manufacturer’s instructions. 2.10. Apoptosis assay After treatment with 4 ␮M cisplatin in the presence or absence of 0.5 ␮M belinostat for 48 h, parental H460 or cisplatin-resistant H460 cisR cells were collected. The proportion of apoptotic cells was determined by using the APC Annexin V Apoptosis Kit (BD Bioscience) according to the manufacturer’s instruction. 2.11. Statistical analysis All experiments were repeated at least three times. The statistical software SPSS16.0 (IBM, Armonk, NY, USA) was used for data analysis. Statistical significance was determined at p < 0.05 by the Student’s t-test. 3. Results

2.5. Flow cytometric analysis of ABCC2 transporter activity

3.1. Combination of cisplatin and belinostat was synergistic in NSCLC cells

A flow cytometric assay was employed to study the inhibition of ABCC2 transport activity by belinostat in ABCC2-stably transfected HEK293 cells as described previously [11] by measuring the cellular retention of a fluorescent ABCC2 probe substrate (0.2 ␮M carboxy2 ,7 -dichlorofluorescein diacetate (CFDA)). Benzbromarone was used as a specific ABCC2 inhibitor for comparison. CFDA fluorescence was detected with a 488-nm argon laser and a 530-nm bandpass filter on LSRFortessa Cell Analyzer (BD Biosciences, San Jose, CA, USA).

The growth inhibitory effect of cisplatin and belinostat on two pairs of parental (H460 and A549) and cisplatin-resistant NSCLC (H460 cisR and A549 cisR) cell lines were examined by SRB assay. The IC50 of the two drugs in the cell lines are summarized in Supplementary Table 2. Both H460 cisR and A549 cisR were remarkably less responsive to cisplatin (relative resistance = 36-fold and 35-fold, respectively) than the parental cells. On the other hand, belinostat exhibited similar growth inhibitory effect in the parental and cisplatin-resistant cells.

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Fig. 1. Combination of cisplatin and belinostat gave rise to synergistic anticancer effect. (A) Combination index (CI) plot showing anticancer effect of combination of cisplatin and belinostat in two pairs of parental (H460 and A549) and cisplatin-resistant (H460 cisR and A549 cisR) NSCLC cell lines. CI values were plotted as a function of fractional growth inhibition (Fa). CI < 1, =1, and >1 suggests synergism, additivity and antagonism, respectively. (B) Belinostat sensitized H460 and H460 cisR cells to apoptosis. Cells were exposed to cisplatin alone (4 ␮M), belinostat alone (1 ␮M), or their combination for 48 h before harvest for apoptosis assay. A representative set of data from three independent experiments is shown. (C) Summary of apoptosis assay data in (B) from three independent experiments. Data are presented in histogram as means ± SD.

