Histone deacetylase (HDAC) inhibitors represent a novel class of anticancer agents, which can be used to inhibit cell proliferation and induce apoptosis in several types of cancer cells. In this study, we investigated the anticancer activity of MHY4381, a newly synthesized HDAC inhibitor, against human prostate cancer cell lines and compared its efficacy with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. We assessed cell viability, apoptosis, cell cycle regulation, and other biological effects in the prostate cancer cells. We also evaluated a possible mechanism of MHY4381 on the apoptotic cell death pathway. The IC50 value of MHY4381 was lower in DU145 cells (IC50=0.31 μM) than in LNCaP (IC50=0.85 μM) and PC-3 cells (IC50=5.23 μM). In addition, the IC50 values of MHY4381 measured in this assay were significantly lower than those of SAHA against prostate cancer cell lines. MHY4381 increased the levels of acetylated histones H3 and H4 and reduced the expression of HDAC proteins in the prostate cancer cell lines. MHY4381 increased G2/M phase arrest in DU145 cells, and G1 arrest in LNCaP cells. It also activated reactive oxygen species (ROS) generation, which induced apoptosis in the DU145 and LNCaP cells by increasing the ratio of Bax/Bcl-2 and releasing cytochrome c into the cytoplasm. Our results indicated that MHY4381 preferentially results in antitumor effects in DU145 and LNCaP cells via mitochondria-mediated apoptosis and ROS-facilitated cell death pathway, and therefore can be used as a promising prostate cancer therapeutic.
Prostate cancer is the most common type of cancer diagnosed in men. In 2018, prostate cancer represented 19% of all cancer diagnoses in men in the United States, which is the highest in the entire world (Siegel
Several studies have indicated that epigenetic aberrations can contribute to cancer development (Ducasse and Brown, 2006; Dokmanovic
The aim of this study was to evaluate the differential anticancer efficacy of the new HDAC inhibitor MHY4381 in both androgen-sensitive and androgen-independent cancer prostate cells. Our results suggest that the MHY4381 preferentially results in antitumor effects in DU145 and androgen-dependent LNCaP cells via both mitochondria-mediated apoptosis and reactive oxygen species (ROS)-facilitated cytotoxicity.
Suberoylanilide hydroxamic acid (SAHA) and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). MHY4381 (purity, 98.5%) was synthesized by Prof. Moon (College of Pharmacy, Pusan National University, Busan, Korea). Stock solutions (10 mM) of drugs were prepared in sterile dimethyl sulfoxide (DMSO) and stored at −20°C until use. Culture media and fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Primary antibodies against acetyl-H3, acetyl-H4, Bax, poly-ADP-ribose polymerase (PARP), Bcl-2, p53, cytochrome C, cyclin A, cyclin B, cyclin D, HDACs, caspase-3 and 9, and β-actin were purchased from Cell Signaling (Beverly, MA, USA). Antibodies against p27 and p21 and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) and DAPI (4′-6-diamidino-2-phenylindole) were purchased from Thermo Fisher Scientific, Inc (Invitrogen, MA, USA), while DCFH-DA (2′,7′-dichlo rofluorescein-diacetate) was obtained from Sigma-Aldrich.
Human prostate cancer cell lines (LNCaP, PC-3, and DU145) were obtained from the American Type Culture Collection (Manassas, VA, USA). DU145 and LNCaP cells were grown in RPMI 1640 (with 10% FBS), and PC-3 cells were maintained in F12 media supplemented with 4 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained as monolayers in a humidified atmosphere containing 5% CO2 at 37°C, and the culture medium was replaced every two days.
HDAC enzyme activity was assessed using an HDAC fluorogenic assay kit (BPS, Bioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, HDAC enzymes were incubated with vehicle, various concentrations of MHY4381, or SAHA at 37°C for 30 min in the presence of an HDAC fluorometric substrate. Fluorescence was measured using VICTOR 3 (PerkinElmer, MA, USA) with excitation at 360 nm and emission at 460 nm. Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA).
