Insufficient Effective Time of Suberanilohydroxamic Acid, a Deacetylase Inhibitor, Treatment Promotes PC3 Cell Growth

Abstract

Castration-resistant prostate cancer (CRPC) contributes mostly to prostate cancer-specific mortality, and conventional castration therapy is almost ineffective, new therapies are needed. As a new potential anti-cancer drug, histone deacetylases (HDACs) inhibitors were demonstrated to be effective in inhibiting drug-resistance cancers in preclinical studies, but the results from clinical trials on CRPC patients were disappointing, and the reasons are unknown. In this study, we investigated the effect of suberanilohydroxamic acid (SAHA), a broad-spectrum pan-HDAC inhibitor, on proliferation, apoptosis, cell cycle progression in PC3 cells, and found that, unlike significant inhibiting effects at high-dose, low-dose SAHA significantly promoted PC3 cell growth. Further colony formation assay showed that the inhibitory effect of SAHA is also dependent on the treatment time, high-dose SAHA also exhibited promoting effect on PC3 cells when the treatment time was insufficient. However, this effect was not observed in another CRPC cell line, DU145, or another HDAC inhibitor, Trichostatin A (TSA). Our results indicate that, instead of inhibitory effect, SAHA would promote PC3 cell growth if the dose is low or the treatment time is insufficient, but this effect has not been observed in other CRPC cell line or HDAC inhibitors.

INTRODUCTION

With the socio-economic development and aging population, prostate cancer (PC) has become the second most common cancer in the world^1,2) and contributes a lot to male mortality.^3,4) Localized PC is highly curable, but the lethal phenotype, advanced PC evolves to metastatic castration-resistant PC (CRPC)^5–7) are main contributor to prostate cancer-specific mortality.⁸⁾ CRPC will occur in most PC patients after 18–24 months of treatment with a poor prognosis,^9–11) and require systemic therapies, including hormonal inhibition, chemotherapy, and targeted agents,¹²⁾ but drug resistance make it very difficult to achieve an ideal prognosis,¹³⁾ new and effective therapies are needed.^7,10)

Aberrant gene expression is a driver for tumor progression, most of which are caused by the disruption of the ‘epigenetic code’ and post-translational modifications.^14,15) As primary component of chromatin, histone proteins modifications directly affect the behavior of the genetic material,¹⁶⁾ to which, acetylation is one of the most majority modifications.¹⁶⁾ In most cancer cells, histone is deacetylated due to the overactivated histone deacetylases (HDACs)^14,15) that represses anti-tumor gene transcription and then provokes the proliferation, cell cycle progression and inhibition of apoptosis and autophagy.¹⁴⁾ Thus, using HDAC inhibitors (HDACis) to reverse the HDAC-mediated transcriptional repression was suggested to be the potential way for refractory cancer therapy.^17,18)

Suberanilohydroxamic acid (SAHA), also name vorinostat, is a broad-spectrum pan-HDACi that has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma.¹⁹⁾ SAHA inhibits class I, II, IV HDACs that display Zn²⁺-dependent deacetylase activity²⁰⁾ to prevent the deacetylation of key autophagic markers and thus interferes with autophagic cell death.¹⁹⁾ Due to the great ability on inducing cell cycle arrest, apoptosis, and DNA damage repair, thereby inhibiting metastasis and angiogenesis, several reports have implied the potential of SAHA as an anticancer drug.²⁰⁾ Besides, multi-pharmacological targets (epidermal growth factor receptor (EGFR), RTKs and synergistic agents) activation making it a more effective drug against heterogeneous tumors.²¹⁾ However, though satisfactory effect have been observed in pre-clinical models,²²⁾ results of clinical trials using these agents in patients with CRPC were disappointing.^23–25) The reasons have not yet fully understood, continued interest in understanding the effect of HDACis on prostate cancer cell remains.

In this study, we did an in vitro study on the inhibitory effect of SAHA on PC3 cells, and contractor to our expectations, the results showed that SAHA have an effect in promoting PC3 cell proliferation when the treatment time is insufficient. However, these effects were not observed in another CRPC cell line, DU145, or another HDACi, Trichostatin A (TSA), more studies are required.