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The cells were also treated with concomitant combination of cisplatin and belinostat at fixed molar ratio for 72 h. The ratios of cisplatin and belinostat were set according to the IC50 of the drugs in each cell line. The effects of the drug combinations are shown in the combination index (CI)-fraction affected (Fa) plot (Fig. 1A). For all cell lines tested, a CI index of < 1 was observed over a wide range of growth inhibition levels. In both cisplatin-resistant cell lines, the CI at 50% growth inhibition level was around 0.25, indicating strong synergistic effect. To determine whether the sequence of drug addition affects the resulting synergy, cisplatin-resistant H460 cisR cells were treated for 4 h (or 24 h) with belinostat, followed by drug-free washout, and treatment for 4 h with cisplatin, or vice versa (Supplementary Fig. 1A). The elimination half-life of belinostat was reported to be 0.3–1.3 h but increased histone hyperacetylation was observed for 4–24 h after each infusion [16]. Therefore, 4-h or 24-h treatment of belinostat was tested. On the other hand, cisplatin is usually given as infusion over a few hours. Thus, 4-h treatment of cisplatin was tested in the combination study. The results showed that 4-h (or 24-h) exposure to belinostat followed by a 4-h exposure of cisplatin and the concomitant treatment led to a strong synergistic anticancer effect in H460 cisR (Supplementary Fig. 1B), with a CI of around 0.25 at 50% growth inhibition level. On the contrary, the reverse sequence (cisplatin followed by belinostat) only resulted in an additive effect (Supplementary Fig. 1B). 3.2. Belinostat increased apoptosis in cisplatin-treated NSCLC cells H460 and H460 cisR were treated with a combination of cisplatin (4 ␮M) and belinostat (0.5 ␮M) for 48 h, after which the extent of apoptosis was measured. While belinostat alone only caused very mild apoptosis at 0.5 ␮M (∼10%), its combination with cisplatin was found to dramatically increase the proportion of apoptotic cells especially in cisplatin-resistant H460 cisR (47.9 ± 5.2% for drug combination versus 2.3 ± 0.4% for cisplatin alone; p < 0.01) (Fig. 1B & C). 3.3. Belinostat inhibited ABCC2 transport activity ABCC2 is a major efflux transporter for Pt drugs and its overexpression is known to mediate Pt resistance [17]. Since belinostat was found to enhance the anticancer activity of cisplatin, we hypothesized that belinostat may inhibit ABCC2 transport activity to enhance the cellular cisplatin accumulation and increase drug response. Belinostat was first evaluated for its inhibition of ABCC2 transport activity. Using specific fluorescent ABCC2 probe substrate (CFDA), the inhibition of ABCC2 efflux was evaluated in ABCC2-stably transfected HEK293 cells (Fig. 2). Inhibition of the transporter-mediated efflux is indicated by a shift to higher intracellular fluorescence retention. As illustrated in Fig. 2, belinostat was found to inhibit ABCC2 transport activity in a concentrationdependent manner. No effect on the intracellular fluorescence signal was observed in the backbone vector (pcDNA3)-transfected cells. 3.4. Inhibition kinetic of ABCC2-mediated drug efflux by belinostat To elucidate the mode of interaction between belinostat and ABCC2, the efflux of the CFDA (1, 2, or 5 ␮M) was studied in the presence of different concentration of belinostat. As demonstrated in the Dixon plot (Fig. 3A), all curves intersect above the x-axis, suggesting competitive inhibition. The Ki values was estimated to

Fig. 2. Inhibition of ABCC2-mediated efflux of fluorescent probe substrate (CFDA) by belinostat. (Top panel) Representative histogram from three independent experiments is shown. HEK293 cells stably transfected with ABCC2 or the backbone vector pcDNA were incubated with ABCC2 fluorescent probe substrate CFDA (0.2 ␮M) alone (black), the fluorescent probe in the presence of belinostat at the indicated concentrations (various colors), or the fluorescent probe in the presence of ABCC2 specific inhibitor 50 ␮M benzbromarone (BB) (red) at 37 ◦ C for 30 min. Retention of the fluorescent probe substrate in the cells after a 1-h substrate-free efflux was measured by flow cytometry. (Bottom panel) The relative fluorescent probe retention was quantified by setting the value in ABCC2-overexpressing cells in the absence of belinostat as 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

be 1.85, indicating a fairly strong interaction between belinostat and ABCC2. 3.5. Modulation of ABCC2 ATPase activity by belinostat ABC transporters use energy generated from ATP hydrolysis to actively pump their substrate drugs out of the cells. The extent of ATP consumption is modulated in the presence of their substrates or inhibitors. To understand further the mechanism of ABCC2 inhibition by belinostat, ABCC2-mediated ATP hydrolysis in the presence of different concentration of belinostat was measured (Fig. 3B). ABCC2 ATPase activity was stimulated by belinostat until it reached a plateau about 32 nmol Pi/min/mg protein and it remained steady at belinostat concentration >10 ␮M. 3.6. Belinostat increased cellular cisplatin accumulation by reducing ABCC2 expression in cisplatin-resistant cells Consistent with commonly-observed Pt resistance mechanism, cisplatin accumulation in our cisplatin-resistant H460 cisR was remarkably decreased relative to that in the parental H460 (Fig. 4,