The cytotoxic effects of MHY4381 and SAHA were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 5 mg/mL). Approximately, 3×103 cells per well were seeded into a 96 well plate. After 24 h, cells were treated with a range of drug concentrations (0.01–10 μM) for 24 and 48 h. One hundred microliters of MTT solution was added to each well at the end of the experiment and incubated for 4 h at 37°C in the dark. A VERSA Max Microplate Reader (Molecular Devices Corp., CA, USA) was then used to measure the optical density at 540 nm. The data obtained from three independent experiments were used to calculate the IC50 from sigmoidal dose-response curves using SigmaPlot 10 software (Jandel Scientific, San Rafael, CA, USA).
A total of 500 cells/well were seeded into 6 well plates and then incubated with MHY4381 (1 μM) or SAHA (1 μM) for 14 days. Viable colonies were fixed with methanol, stained with 0.05% crystal violet for 20 min, washed with phosphate buffered saline (PBS), and air dried. Colonies with more than 30 cells were counted and then normalized to the numbers in the control group (Franken
After 48 h of treatment with MHY4381 (0.1, 0.5, or 1 μM) or SAHA (1 μM), cells were collected and washed with cold PBS. Total protein was isolated using a PRO-PREP™ protein extraction solution (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (20 μg) were loaded onto 6–15% SDS polyacrylamide gels and then transferred to PVDF membranes. The membranes were then incubated with the desired primary antibodies at 4°C overnight, washed with TBS buffer, and then incubated with HRP-conjugated anti-mouse or anti-rabbit antibodies. The immunoreactive bands were visualized using Chemi Doc™ (Bio-Rad, Hercules, CA, USA), and the blots were quantitatively analyzed using an image analyzer (LAS-4000; Fuji film, Tokyo, Japan).
Cells were seeded and allowed to attach overnight, and were then treated with MHY4381 (0.1, 0.5 or 1 μM) or SAHA (1 μM) for 48 h. The total number of cells, including the ones in suspension and those adhered to the walls of the wells, were harvested separately to identify sub-G1 or other cell cycle stages, respectively, and were washed in 1% bovine serum albumin (BSA) before fixing in 95% ice-cold ethanol containing 0.5% Tween-20 for 1 h. Cells (1×106) were washed in a solution containing 1% BSA, stained with cold propidium iodide (PI) staining solution (10 μg/mL PI and 100 μg/mL RNase in PBS), and incubated in the dark for 30 min at room temperature. Data were analyzed using a flow cytometer (Guava EasyCyte flow cytometer, Millipore, Billerica, MA, USA).
The AnnexinV-FITC binding assay was carried out using the AnnexinV-FITC detection kit (BD, Bioscience). The cells were treated with MHY4381 (0.1, 0.5, or 1 μM) or SAHA (1 μM) for 24 and 48 h. The samples were prepared according to the manufacturer’s instructions and analyzed by flow cytometry (Guava EasyCyte flow cytometer, Millipore).
Morphological changes in the nuclear chromatin in apoptotic cells were assessed by DAPI staining. Cells (1×104) were seeded into a 6-well plate dish and treated with MHY4381 (0.1, 0.5, or 1 μM) or SAHA (1 μM) for 48 h, followed by fixation with methanol and staining with DAPI solution (1 μg/mL). The staining solution was discarded, and the cells were washed with cold PBS. Confocal microscopy was performed using an FV10i microscope (40x, Olympus, Tokyo, Japan) to visualize apoptotic cells.
A non-fluorescent 2′,7′-dichlo rofluorescein-diacetate (DCFH-DA, 10 μM) intracellular probe was used to detect intracellular ROS formation. Approximately 1×103 cells/well were seeded into a 96-well plate. After 24 h, the cells were exposed to MHY4381 (1 μM) or SAHA (1 μM) for 12 h followed by incubation with 10 μM DCFH-DA in serum-free media for 30 min at 37°C. The cells were washed twice with PBS, and DCF fluorescence was detected using a fluorometric imaging plate reader at excitation and emission wavelengths of 488 nm and 520 nm, respectively. To observe the fluorescence image, the cells were seeded into a confocal dish and treated as described previously with the given compounds (MHY4381 or SAHA, each 1 μM) and DCFH-DA in serum-free media for 30 min at 37°C. After washing the cells with PBS, their images were captured using an FV10i microscope (Olympus).
