Class IIb HDAC Inhibition Enhances the Inhibitory Effect of Am80, a Synthetic Retinoid, in Prostate Cancer

Abstract

Combination therapy is often an effective strategy to treat cancer. In this study, we examined the growth-inhibitory effects of Am80 (tamibarotene), a specific retinoic acid receptor (RAR) α/β agonist, in combination with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), or a DNA methyl transferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine, on androgen receptor (AR)-positive and AR-negative prostate cancer cell lines (LNCaP and PC-3, respectively). We found that the combination therapy of SAHA and Am80 showed an enhanced growth-inhibitory effect on LNCaP cells. Further studies with various HDAC isotype-selective inhibitors showed that SAHA and KD5170 (a selective class I and II HDAC inhibitor) each increased the RARα protein level in LNCaP cells. Our results indicate that the target of the enhancing effect belongs to the Class IIb HDACs, especially HDAC6. Dual targeting of Class IIb HDAC and RARα may be a candidate therapeutic strategy for prostate cancer.

INTRODUCTION

More than 10000 clinical trials of combination therapies are currently registered worldwide for the treatment of cancer, infectious diseases, metabolic diseases, cardiovascular diseases, autoimmune diseases, and neurological diseases. To determine the efficacy of combination therapy, it is important not only to evaluate the therapeutic effects when the drugs are used individually as single agents and in combination, but also to compare them with previously reported combination therapies. One potential target for combination therapy is prostate cancer. The current strategies employed for prostate cancer treatment are surgery to remove the tumor, radiation therapy to kill cancer cells or control their proliferation, and hormone therapy to inhibit progression. However, these treatment methods have significant side effects, such as impaired sexual function, weakened bones, diarrhea, nausea, and itching.^1–3) Therefore, more effective treatments are needed.

Retinoids have been reported to induce apoptosis in androgen-dependent and independent prostate cancer cells,^4–7) and the combination of retinoids with an organic arsenical or a histone deacetylase (HDAC) inhibitor induced apoptosis in several human and rodent prostate carcinoma cell lines in vitro and in vivo.^8,9) The reported molecular mechanisms of induction of apoptosis by retinoids in prostate cancer cells include down-regulation of Bcl-2 expression,^6,7,10,11) induction of insulin-like growth factor-binding protein-3 (IGFBP-3),¹²⁾ and accumulation of tissue transglutaminase in cells.⁶⁾

Epigenetic modifications of chromatin play an essential role in normal development, growth, and differentiation of cells by transcriptionally silencing specific control regions. Two major epigenetic modifications, histone deacetylation and DNA methylation, contribute to the progression and drug resistance of many cancers.¹³⁾ Recent studies have shown that HDAC and DNA methyl transferase (DNMT) inhibitors can restore the expression of numerous silenced nuclear receptors (NRs), including estrogen receptor α and retinoic acid receptor (RAR) β.^14,15) Therefore, combinations of NR-binding drugs with HDAC and/or DNMT inhibitors may be promising strategy for cancer treatment. Indeed, this has been tried for some cancers,^16,17) but little work has been done with prostate cancer.¹⁸⁾ Besides, these studies lead to better understanding of the biological action mechanisms of drugs and help in the discovery of new drug targets.

In this study, therefore, we examined the growth-inhibitory effects of a synthetic retinoid, Am80 (tamibarotene), in combination with HDAC and DNMT inhibitors on androgen receptor (AR)-positive prostate cancer (LNCaP) and AR-negative prostate cancer (PC-3) cells. Am80 is a specific RARα/β agonist that exhibits anticancer activity against acute promyelocytic leukemia (APL) cells and was approved for the treatment of intractable and relapsed APL in Japan in 2005.¹⁹⁾ It also exhibits antiproliferative effects against various tumor cell lines.²⁰⁾ As a DNMT inhibitor, we chose 5-aza-2′-deoxycytidine (5-AzadCyD), which is a pyrimidine nucleoside analog of cytidine that is incorporated into DNA, where it blocks the action of DNMT; it was approved by the United States Food and Drug Administration (FDA) in 2006 for the treatment of myelodysplastic syndromes.²¹⁾ As an HDAC inhibitor, we chose suberoylanilide hydroxamic acid (SAHA), which is a pan-HDAC inhibitor that was approved by the FDA, also in 2006, for the treatment of cutaneous T cell lymphoma (CTCL).²²⁾ As target cell lines, we used AR-positive prostate cancer cell line LNCaP and AR-negative prostate cancer cell line PC-3, which are considered representative of the early androgen-dependent stage and the advanced androgen-independent stage of the disease, respectively.