MATERIALS AND METHODS

Cell Culture and Chemicals

PC3 and DU145 cells were purchased from American Type Culture Collection (ATCC). PC3 cells were cultured with in Dulbecco’s modified Eagle medium (DMEM, 2492923, Gibco, Grand Island, NY, U.S.A.) containing 10% fetal bovine serum (FBS, 1943609-65-1, Sigma-Aldrich, St. Louis, MO, U.S.A.). DU145 cells were cultured with in Roswell Park Memorial Institute (RPMI) 1640 (11875093, Gibco) containing 10% FBS. Both cells were cultured in an incubator at 37 °C 5% CO₂. Cells were subculture into 6-well plate (Corning, NY, U.S.A.) at a density about 1 × 10⁵/well, and the cells were applied for further experiments after 24 h culture. SAHA (0000084785, Sigma-Aldrich) and TSA (HY-15144, MedChemExpress, Shanghai, China) were diluted in dimethyl sulfoxide (DMSO, Q4199, MP Biomedicals, Irvine, CA, U.S.A.) at the concentration of 10 mM for storage and 1 mM for use.

Cell Counting Assay

After treatment, cell culture images of each well at 6 points on the bottom surface were taken under a photographic microscope (AX10, Zeiss, Oberkochen, German), and the cell number of each image were counted.

Cell Proliferation Rate Assay

Cell proliferation rate was determined by using Click-iT EdU Alexa Fluo 488 kit (C10425, Invitrogen, Shanghai, China). After treatment cells were incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU) for 1 h, and then, cells were harvested and fixed. Then, the EdU inside cells were labeled and detected by flow cytometry (Cytoflex, Beckman Coulter, Shanghai, China). More detail can be found in the manufacturer’s instruction.

Cell Viability Assay

Cells were sub-cultured (100 µL/well) to a 96-well plate (Corning). After treatment, 10 µL cell counting Kit-8 (CCK-8) agent (K1018, APExBio, Shanghai, China) were added into each well, and the 450 nm light absorbance were determined by a microplate photometer (Spark 20M, Tecan, Shanghai, China) 1.5 h later.

Colony Formation Assay

Cells were cultured into a 6-well plate (800/well), SAHA or TSA were added into the culture medium after cell adhesion (about 2 h). Then the culture medium was changed every 3 d. After 1 week, the cells were fixed with 4% polyformaldehyde (22256161, Biosharp, Hefei, China) for 30 min and then stained by crystal violet (C0121, Beyotime, Shanghai, China) for 20 min. The images were taken by an iphone 14 and the clone for each well were counted.

Cell Apoptosis Analysis

After treatment, cells were digested by 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) and resuspended in 1 mL culture medium and then collected into flow tube (20622601, FALCON, Shenzhen, China). The culture medium was removed by centrifuge and cells were incubated with apoptosis analysis kit agent (A211-02, Vazyme, Nanjing, China) at room temperature for 30 min. The signal of propidium iodide (PI) and Annexin V were determined via flow cytometry (Cytoflex, Beckman Coulter).

Cell Cycle Analysis

After treatment, cells were digested by trypsin–EDTA (0.25%) and resuspended in 1 mL culture medium and then collected into flow tube (FALCON). The culture medium was removed by centrifuge and cells were fixed with 70% ethanol at −20 °C overnight. Then, cells were washed with phosphate buffered saline (PBS) and then incubated with 50 µg/mL propidium iodide (PI, ST512, Beyotime) and 50 µg/mL RNase A (EN0531, ThermoFisher Scientifc, Waltham, MA, U.S.A.) diluted in PBS at room temperature for 30 min. The PI signal was detected by flow cytometry and cell cycle distribution analyzed by a software (Modifit 3.0, Verity Software House, Topsham, ME, U.S.A.), percentage of G0/G1, S, and G2/M phase were determined.

Intracellular Reactive Oxygen Species (ROS) Determination

A ROS assay kit (S0033, Beyotime) was used to detected intracellular ROS. Cells were incubated with 2-7'-dichlorodihydrofluorescin diacetate (DCFH-DA), probe for ROS, diluted in DMEM culture medium without FBS for 20 min, and then cells were washed with PBS for 3 times. The fluorescence signal of ROS was determined by flow cytometry.