All the data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s test. Analysis was performed using Graph Pad Prism Software Version 5.0 (GraphPad Software). A
The chemical structures of MHY4381 and SAHA are shown in Fig. 1A. Both MHY4381 and SAHA treatment significantly inhibited HDAC enzyme activity in a concentration-dependent manner, and the IC50 value (0.31 μM) for MHY4381 was considerably lower than that of SAHA (1.21 μM) (Fig. 1B). The effect of MHY4381 on the expression levels of acetylated histone proteins and HDACs was measured in three prostate cancer cell lines (DU145, LNCaP, and PC-3) by western blotting. MHY4381 treatment markedly increased acetylated H3 and H4 protein levels in both DU145 and LNCaP cells at 0.5 μM and 1 μM (Fig. 1C). In PC-3 cells, hyperacetylation of H3 and H4 was observed at 1 μM MHY4381 (Fig. 1C). After 48 h of MHY4381 treatment, class I HDACs (1–3 and 8) were noticeably downregulated in the three prostate cancer cell lines. In contrast, the expression of HDAC4 (class II) was downregulated only in DU145 cells but was unchanged in both LNCaP and PC-3 cells by MHY4381 treatment. The expression of the other class II HDACs (5, 6, and 7) was unaffected by treatment with MHY4381 in all three prostate cancer cell lines (Fig. 1D).
The cytotoxicity of MHY4381 in the human prostate cancer cell lines (DU145, LNCaP, and PC-3) was assessed by MTT assay. Both MHY4381 and SAHA prevented cancer cell proliferation in a concentration-and time-dependent manner. MHY4381 was more potent than SAHA (IC50, 5.48 μM) with the following IC50 values in the different cell lines after treatment: DU145 (IC50=0.31 μM), LNCaP (IC50=0.85 μM), and PC-3 (IC50=5.23 μM) (Fig. 2A). All three human prostate cancer cell lines showed different growth inhibition responses when exposed to MHY4381. Prominent morphological changes included cellular elongation induced by MHY4381 in both DU145 and LNCaP cells after 48 h of treatment (Fig. 2B).
To evaluate the anti-tumor effects of MHY4381, a colony formation assay was performed. The ability to form colonies is a fundamental characteristic of cancer cells (Nair
HDAC inhibitors reduce cellular growth by arresting the cell cycle at various checkpoints. The three prostate cancer cell lines were treated with MHY4381 (0.1, 0.5, or 1 μM) or SAHA (1 μM) for 48 h, and their DNA content was measured by flow cytometry. As shown in Fig. 3A, MHY4381 significantly induced G2/M accumulation in DU145 cells and simultaneously decreased the number of cells in S phase. In comparison, MHY4381 arrested cells at the G1 phase of the cell cycle in LNCaP cells. A similar effect was observed with SAHA treatment and occurred in a concentration-dependent manner. Both MHY4381 and SAHA arrested cells at the G2/M phase of the cell cycle in PC-3 cells, but this was not significant. The relative distribution of the effects on different phases of the cell cycle is shown in Fig. 3B. The effect of MHY4381 on the expression of cell cycle proteins was studied by western blotting. The expression levels of cyclin B and cyclin A were markedly decreased, whereas the expression of cyclin D was increased in DU145 cells after MHY4381 treatment. In LNCaP cells, the expression of cyclin D was reduced after MHY4381 treatment. MHY4381 considerably enhanced both p21WAF1/CIP1 and p27 protein levels in DU145 and LNCaP cells, and to a lesser extent in PC-3 cells, in a concentration-dependent manner. Furthermore, p53 was also upregulated in DU145 and LNCaP cells, whereas the expression levels of p53 did not noticeably change in PC-3 cells after MHY4381 treatment (Fig. 3C).
AnnexinV/PI double staining and western blot analysis were performed to understand the mechanism of prostate cancer cell growth inhibition after MHY4381 treatment. In both DU145 and LNCaP cells, the number of late stage apoptotic cells were increased in a concentration-dependent manner after MHY4381 treatment when compared to the control (Fig. 4A, 4B). The expression of apoptosis-related proteins was measured by western blotting. Treatment with MHY4381 increased the levels of cleaved PARP. MHY4381 also caused the cytoplasmic release of cytochrome c and decreased Bcl-2 expression levels in both DU145 and LNCaP cells in a concentration-dependent manner (Fig. 4C). Next, we used DAPI staining to analyze the apoptotic cells by confocal microscopy. The number of apoptotic cells with enhanced fluorescence increased after DAPI staining of MHY4381 treated cells (Fig. 4D). PC-3 cells did not show any significant increase in apoptotic cell death with FACS analysis.