We found that the combination therapy of Am80 with SAHA showed an enhanced growth-inhibitory effect on LNCaP cells, and we examined the mechanism involved. Our results suggest that dual targeting of Class IIb HDAC and RARα might be a candidate therapeutic strategy for prostate cancer.

MATERIALS AND METHODS

Chemicals

5-AzadCyD and SAHA were purchased from Tokyo Chemical Industry (Japan). Selective HDAC inhibitors were purchased from the indicated sources: MGCD0103 (Adooq Bioscience, CA, U.S.A.), R306465 (Adooq Bioscience), KD5170 (Santa Cruz Biotechnology, CA, U.S.A.). These drugs were dissolved in dimethyl sulfoxide (DMSO); in all cases, the final concentration of DMSO was 0.1% or less.

Cell Lines and Culture Conditions

LNCaP and PC-3 cell lines were purchased from RIKEN BRC (Japan) and cultured in RPMI 1640 medium (Gibco, Life Technologies, CA, U.S.A.) supplemented with 10% fetal bovine serum (Sigma-Aldrich, MO, U.S.A.) at 37°C in an atmosphere of 5% CO₂ in humidified air. Cells were harvested using trypsin–ethylenediaminetetraacetic acid (EDTA) at 70–80% confluency and collected by centrifugation (300 × g for 5 min at room temperature (r.t.)). The cell pellets were resuspended in fresh medium and subcultured in T75 flasks or 96-well plates, as required.

Measurement of Growth-Inhibitory Potency

The concentrations of single agents resulting in IC₅₀ were determined by dose–effect analysis. The cells were plated at a density of 0.5 × 10⁴ cells per well in 96-well plates and incubated for 24 h. Then, the cells were further incubated with drugs in the concentration range of 0.1–100 µM (final concentration) for 72 h. Cell proliferation was evaluated using the WST-8 Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). The absorbance at 450 nm was measured using a microplate reader.

Apoptosis Assay

For staining of apoptotic cells, an Annexin V-PI Assay Kit (Nacalai Tesque, Japan) was used according to the manufacturer’s protocol. After staining, the samples were analyzed by using NovoCyte (ACEA Biosciences, CA, U.S.A.), and the data were analyzed using NovoExpress (ACEA Biosciences).

Immunoblot Analysis

Following single or combination drug treatment, the cells were washed twice with phosphate-buffered saline, dissociated using trypsin–EDTA, and pelleted by centrifugation (9000 × g for 10 min at 4°C). Cell lysates were obtained using M-PER with a protease inhibitor cocktail (Thermo Fisher Scientific, MA, U.S.A.), according to the manufacturer’s protocol. Total protein concentrations were determined using the BCA Protein Assay Kit (TaKaRa, Japan), according to the manufacturer’s protocol. Cell lysates were mixed with an equal volume of 2× loading dye (125 mM Tris–HCl pH 6.8 at 25°C, 4% sodium dodecyl sulfate (SDS) (w/v), 20% glycerol, and 0.02% bromophenol blue (w/v)). The samples were loaded on 5–20% acrylamide/bis-acrylamide gels immersed in a running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) and electrophoresed until they had migrated across the entire gel. Proteins were electroblotted onto methanol-activated polyvinylidene difluoride membranes (MilliporeSigma, MA, U.S.A.) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol (v/v)). The membranes were blocked for 2 h with 1% bovine serum albumin (BSA) in TBST (137 mM sodium chloride, 20 mM Tris, 0.05% Tween-20) and incubated for 2 h with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GeneTex, CA, U.S.A.) or anti-RARα (Abcam, Cambridge, U.K.) primary antibody in 1% BSA in TBST. The membranes were then washed three times with TBST and further incubated for 1 h with horseradish peroxidase (HRP)-conjugated mouse anti-rabbit immunoglobulin G (IgG) (H + L) or HRP-conjugated sheep anti-goat IgG(H + L) secondary antibody (Jackson ImmunoResearch, PA, U.S.A.), as appropriate, in 1% BSA in TBST. The membranes were washed again, and EzWestBlue (ATTO, Japan) was used for color development. Quantitation of western blots was performed using ImageJ software (NIH, Bethesda, MD, U.S.A.).

Colorimetric HDAC Inhibition Assay

Whole-cell fractions were obtained using the EpiQuik whole-cell Extraction Kit (Epigentek, NY, U.S.A.) according to the manufacturer’s protocol. The extracted samples were frozen at −80°C until use. Inhibition of HDAC activity was measured using the HDAC colorimetric assay kit (Epigentek), and calculated HDAC activity (%) following the manufacturer’s protocol.

