Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer

Human NSCLC established cell lines (H1299, H460, A549, H1650, Calu-1) were cultured in 10% inactivated foetal bovine serum (HyClone, Termoscientific, South Logan, UT) in RPMI medium (Invitrogen, Carlsbad, CA). H1299 short hairpin (sh) Beclin1, shControl, EGFP-LC3B and ptf-LC3 stable clones were generated as previously described[37] and cultured in the presence of geneticin (800 μg/ml, Sigma-Aldrich, St. Louis, MO).

Patient-derived LCSC18, LCSC36, LCSC136 and LCSC143 cell lines were isolated as described[38] and cultured in serum-free medium containing 50 μ g/ml insulin (Sigma-Aldrich), 100 μ g/ml apo-transferrin (Sigma-Aldrich), 10 μ g/ml putrescine (Sigma-Aldrich), 0.03 μ M sodium selenite (Sigma-Aldrich), 2 μ M progesterone (Sigma-Aldrich), 0.6% glucose (Sigma-Aldrich), 5mM HEPES (Euroclone, Pero, Italy), 0.1% sodium bicarbonate (Euroclone), 0.4% BSA (PAA Healthcare, Milan, Italy), glutamine (Euroclone), and antibiotics (Euroclone), dissolved in DMEM–F12 medium (Invitrogen) and supplemented with 20 n g/ml EGF (Peprotech, Princeton, NJ) and 10 n g/ml bFGF (Peprotech). Flasks non-treated for tissue culture were used to reduce cell adherence and support growth as undifferentiated tumor spheres.

Pooled small interfering RNA (siRNA) oligonucleotides against TS and ATG7 were purchased from Dharmacon RNA Technologies (siGENOME SMART pool, Lafayette, Colorado). For siRNA transfection, cells were seeded and after 24 h transfected with 100 nM pooled oligonucleotides mixture by using Lipofectamine2000 (Invitrogen) following manufacturer’s protocol. 24 h after transfection, media were removed and cells were treated with Pemetrexed (Alimta, formerly LY231514, Eli Lilly and Company) and ITF2357 (Givinostat, Italfarmaco) alone or in combination. Gene silencing efficacy by siRNA was assessed by Western blot or qRT-PCR analyses.

Cells were treated with ITF2357 and pemetrexed, either alone or in combination, as follows: (a) ITF2357; (b) pemetrexed; (c) ITF2357 and pemetrexed, simultaneously; (d) ITF2357 followed by pemetrexed; (e) pemetrexed followed by ITF2357. Both detached and adherent cells were collected, and differentially processed according to analyses performed.

3-Methyladenine (3MA, 1mM) (Enzo Life Science, Plymouth Meeting, PA, USA), and the pan-caspase inhibitor zVAD-fmk (zVAD, 50μM, Sigma-Aldrich) were dissolved in DMSO. Chloroquine diphosphate (CQ, 25μM, Sigma-Aldrich) was dissolved in water.

The inhibitory effect of different drugs was assessed following manufacturer’s protocol on i) NSCLC cell growth by measuring 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide inner salt (MTT, Sigma-Aldrich) dye absorbance of cells, and ii) LCSC cell growth by quantitation of the ATP present in metabolically active cells using CellTiter-Glo® Luminescent (Promega, Southampton, UK). Data were analyzed by the Chou-Talaly method (CalcuSyn software, Biosoft, Cambridge) to determine the combination index (CI), a well-established index of the interaction between two drugs. CI values of <1, =1, and >1 indicate synergistic, additive, and antagonistic effects, respectively.

Flow cytometric analysis (BD Accuri™ C6, BD biosciences) was performed to evaluate cell cycle distribution by propidium iodide (PI) staining, apoptosis by AnnexinV-FITC/PI staining, and to detect acidic vesicular organelles (AVOs) by acridine orange staining, as previously described[39, 40]. Active Caspase-3 Apoptosis Kit (BD biosciences) was used to detect the heterodimer of 17 and 12 kDa subunits, which is derived from the pro-enzyme.