Evidence has shown that intracellular ROS play an important role in cell death by inducing apoptosis (Robert and Rassool, 2012). Accordingly, we measured intracellular ROS formation in prostate cancer cell lines after MHY4381 treatment. As shown in Fig. 5A, the intracellular ROS levels increased about 3.4-fold compared to the control in DU145 cells after MHY4381 treatment, whereas in LNCaP cells, ROS levels increased by about 2.4-fold. In PC-3 cells, there was no significant generation of intracellular ROS. These data support the hypothesis that MHY4381 treatment increases ROS production, and that it could be involved in inducing apoptosis in prostate cancer cell lines (Fig. 5B). To further confirm the role of ROS in inducing apoptosis, an ROS scavenger (NAC) was used. Pretreatment with NAC completely erased the production of ROS induced by MHY4381 treatment in the prostate cancer cell lines. In parallel, NAC also significantly attenuated MHY4381-mediated apoptosis. These data support the notion that the generation of ROS by MHY4381 treatment may play a role in inducing apoptosis (Fig. 5C, 5D).
In both androgen-dependent and independent prostate cancer cells, AR plays an important role in growth and proliferation. The effect of MHY4381 on AR expression levels and its target gene PSA was examined in cell lysates. As shown in Fig. 6, high concentrations of MHY4381 caused a significant reduction in both AR and PSA levels in DU145 and LNCaP cells, although not in PC-3 cells. The data show that there is an inhibitory effect of MHY4381 on the AR signaling pathway in prostate cancer cells which may affect the overall biological function of the cells.
HDAC inhibitors induce cancer cell cycle arrest, apoptosis, and autophagy through the regulation of target gene expression by altering the acetylation status of chromatin and other non-histone proteins. Currently, there are a number of HDAC inhibitors in various stages of clinical trials, and some have been approved for use in cancer patients (Marks, 2010; Eckschlager
It has become clear that in antiandrogen-resistant cancers, the androgen receptor (AR) signaling axis is intact and is required for prostate cancer growth. Thus, there is a heightened interest in developing new drugs that function in part by down-regulating AR expression in prostate tumors. In the present study, we evaluated the anticancer effects of a novel HDAC inhibitor MHY4381 against three types of prostate cancer cell lines (DU145, LNCaP, and PC-3). Our data show that MHY4381 has potent anticancer activities at low doses, not only inhibiting HDAC activity, but also inducing apoptosis through cell cycle arrest and ROS generation in different types of prostate cancer cells. Histone protein acetylation was induced in DU145 and LNCaP cells by MHY4381 treatment at 0.5 and 1 μM, whereas in PC-3 cells, this effect was only observed at the highest concentration (1 μM). HDAC enzymes control the balance between the acetylation and deacetylation of histone and non-histone proteins, which normally regulate the expression of transcriptional factors. It has been reported that both class I and class II HDACs are present in prostate cancer cells at various levels (Waltregny
Previous research has shown that different types of HDAC inhibitors induce cell growth arrest via different intracellular signaling pathways; they discontinue the cell cycle at the G0/G1 or G2/M phase (Park
MHY4381 possesses the ability to stimulate ROS production and cytochrome c release to activate cell death, similar to what is seen for SAHA (Ruefli
It has been reported that AR is involved in regulating the cell cycle, and that inhibiting AR expression results in cell cycle arrest in cancer cells (Balk and Knudsen, 2008; Koryakina
In summary, our data demonstrated that the MHY4381 could inhibit growth of human prostate cancer cells through mitochondria-mediated apoptosis and a reduction in AR expression in DU145 and LNCaP cells. These cellular events are accompanied by modifying the activity of cell regulatory proteins via ROS production. Therefore, we suggest that MHY4381 could be used as an anticancer agent for the treatment of prostate cancer.
The authors declare no conflicts of interest.
This work was supported by grants from the Natio nal Research Foundation (NRF) of Korea (NRF-2016R1A2B 2011071, NRF-2018M3A9C8021792, and NRF2018R1D1A 11307048591), which is funded by the Korean Government.