Quantification of Prostate-Specific Antigen (PSA) in Cell Culture Supernatant

PSA levels were measured according to the procedure described in our previous report.²³⁾ Briefly, after drug treatment for 72 h, the supernatant was collected and the amount of PSA in the supernatant was quantified using the f-PSA enzyme immunoassay test kit (Immunospec Corp., CA, U.S.A.), according to the manufacturer’s protocol. Significance was confirmed by the t-test.

RESULTS

Determination of IC₅₀ Values for Growth Inhibition of LNCaP and PC-3 Cells

To determine the appropriate concentrations of Am80 and epigenetic inhibitors for combination experiments, we first determined the IC₅₀ values for the individual compounds, Am80, SAHA, and 5-AzadCyD, in cell viability assays (Table S1). These compounds all dose-dependently inhibited the proliferation of LNCaP and PC-3 cells, and the IC₅₀ values were consistent with previously reported values. In the combination studies, all compounds were used at concentrations equal to their IC₃₅ and IC₂₀ values.

Enhanced Effects of Am80 in Combination with Epigenetic Inhibitor

The enhanced effects of epigenetic inhibitors at various concentrations in the presence of a fixed concentration of Am80 were examined in LNCaP and PC-3 cells. As shown in Fig. 1, enhanced growth-inhibitory effects were observed in LNCaP cells treated with 25 µM Am80 and 1.6, 2.1 or 2.6 µM SAHA, whereas treatment with the individual agents did not significantly inhibit cell growth. In the case of the PC-3 cell line, the effect of the combination was not remarkable (Fig. S1). PSA has been used as a serum marker for diagnosis and prognosis of prostate cancer, and its decrease indicates attenuation of LNCaP cell activity.²⁴⁾ The concentration of PSA was decreased by treatment of each drug or the combination (Fig. S2). Therefore, we focused on the LNCaP cell line for further experiments.

Fig. 1. The Enhanced Growth-Inhibitory Effect of Am80 with HDAC or DNMT Inhibitor on LNCaP Cells

LNCaP cells were treated singly or in combination as indicated for 72 h, and cell viability was determined by MTT assay. The absorbance of the vehicle control wells was taken as unity. Data are expressed as means ± standard deviation (S.D.). Experiments were performed in triplicate. n.s., Not significant, * p < 0.05; versus the vehicle; Student’s t-test.

Profile of HDAC Isotype Selectivity

SAHA is a pan-HDAC (Class I, IIb, and IV) inhibitor.²²⁾ Therefore, to identify the class of HDAC that mediates the growth-inhibitory effect observed in our study, we tested selective HDAC inhibitors. Results similar to those observed with SAHA were obtained when the isotype-selective HDAC inhibitor KD5170 (Class I, IIa, and IIb) was used in combination with Am80. The IC₅₀ value of KD5170 growth inhibition in LNCaP cells changed from 0.51 µM in the absence of Am80 to 0.11 µM in combination with 25 µM Am80 (Table S2). Other isotype-selective HDAC inhibitors MGCD0103 (Class I and IV)²⁵⁾ and R306465 (Class I)²⁶⁾ did not show an enhanced growth-inhibitory effect (Table S2).

Effect of HDAC Inhibitors on Apoptosis

Since enhancement in growth inhibition of LNCaP cells was observed with Am80 and HDAC inhibitor, we quantified the percentage of apoptotic cells after 72 h by means of annexin V staining. As expected, there was an increase in the percentage of apoptotic cells in cells exposed to the combination of Am80 and SAHA (38.1%), compared to those in cells exposed to SAHA (26.4%) or Am80 (35.5%) alone (Fig. 2). The combination of Am80 with KD5170 also showed an increase in apoptosis compared with the individual agents.

Fig. 2. Apoptosis-Inducing Effect of Am80 in Combination with HDAC Inhibitors on LNCaP Cells

LNCaP cells were treated with the indicated drugs for 72 h. The proportion of apoptotic cells (expressed as a percentage in each plot) was evaluated by flow cytometric analysis using annexin V-FITC staining.

Effect of HDAC Inhibitors on HDAC Activity

Almost all HDACs are distributed in the nucleus; however, Class IIb HDACs, which may be inhibited by SAHA and KD5170, are distributed in cytosol. To confirm the inhibition of cellular HDAC activity, we determined the HDAC activity in whole-cell lysates. SAHA and KD5170 inhibited HDAC activity by 67.0 and 42.0%, respectively, compared to the vehicle control. Interestingly, SAHA showed higher inhibitory activity than KD5170, even though KD5170 showed more potent cell growth inhibition (Fig. 3).