Cells grown on glass coverslips were fixed in 2% formaldehyde for 10 minutes at room temperature. Detection of autophagosomal structures was performed by fluorescence microscopy observing LC3B puncta in EGFP-LC3B expressing cells[41]. Autophagic flux was analyzed by fluorescence microscopy monitoring the distribution and alteration of mRFP-GFP-LC3B fluorescent signals[41]. Typically, at least 200 cells were counted, and cells with more than 10 puncta were considered autophagy positive. Images were scanned under a × 63 oil immersion objective and, to avoid bleed-through effects, each fluorescent signal was scanned independently by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera, elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany) and processed using Adobe PhotoShop software to adjust image brightness and contrast.

qRT-PCR was also performed by using tumor thick sections (10 mm) for RNA extraction. The sections were serial to other 4 mm-thick sections, from the same formalin-fixed paraffin embedded tumor block, used for H&E staining, to select appropriate neoplastic areas and for TS immunohistochemistry. The 10 μm-thick sections were dried overnight at 56°C, de-paraffinated and stained with Nuclear Fast Red solution (Sigma-Aldrich), then rehydrated through graded alcohols and dissected using a scalpel. RNA isolation and retrotranscription were performed as previously reported[42]. An ABI PRISM 7900HT Sequence Detection System (Life Technologies, Applied Biosystems Division, Carlsbad, CA, USA) in 384-wells plate was used. All qPCR mixtures contained 1μl of cDNA template (approximately 40 ng of retrotranscribed total RNA) diluted in 9 μl of distilled-sterile water, 1200 nM of each primer, 200 nM of internal probe and TaqMan Gene Expression Master Mix (Life Technologies) to a final volume of 20 μl. The sequences of primers and probes used for qPCR analyses were previously published[42].

Cycling conditions were 50°C for 2 minutes, 95°C for 10 minutes followed by 46 cycles at 95°C for 15 seconds and 60°C for 1 minute. Baseline and threshold for cycle threshold calculation were set manually with ABI Prism SDS 2.4 Software. A mixture containing Human Total RNA (Stratagene, La Jolla, CA) was used as control calibrator on each plate. β-actin was used as internal reference gene. The fold change in gene expression levels, expressed in unitless values, was evaluated using the 2^–ΔΔCt method[43].

Total protein extracts were fractionated by SDS-PAGE, transferred to a nitrocellulose filter and subjected to immunoblot assay. Immunodetection was performed using antibodies directed to: H3 acetylated histone (Millipore, Billerica, MA), TS (ab58287, Abcam), p62/SQTSM1 (Santa Cruz Biotechnology, Santa Cruz, CA), HSP72/73 (Calbiochem, San Diego, CA), LC3B and acetyl-α-tubulin (K40, Sigma-Aldrich), ATG7 (Millipore), caspases 3 and PARP (Santa Cruz). Anti-mouse or anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Cell Signaling, Amersham Biosciences, Freiburg, Germany) were used as secondary antibodies at 1:10000 dilution. Antibody binding was visualized by enhanced chemiluminescence method (Amersham Biosciences) according to manufacturer’s specification and recorded on autoradiographic film (Amersham Biosciences). Developed films were acquired using GS-700 Imaging Densitometer (Biorad Laboratories, Hercules, CA) and processed with Adobe PhotoShop software. Densitometric evaluation was performed using Molecular Analyst Software (Biorad) and normalized with relative controls depending on the analysis.

To evaluate the effect of different treatments on in vivo tumor growth, 5 × 10⁶ cells were injected intramuscularly into 6-8 week-old female immunodeficient athymic mice (10 for each group). Weekly intraperitoneal treatment with pemetrexed (1000 mg/Kg), and oral administration with ITF2357 (100 mg/Kg) every 24 h for 4 days started when tumors were palpable (about 7 days after cell injection), and stopped when the animals were sacrificed. Animals were observed daily and tumor volume (mm³) calculated as length × width² × π/6. Mice survival was calculated by euthanizing the animals when the tumors reached 2.5 g. The experiments were repeated twice. All procedures involving animals and their care were authorized and certified by the decree n. 67/97A of the Italian Minister of Health and protocol 2560/97 of the Rome Health Service Unit (ASL – RMB).