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Fig. 3. Inhibition kinetic of ABCC2 transport activity and ATPase activity by belinostat. (A) Dixon plot analysis of the inhibition kinetic of ABCC2-mediated fluorescent probe efflux by belinostat. HEK293/ABCC2 cells were incubated with different concentration of CFDA (1, 2, and 5 ␮M) in the presence of four concentrations of belinostat (0.25, 0.5, 2, or 4 ␮M) for 1 h. After a brief wash, the cells were allowed to incubate in probe-free medium continuing with or without belinostat incubation to allow for efflux. The quantity of the fluorescent probe efflux was measured for 10 min by flow cytometry, which was calculated by subtracting the fluorescence signal obtained at 37 ◦ C from that at 0 ◦ C. The reciprocal of the fluorescent probe efflux rate is plotted against belinostat (i.e. the inhibitor) concentration using the Dixon plot. Since the lines converge above the x-axis, belinostat is likely competitive inhibitor of ABCC2 for the transport of CFDA. Each data point is presented as the mean ± standard deviation from three independent experiments. (B) Effect of belinostat on the ATPase activity of ABCC2. The vanadate-sensitive ATPase activity of ABCC2 in the recombinant ABCC2 protein obtained from cell membrane fraction was determined at different concentrations of belinostat. ATP hydrolysis was monitored by measuring the amount of inorganic phosphate released using a colorimetric assay.

top). Belinostat was found to increase the cellular cisplatin accumulation, more significantly in cisplatin-resistant cells. At 2 ␮M, belinostat was found to recover cisplatin accumulation in cisplatinresistant H460 cisR to a similar level attained in the parental H460 (Fig. 4, top). The reversal of cisplatin resistance by belinostat may also be associated with change in ABCC2 expression in the treated cells. Therefore, real-time PCR was performed in H460 and H460 cisR treated with belinostat (0, 0.5, 1 or 2 ␮M) for 16 h. Belinostat was found to significantly reduce ABCC2 expression in cisplatinresistant H460 cisR (Fig. 4, bottom). The reduction of ABCC2 expression and increase in Pt accumulation by belinostat was also similarly observed in another pair of parental A549 and cisplatinresistant A549 cisR cells (Supplementary Fig. 2).

Fig. 4. Belinostat increased cellular accumulation of cisplatin in cisplatin-resistant H460 cisR cells by downregulating ABCC2 expression. (Top panel) Cisplatin accumulation in belinostat-treated H460 and H460 cisR cells. Relative to the parental H460 cells, there was a remarkable reduction in cisplatin accumulation in H460 cisR cells. (Bottom panel) Real-time PCR analysis of ABCC2 mRNA expression in belinostattreated H460 and H460 cisR cells. The value in H460 without belinostat treatment was set as 1 for comparison. The mean value from three independent experiments is shown. *p < 0.05, **p < 0.01, compared with untreated H460 cisR cells.

3.7. Downregulation of ABCC2 by belinostat was associated with increased binding of NC2 but reduced association of TFIIB with the ABCC2 promoter Belinostat was found to induce histone acetylation in both H460 and H460 cisR cells as expected (Fig. 5A). Upon close examination of the ABCC2 promoter (GenBank accession no. AJ005200), we identified a TATA-box binding protein (TBP) binding site upstream of the transcription start site (Fig. 5B). ChIP assay was performed to investigate whether increased binding of a repressor negative cofactor 2 (NC2) to the ABCC2 promoter is involved in ABCC2 downregulation by belinostat. DNA purified after immunoprecipitation with anti-NC2 antibody was evaluated by PCR using primers targeting the ABCC2 promoter (Fig. 5B). The relative amount of promoter-associated NC2 was determined by quantifying the PCR products thus obtained followed by normalization with the input. No signal was obtained from immunoprecipitated samples when normal IgG was used (Fig. 5C). Compared with untreated H460 cisR, an increased association of NC2 with the ABCC2 promoter was observed in belinostat-treated H460 cisR, consistent with the downregulation of ABCC2 in the latter. Of note, an increase association of the transcription factor TFIIB was observed in H460 cisR relative to the parental H460 (Fig. 5C), apparently contributing to the overexpres-