DNA Damage Detection

DNA damage in cells were determined by alkaline comet assay. The detail of this method can be found in a previously publication.²⁶⁾ In brief, cells were harvested and packed at 0.65% agarose gel and spread on a slide. Cells membranes were digested in lysis buffer containing 1% Trition X-100 at 4 °C for 1 h and the protein were digested in lysis buffer containing 0.5 mg/mL deoxyribonuclease (DNase)-free proteinase K (Amresco, Solon, OH, U.S.A.) at 37 °C for 2 h. After DNA were unwounded in ice-cold alkaline electrophoresis solution for 20 min, electrophoresis was proceeded at 100 V/m for 20 min. The DNA in gel were stained with Gel-red and photographed by using a fluorescence microscope (Nikon, Tokyo, Japan). Each comet was analyzed by using the CASP 1.2.2 software (Krzysztof Konca, Wroclaw, Poland).

Western Blot Analysis

After treatment, cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime) containing 1 × protease inhibitor cocktail (4693116001, Roche Diagnostics, Indianapolis, IN, U.S.A.) and 1 × phosphatase inhibitor cocktail (HY-K0021, MedChemExpress) on ice. About 10 µg sample protein per well were separated in sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to a polyvinylidene fluoride (PVDF) membrane (1620177, Bio-Rad Laboratories, Shanghai, China). Membranes were blocked in 5% milk (A600669, Sangon Biotech, Shanghai, China) diluted in Tris buffer solution (TBS, B548105, Sangon Biotech) with 0.5% Tween 20 (1247ML100, BioFroxx, Einhausen, Germany). Then, incubated with primary antibody, anti-β-Actin (P30002, Abmart, Shanghai, China), anti-Acetyl Histone H3 (ET1706-28, HUABIO, Hangzhou, China), and anti-Histone H3 (M1309-1, HUABIO) for 2 h at room temperature, and with HRP-conjugated goat anti-mouse or goat anti-rabbit immunoglobulin G (IgG) (HA1006/HA1001, HUABIO) for 1 h at room temperature. Blots were visualized in a chemiluminescence imager (ChemiDoc XRS+, Bio-Rad) and quantified by using ImageJ 1.53e software.

Statistical Analysis

The number of samples (N) per each group were labeled in the statistical graphs, statistical analysis was done in GraphPad Prism software (8.0.1, GraphPad Software, CA). One-way ANOVA or Two-way ANOVA multiple comparisons were applied to compare the difference between each two group, p < 0.05 were considered as significant different, and the exact p value were labeled in the statistical graphs.

RESULTS

Low Dose SAHA Promoted PC3 Cell Proliferation

Treated with high dose (5–20 µM) SAHA for 24 h significantly decreased cell counts and cell viability in PC3 cells compared to control group (Fig. 1A), but low dose (2 µM) SAHA significantly increased cell counts and cell viability in PC3 cells (Fig. 1B), indicated that low dose SAHA have an effect in promoting PC3 cell proliferation. EdU assay were applied to determine whether low dose SAHA have an effect in promoting cell proliferation. The results showed that treated with 2 µM SAHA for 24 h significantly increased the ratio of EdU positive cells at relatively low-density cultivation conditions (0.06–0.12 × 10⁵/dish), but inhibited the rate of EdU positive cells at relatively high-density cultivation conditions in PC3 cells (Fig. 2), suggested that low-dose SAHA have an effect in promoting PC3 cell proliferation under certain conditions. To further determine the effect of SAHA on PC3 cell growth, we did colony formation assay. The results showed 3- or 7-d treatment with SAHA significantly inhibited colony formation even at low dose (2 µM) (Fig. 3), but 1-d treatment with SAHA significantly promoted colony formation even at high dose (5 µM) (Fig. 3A), suggested SAHA would promote PC3 cell proliferation when the treatment time is insufficient.

Fig. 1. The Effect of SAHA on Cell Proliferation of PC3 Cells

(A) Cell counts and viability of PC3 cells treated with 0 to 20 µM SAHA for 24 h. (B) Cell counts and viability of PC3 cells treated with 0 to 5 µM SAHA for 24 h.