We first investigated in vitro effects of the HDAC inhibitor ITF2357 in four NSCLC cell lines. ITF2357 dose-dependently inhibited the growth of all cell lines tested, with IC₅₀ values ranging from 3 to 20 μM after 72 h of drug exposure (Figure 1A). Increased acetylation of both histone H3 and α-tubulin was observed by Western blot analysis after 24 h treatment with 0.5 and 1 μM ITF2357 in all cell lines tested (Figure 1B).

Next, we examined the levels of TS mRNA and protein expression following ITF2357 treatment. ITF2357 (0.5 and 1 μM) significantly reduced TS expression at both the mRNA (Figure 1C) and protein (Figure 1B) level in all four NSCLC cell lines.

Since TS expression may affect sensitivity to pemetrexed[13], we assessed whether ITF2357 could modulate pemetrexed cytotoxicity. To this purpose, combination studies were performed using a fixed-ratio experimental design (1:1) in H1299, A549 and Calu-1 cell lines using three different schedules: simultaneous treatment with the two drugs, ITF2357 followed by pemetrexed, and pemetrexed followed by ITF2357. As shown by growth inhibition curves (Additional file1: Figure S1A) and analysis of drug interactions (Additional file1: Figure S1B), simultaneous treatment with equipotent doses of pemetrexed and ITF2357 for 72h resulted in an antagonistic effect (H1299, CI = 1.7; A549, CI = 2.97, Calu-1: CI = 7.26) in all cell lines analyzed. The sequence of ITF2357 followed by pemetrexed had synergistic growth inhibitory effects in H1299 and Calu-1 cells (H1299: CI = 0.54; Calu-1: CI = 0.37), whereas it had an effect comparable to that of ITF2357 alone in A549 cells (Additional file1: Figure S1A,B). Of note, the sequence of pemetrexed followed by ITF2357 was the most effective, with strikingly synergistic drug interactions and CI values < <1 in all cell lines studied: H1299, CI = 0.21; A549, CI = 0.15; Calu-1, CI = 0.02 (Figure 1D,E). A strong growth-inhibitory effect in vitro and a synergistic drug interaction was also observed in three other NSCLC cell lines (H1975, CI = 0.21; H460, CI = 0.12; H1650 CI = 0.59; Additional file2: Figure S2A,B) treated with this schedule.

Next, we explored the putative mechanisms of synergistic growth-inhibitory interactions induced by pemetrexed followed by ITF2357 in the A549 and H1299 representative cell lines. As shown in Figure 2A, less than 10% of cells with sub-G1 DNA content were detected after either drug alone; in contrast, when cells were exposed to pemetrexed followed by increasing concentrations of ITF2357, the percentage of sub-G1 cells dose-dependently increased up to 32% and 23% in A549 and H1299 cells, respectively. Apoptosis induction was confirmed by cytofluorimetric analysis of AnnexinV/PI staining (Figure 2B). Conversely, combined treatment with ITF2357 given simultaneously or before pemetrexed did not increase apoptosis induction in H1299 cells, as compared with treatment with individual drugs (data not shown). Consistently, pro-caspase 3 activation was observed in H1299 cells exposed to pemetrexed followed by ITF2357, but not in cells treated with either drug alone (Additional file3: Figure S3F).

We also analyzed the effect of each agent, alone or in combination, on TS mRNA and protein expression in the H1299 cell line. After exposure to pemetrexed (24 h) followed by wash out of the drug (24 h), TS expression was upregulated at both mRNA (Figure 3A) and protein (Figure 3B) levels; conversely, ITF2357 profoundly downregulated TS mRNA and protein expression (Figure 1B,C and Figure 3A,B) and, in combination treatment, completely prevented pemetrexed-induced TS upregulation, when ITF2357 was used at high doses (Figure 3A,B). To determine if ITF2357-induced TS modulation is causally involved in the observed synergism, we next evaluated whether TS knockdown by a specific siRNA (Additional file3: Figure S3A) affects ITF2357/pemetrexed-mediated apoptosis in H1299 cells. TS silencing sensitized cells to treatment with both agents, alone or in combination, in terms of both viability (Figure 3C) reduction and apoptosis induction (Figure 3D).