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Fig. 5. Belinostat induced global histone acetylation but reduced the association of TFIIA on ABCC2 promoter in H460 cisR cells. (A) Western blot analysis showing the increase in histone H3 acetylation by belinostat in H460 and H460 cisR cells in a concentration dependent manner. (B) A schematic representation of the ABCC2 promoter studied by ChIP analysis. A pair of primers were used to amplify the ABCC2 promoter flanking a putative TBP binding site. Nucleotide positions are numbered relative to the translation start site (+1) defined in the published DNA sequence (GenBank accession no. AJ005200). Big arrowhead: transcription start site. (C) ChIP assay showing the increased recruitment of NC2 but reduced association of TFIIB with the ABCC2 promoter in belinostat-treated H460 cisR cells. Input, DNA isolated from the lysate before immunoprecipitation (only 10% of the total chromatin was used for PCR reactions); IgG, ChIP using normal IgG for immunoprecipitation. Average data from three independent immunoprecipitations is shown.

sion of ABCC2 in the resistant cells. Coincidence with the increased association of the repressor NC2 to the ABCC2 promoter by belinostat, the binding of TFIIB to the promoter was decreased by belinostat treatment. 3.8. Belinostat increased DNA platination after cisplatin treatment by downregulating the DNA repair gene ERCC1 Cisplatin was found to attain remarkably lower DNA platination in resistant H460 cisR than in parental H460 (Fig. 6A, top), suggesting that DNA repair also played an important role in the observed cisplatin resistance. Importantly, belinostat was found to increase DNA platination after cisplatin treatment in both H460 and H460 cisR, albeit more significantly in the resistant H460 cisR (Fig. 6A, top). Consistent with the reduced DNA platination in H460 cisR, the expression of ERCC1 was remarkably elevated in H460 cisR than in H460. Importantly, belinostat was found to downregulate ERCC1 expression in H460 cisR in a concentration-dependent manner, parallel to the increase in DNA platination in belinostat-

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Fig. 6. Belinostat increased DNA platination in cisplatin-treated H460 cisR cells by downregulating ERCC1 expression. (A) Top panel: DNA platination in H460 and H460 cisR cells treated with combination of cisplatin and belinostat. Relative to the parental H460 cells, there was a remarkable reduction in DNA platination in cisplatin-incubated H460 cisR cells. Lower panel: Real-time PCR analysis of ERCC1expression in belinostat-treated H460 and H460 cisR cells. The value in H460 without belinostat treatment was set as 1 for comparison. The mean value from three independent experiments is shown. *p < 0.05, **p < 0.01, compared with untreated H460 cisR cells. (B) DNA repair capacity in H460 and H460 cisR cells with or without belinostat. Retention of DNA platination after recovery in drug-free medium was measured.

treated cells (Fig. 6A, bottom). Similar findings were also observed in another pair of parental A549 and cisplatin-resistant A549 cisR cells (Supplementary Fig. 3). The DNA repair capacity was also examined by measuring the amount of DNA platination retained in cisplatin-treated cells after recovery in drug-free medium. After cisplatin alone treatment, DNA platination was significantly lower in H460 cisR than in H460. Moreover, the decrease in DNA platination level upon recovery in drug-free medium was more dramatic in H460 cisR than in H460 (Fig. 6B, left). However, the extent of DNA platination upon combination of cisplatin and belinostat was much less reduced in H460 cisR, compared with cisplatin incubation alone (Fig. 6B, right). Moreover, rate of decrease in DNA platination upon recovery in drug-free medium was very mild and similar in both H460 and H460 cisR (Fig. 6B, right). 4. Discussion Although a number of novel molecular-targeted drugs have been developed for NSCLC treatment in recent years, Pt-based reg-