Fig. 3. The Effect of HDAC Inhibitors on HDAC Activity of LNCaP Cells

HDAC activity was evaluated in whole-cell lysates after treatment as indicated for 72 h. HDAC activity is calculated as follows; [[OD₄₅₀ (control − blank) − OD₄₅₀ (sample − blank)]/OD₄₅₀ (control − blank)] × 100, where OD₄₅₀ is absorbance at 450 nm. Data are expressed as means ± S.D. Experiments were performed in triplicate.

Effect of HDAC Inhibitors on Protein Levels of RARα

Expression of numerous NRs, including ERα and RAR β, is silenced in several cancer cell lines,^14,15) and RARα is activated by agonists, such as all-trans-retinoic acid (ATRA) and Am80, following degradation.²⁷⁾ That is, the existing NR level tends to be insufficient. Thus, clarifying the existence of RARα provides important information to elucidate this enhancement mechanism. We examined the levels of RARα in LNCaP cells by means of immunoblotting. SAHA and KD5170 increased the RARα protein level, and Am80 also had same effect. However, the RARα protein level was decreased by combination treatment of Am80 with either SAHA or KD5170 (Fig. 4).

Fig. 4. Changes of RARα Protein Expression in LNCaP Cells

Western blot analysis was performed with antibodies against RARα in LNCaP cells treated as indicated for 48 h. GAPDH was used as the loading control. The relative intensity of the RARα that have been normalized to GAPDH protein was calculated using ImageJ software, and the values are presented in the lower panel.

DISCUSSION

In this study, we aimed to explore the epigenetic inhibitors that effectively inhibit prostate cancer cell growth in combination with the RARα agonist, Am80. We found that SAHA and KD5170 enhanced inhibitory effect of Am80 and investigated the mechanism underlying it.

Drug discovery is a time-consuming and costly process, and therefore there is increasing interest in drug repositioning, i.e., finding new medical uses for existing drugs. In such cases, regulatory approval can be speeded up because data on pharmacokinetics, side effects, and so on are already available. Prostate cancer is a promising target for such efforts, as it is the second most common cancer in men worldwide.²⁸⁾ In the early stages, androgen-deprivation therapy is effective, but it remains effective only for a short time, resulting in a poor prognosis. Therefore, new therapies or new target molecules are needed.

Here we focused on the potential value of approved epigenetic inhibitors for combination therapy with Am80. We were particularly interested in HDAC inhibitors, because they cause the accumulation of acetylated histones and activate the transcription of genes whose expression induces apoptosis. Also, HDACs are upregulated in various human prostate cancer cell lines in comparison between human prostate cancer cell lines and non-malignant prostate tissues same as in most cancers.^29,30) Indeed, we found an enhanced growth-inhibitory effect of the pan-HDAC inhibitor SAHA with Am80 in the AR-positive prostate cancer cell line LNCaP. Further studies with various isotype-selective HDAC inhibitors revealed that KD5170 showed a similar effect, and overall, the results suggested the involvement of Class IIb HDAC(s). KD5170 has the highest specificity for HDAC6,³¹⁾ and therefore we speculate that inhibition of HDAC6 may be a major target of the effect. Thus, Class IIb HDACs (HDAC6, and/or HDAC10) may be potential targets for treatment of prostate cancer.

The expression of NRs is often silenced in cancer cells, and moreover, RARα is degraded after activation through agonist binding.²⁷⁾ That is, the existing NR level tends to be insufficient, and the RARα levels in acute myelogenous leukemia cells decline further after treatment with Am80 (unpublished data). Here, we found that either KD5170 or SAHA alone increased the RARα protein level in LNCaP whole-cell lysate, though the combination treatment with KD5170 and SAHA decreased RARα protein (Fig. 4). It is well known that RARα translocates to the nucleus after binding with its agonist, retinoic acid.³²⁾ In this context, deacetylation of HSP90 by HDAC6 results in the retention of glucocorticoid receptor (GR) in the cytoplasm, failure of GR maturation, and decreased transcription of target genes.^33,34) Thus, it seems plausible that the HDAC inhibitors KD5170 and SAHA promote RARα maturation in LNCaP cells. We are currently investigating whether this is indeed the case.

CONCLUSION

Combination treatment of Am80 with HDAC inhibitors enhanced the growth-inhibitory effect in LNCaP cells, accompanied with increased apoptosis and reduced PSA secretion. Studies with isotype-selective HDAC inhibitors suggested that Class IIb HDACs are involved in the effect. Dual targeting of Class IIb HDAC and RARα may be a promising therapeutic strategy for prostate cancer.

Acknowledgments

This work was partially supported by TMRC Co., Ltd. Also it was supported by JSPS KAKENHI Grant No. 18K06572 and Tokyo Kasei Chemical Promotion Foundation for financial support.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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