To assess the role of apoptosis in the growth inhibitory synergism observed with pemetrexed followed by ITF2357, we performed experiments in the absence or presence of the pan-caspase inhibitor zVAD. First, we analyzed apoptotic and non-apoptotic populations for active caspase-3, using A549 cells (Additional file2: Figure S2C). We found that untreated, as well as pemetrexed-treated cells were primarily negative for the presence of active caspase-3, whereas about 20% of ITF2357 treated cells were positive for active caspase-3 staining. This percentage increased up to about 36% when cells were exposed to combined treatment and, as expected, the pan-caspase inhibitor zVAD completely inhibited caspase-3 activation. Similar results were observed in H1299 cells (data not shown). zVAD also almost completely inhibited apoptosis induced by combined treatment in H1299 cells, as demonstrated by the percentage of AnnexinV positive cells (Figure 3F), but had very limited, albeit statistically significant, effects on loss of viability induced by the combination (Figure 3E). Similar results were obtained with A549 cells (data not shown).

Since both HDACi and pemetrexed can induce autophagic flux[28, 44, 45], we investigated autophagy as a potentially alternative mode of cell death activated by combined pemetrexed followed by ITF2357. As shown in Figure 4A, vital staining of H1299 and A549 cells with acridine orange showed prominent AVOs formation after exposure to pemetrexed followed by ITF2357, while a much lower percentage of AVOs positive cells was observed after treatment with individual drugs. Autophagy induction was further confirmed by analysis of microtubule associated protein I light chain 3 (LC3) redistribution in H1299 cells stably expressing an EGFP-LC3B fusion protein. As shown in Figure 4B,D, diffuse cytoplasmic distribution of green fluorescence was observed in both control and pemetrexed-treated cells, whereas an increase in the characteristic redistribution of EGFP-LC3B into punctate vesicular structures was observed in cells exposed to ITF2357 and, to a much greater extent, in cells exposed to sequential pemetrexed followed by ITF2357.

To obtain clearer results about the effect of drug combination on autophagic flux, we analysed the distribution and alteration of mRFP-GFP-LC3B fluorescent signals using H1299 cells stably expressing mRFP-EGFP-LC3 reporter (H1299/ptf-LC3)[37, 41] (Figure 4C,D). As GFP but not mRFP fluorescence is lost in acidic compartments, mRFP-GFP-LC3B labels nonacidic autophagosomes as yellow fluorescence (positive for both green and red) but acidic autophagolysosomes as red fluorescence only[41]. The increase of red fluorescence observed under ITF2357 and combination treatment indicates that autolysosome maturation proceeds normally. By contrast, as expected a prominent increase in the yellow fluorescence vesicles was observed in CQ-treated cells, indicating incomplete/impaired autophagosome maturation into lysosome (Additional file4: Figure S4).

Consistent with these data, Western blot analysis performed in H1299 cells showed increased processing of the membrane-bound form of microtubule-associated protein LC3BII, decreased p62/SQTSM1 protein levels, and increased Beclin1 expression in ITF2357-treated cells, particularly after sequential exposure to pemetrexed followed by ITF2357 (Figure 4E). Conversely, no changes in ATG7 expression were observed after single and combined drug treatment (Figure 4E).

As shown in Figures 2,3, and4, exposure of NSCLC cell lines to pemetrexed followed by ITF2357 induced both apoptosis and autophagy; moreover pharmacological caspase inhibition by zVAD completely abolished caspase 3 activation (Additional file2: Figure S2) and apoptosis induction (Figure 3F), but only marginally affected cell viability (Figure 3E). Most importantly, zVAD had no effect on AVOs accumulation induced by drug combination (Figure 5A).

Since autophagy has been implicated in both inhibition and promotion of cell survival[46, 47], we investigated whether a functional relationship exists between apoptosis and autophagy, by silencing the expression of proteins acting at the early steps of autophagy, such as Beclin1 or ATG7, as a genetic approach to inhibit autophagy[41].