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imens are still preferred as the first-line therapy for advanced NSCLC. However, the development of resistance is severely hindering the clinical usefulness of Pt drugs. Different drug combinations have been studied with an aim to reverse Pt resistance. The potentiation of cisplatin anticancer effect by HDAC inhibitors have been reported previously. A few mechanisms have been postulated to contribute to the enhanced anticancer activity of the drug combination [18–20]. To further optimize the combination use of Pt drugs and HDAC inhibitors, a better understanding of the underlying mechanisms is needed. In this study, synergistic combination of cisplatin and a recentlyapproved HDAC inhibitor belinostat was observed in Pt-resistant NSCLC cells. Belinostat was found to potentiate cisplatin-induced apoptosis preferentially in Pt-resistant cells. The cisplatin-resistant cells exhibited overexpression of an efflux transporter ABCC2, upregulation of the DNA repair gene ERCC1 and elevated DNA repair capacity. Interestingly, a novel Pt-belinostat conjugate has been designed and synthesized [21]. This conjugate was found to overcome Pt resistance in ovarian cancer but the precise mechanism was not elucidated. In our Pt-resistant NSCLC cell models, overexpression of the efflux transporter ABCC2 was shown to decrease cellular cisplatin accumulation. Therefore, we speculated that belinostat may inhibit ABCC2 transport activity. The concentration-dependent inhibitory effect of belinostat on ABCC2 efflux activity was demonstrated in an ABCC2 stably-transfected HEK293 cells. The assay was specific because no effect was observed in the backbone vector-transfected cells. Result from the transport kinetic study revealed that belinostat is likely a competitive inhibitor of ABCC2. The small Ki value of belinostat for its inhibition on ABCC2 indicated that it has high affinity to the transporter. ATPase assay is a widely used biochemical assay for the study of MDR transporter-drug interactions [22]. ABC transporters use the energy generated from ATP hydrolysis by ATPase to effectively transport their substrate drugs. The stimulation of ABCC2 ATPase activity by belinostat and the result from the transporter inhibition kinetic study suggest that belinostat is likely a competitive inhibitor of ABCC2. To the best of our knowledge, this study is the first report demonstrating the inhibition of ABCC2 transport activity and the subsequent circumvention of transporter-mediated drug resistance by belinostat. Belinostat was also found to downregulate ABCC2 expression in Pt-resistant cells. HDAC inhibitors have been shown to alter the expression of an estimated 4–12% of genes, presumably due to the relaxation of DNA in the gene promoter by histone acetylation and access to transcriptional regulators [23]. Interestingly, both upregulation and repression of genes have been observed following HDAC inhibitor treatment [24,25]. ABCC2 is an important efflux transporter regulating the cellular accumulation of anticancer drugs including cisplatin [26,27]. Importantly, HDAC inhibitors have been found to downregulate ABCC2 in multidrug resistant cancer cells [28], suggesting their use for chemosensitization. However, the molecular mechanism of ABCC2 downregulation by HDAC inhibitor remains elusive. In HepG2 cells, trichostatin A was found to repress ABCC2 transcription through an upstream region (−517 to −197) [29]. It was speculated that chromatin modification may permit binding of a repressor complex to this promoter region. In this study, belinostat-mediated downregulation of ABCC2 was associated with an increase association of a transcriptional repressor (NC2) to ABCC2 promoter. NC2 is an evolutionarily conserved transcriptional regulator that was originally identified as an inhibitor of basal transcription. NC2 binds to the TATA-binding protein, blocking the recruitment of TFIIA and TFIIB, and thereby inhibiting the preinitiation complex assembly and leading to gene repression [30–32].

The inhibition of DNA repair by belinostat was also studied. Belinostat was found to downregulate ERCC1 expression and inhibit DNA repair preferentially in ERCC1-overexpressing H460 cisR. However, this is in contrast to the study of Luchenko et al., in which Pt adduct formation was not enhanced in cells treated with cisplatin and HDAC inhibitors [19]. The discrepancy may be due to the fact that Pt-resistant NSCLC cells with upregulation of ERCC1 and elevated DNA repair capacity were used in our study whereas sensitive NSCLC cells were used in Luckenko’s study. Thus, our data suggest that belinostat may specifically target Pt-resistant cells with overexpression of ERCC1 and enhanced DNA repair capacity. To this end, the concomitant combination of cisplatin and belinostat was also found to be synergistic in another NSCLC cell line H1299 (Supplementary Fig. 4A). H1299 was chosen because it has been reported to overexpress the DNA repair gene ERCC1 [33]. The drug combination was found to increase cellular Pt accumulation (by downregulating ABCC2) and enhance DNA platination (by reducing ERCC1 expression) (Supplementary Fig. 4B). Of note, it has been recently reported that another new HDAC inhibitor (panobinostat) approved for multiple myeloma could increase cisplatin sensitivity only in NSCLC cell lines with low ERCC1 expression but not those with high ERCC1 expression [33]. Belinostat may thus be a better HDAC inhibitor for combination with Pt drugs because upregulation of ERCC1 is frequently observed in Pt-resistant cells. In conclusion, the combination of belinostat and cisplatin may be adopted as a novel means to circumvent Pt resistance. Further mechanistic investigation and animal studies are warranted to fully understand and optimize the beneficial drug combinations. Conflict of interest statement None declared. Acknowledgements This work was supported by the CUHK Direct Grant for Research (4054207) to Kenneth To. The provision of graduate studentship to Wing-Sum Tong by the Chinese University of Hong Kong is also greatly appreciated. We would like to thank Dr. Susan Bates (National Cancer Institute, NIH) for the NSCLC cell lines (H460 & A549) used in the study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan.2016.11. 019. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66 (2016) 7–30. [2] D.S. Tan, S.S. Yom, M.S. Tsao, H.I. Pass, K. Kelly, N. Peled, et al., The international association for the study of lung cancer consensus statement on optimizing management of EGFR mutation-positive non-small cell lung cancer: status in 2016, J. Thorac. Oncol. 11 (2016) 946–963. [3] D.A. Fennell, Y. Summers, J. Cadranel, T. Benepal, D.C. Christoph, R. Lal, M. Das, F. Maxwell, C. Visseren-Grul, D. Ferry, Cisplatin in the modern era: the backbone of first-line chemotherapy for non-small cell lung cancer, Cancer Treat. Rev. 44 (2016) 42–50. [4] C.A. Rabik, M.E. Dolan, Molecular mechanisms of resistance and toxicity associated with platinating agents, Cancer Treat. Rev. 33 (2007) 9–23. [5] P. Gimsing, Belinostat: a new broad acting antineoplastic histone deacetylase inhibitor, Expert Opin. Investig. Drugs 18 (2009) 501–508. [6] R.M. Poole, Belinostat: first global approval, Drugs 74 (2014) 1543–1554. [7] A. Grassadonia, P. Cioffi, F. Simiele, L. Iezzi, M. Zilli, C. Natoli, Role of hydroxamate-based histone deacetylase inhibitors (Hb-HDACIs) in the treatment of solid malignancies, Cancers (Basel) 5 (2013) 919–942. [8] S. Marsoni, G. Damia, G. Camboni, A work in progress: the clinical development of histone deacetylase inhibitors, Epigenetics 3 (2008) 164–171.

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