Selective knockdown of Beclin1 (Additional file3: Figure S3B) substantially inhibited both AVOs formation (Figure 5A) and apoptosis induction, as measured by AnnexinV binding (Figure 5B), particularly in response to combined pemetrexed/ITF2357 treatment. Moreover, Western blot analysis performed in H1299/shBeclin1 cells exposed to different treatment with or without low concentrations of CQ, a late stage autophagic inhibitor, showed only slightly processing of LC3-II protein and p62 degradation in cells exposed to both single or combination treatment when compared to untreated cells, indicating that autophagic flux is regulated upstream by Beclin1. Of note, the addition of low doses of CQ dramatically promotes the LC3-II protein accumulation and recovery of p62 levels (Additional file4: Figure S4B). Inhibition of both AVOs formation (Figure 5A) and apoptosis induction (Figure 5B), in response to combined pemetrexed/ITF2357 treatment was also observed following ATG7 silencing (Additional file3: Figure S3C). Upon combination treatment selective knockdown of Beclin1 resulted in a decreased percentage of cells positive for active caspase-3 staining, when compared to control-transfected H1299 cells (Additional file3: Figure S3F). Moreover, residual cells positive for active caspase-3 staining in H1299/shBeclin1 cells completely disappeared in the presence of pan-caspase inhibitor (data not shown). As a result, knockdown of either Beclin1 or ATG7 prevented the cytotoxic interaction between ITF2357 and pemetrexed, restoring the viability of cells exposed to the combination to levels comparable to those of cells exposed to ITF2357 or pemetrexed alone (Figure 5C). A similar rescue of cell viability after combined treatment was also observed when cells were exposed to 3MA, an early-stage autophagy inhibitor (Additional file3: Figure S3D). As exposure to 3MA represents a condition that at high concentration can paradoxically induce autophagy by inhibiting class I PI3-kinase[48], the effect of 3MA on Akt/mTOR pathway has been investigated in H1299 cells. Western blot analysis demonstrated that phosphorylation of either Akt at Ser473 or mTOR was not affected by exposure to 3MA (Additional file3: Figure S3E).

Taken together, these findings strongly indicate that the combination of pemetrexed followed by ITF2357 activates an autophagic flux, which, in turn, results in apoptosis induction and overall loss of cell viability.

The sensitivity to ITF2357 and pemetrexed alone or in combination and the modulation of markers of apoptosis and autophagy were also evaluated in patient-derived LCSC models in vitro. As reported in Additional file5: Figure S5A, ITF2357 exposure dose-dependently reduced cell viability in four LCSC cell lines tested, although with different IC₅₀ (0.1 and 0.5 μM in the highly sensitive LCSC136 and LCSC36 lines, as opposed to 5 and >5 μM in the relatively more resistant LCSC18 and LCSC143 cell lines, respectively). Increased histone H3 acetylation and PARP activation were observed in the LCSC136 cell line after exposure to ITF2357 (Additional file3: Figure S3B). Consistent with the findings described above in NSCLC cell lines, ITF2357 decreased TS protein expression in the LCSC143 line (Figure 6A) and synergistically inhibited in vitro cell growth, when administered after pemetrexed exposure (Figure 6B,C). Combined treatment induced both apoptosis and autophagy, as evidenced by PARP cleavage, increased LC3B-II levels, and decreased p62/SQSTM1 protein expression (Figure 6A).

Finally, we investigated the in vivo efficacy of ITF2357 and pemetrexed alone or in combination. To this purpose, established H1650 xenografts in nude mice were treated with vehicle, pemetrexed (1000 mg/kg/weekly), ITF2357 (100 mg/kg/daily for 4 days), or sequential pemetrexed/ITF2357 (weekly pemetrexed followed by daily ITF2357 for 4 days, starting 24 h after pemetrexed administration). As reported in Figure 6D, pemetrexed and ITF2357 had minimal effect on mice survival. In contrast, sequential pemetrexed/ITF2357 administration inhibited tumor growth and prolonged mice survival, as compared to single agent administration. Similar to in vitro experiments, qRT-PCR analyses from tumor xenograft specimens evidenced a 2-fold increase and a 2-fold decrease in the median of TS mRNA relative to control after treatment with pemetrexed and ITF2357, respectively, while similar TS expression was observed in control and combined treatment groups. Such differences did not reach statistical significance (data not shown).

Similar results were also obtained in H1299 xenografts, with 35% inhibition of tumor growth with combined treatment, as compared to about 15% with single treatments, at day 35 after tumor cell injection (data not shown).