Fig. 2. The Effect of SAHA on Cell Proliferation Rate of PC3 Cells

Cell proliferation rate indicated by EdU staining in PC3 cells sub-cultured at different densities after treated with 2 µM SAHA for 24 h.

Fig. 3. The Effect of SAHA on Cell Colony Formation of PC3 Cells

(A) Colony formation of PC3 cells treated with SAHA at 2 or 5 μM for 1, 3, or 7 d. (B) Colony formation of PC3 cells after exposure to 0 to 10 µM SAHA for 7 d.

Dose-Dependent Effect of SAHA on Apoptosis, Cell Cycle Progression, and ROS Generation of PC3 Cells

Then, we determined more effects of low dose SAHA on PC3 cells. The results showed that treated with high-dose (10 µM) SAHA for 24 h induced significant apoptosis, while low-dose (1 to 3 µM) have no significant effect in promoting apoptosis compared to 0 µM group (Fig. 4A). The effects of SAHA on ROS generation and cell cycle progression in PC3 cells were also different between low-dose and high-dose treatment compared to 0 µM group respectively (Figs. 4B, C). We also determined the effect of SAHA on DNA damage, the results showed that SAHA have an effect in decreasing DNA damage levels in PC3 cells, but no typical dose-dependent biphasic effect were observed (Fig. 4D).

Fig. 4. The Effect of SAHA on Apoptosis, ROS Generation, Cell Cycle Progression and DNA Damage of PC3 Cells

(A) Percentage of apoptosis, (B) cell cycle progression, (C) ROS levels, and (D) DNA damage in PC3 cells treated with 0 to 10 µM SAHA for 24 h. ROSup were applied as positive control for ROS detection.

The Effect of Low-Dose SAHA on the Acetylation of Histone H3 in PC3 Cells

Considering HDACis exert their biological effects mainly through inhibiting deacetylase, we examined the effect of low dose SAHA on histone H3 acetylation. The results showed that treated with SAHA at 2 µM for 12 or 24 h significantly increased H3 acetylation compared to control group, while no significant difference between 12 and 24 h treatment group (Fig. 5A), suggested that low dose SAHA is effective in inhibiting histone deacetylation in PC3 cells. But no significant acetylation of histone H3 can be found in PC3 cells 2 d after SAHA removed (Fig. 5B), suggested that the anti-deacetylation effect can only be maintained in the presence of SAHA.

Fig. 5. The Effect of SAHA on Histone H3 Acetylation in PC3 Cells

(A) Histone H3 acetylation in PC3 cells treated with 2 µM SAHA for 12 and 24 h. (B) Histone H3 acetylation in PC3 cells 2 d later after 1-d treatment with SAHA.

The Effect of TSA on PC3 Cell Growth

To determine whether the promoting PC3 cell growth effect is also exists in other HDACis, we treated PC3 cells with TSA. The results showed that exposure to 5 µM TSA for 6 and 12 h do not have significant inhibiting effect, while exposure to 5 µM TSA for 24 h significantly inhibited cell viability of PC3 cells compare to control group respectively (Fig. 6A), suggested treatment-time dependent effect of TSA on PC3 cells. Then we treated PC3 cells with 0–10 µM TSA for 6 h, and found that at this time point, 10 µM TSA had an effect in promoting cell viability (Fig. 6B). To further determine the effect of insufficient treat time of TSA on PC3 cell growth, we did colony formation assay. The results showed that exposure to 5 µM TSA for 6 h also showed significant inhibition of cell colony formation in PC3 cells compared to control group (Fig. 6C), suggested that TSA have no significant effect on promoting PC3 cell growth under insufficient treatment time condition.

Fig. 6. The Effect of TSA on Cell Viability and Colony Formation in PC3 Cells

(A) Cell viability of PC3 cells after treat with 5 µM TSA for 6, 12, and 24 h. (B) Cell viability of PC3 cells after treat with 0–10 µM TSA for 6 h. (C) Colony formation of PC3 cells treated with 5 µM TSA for 6 h or 7 d.

The Effect of SAHA on DU145 Cells

To determine whether the promoting effect SAHA exists in other CRPC cell lines, we did experiments on DU145 cells. The results showed that treated with SAHA at above 1.2 µM for 24 h could induce significant decrease in cell viability of DU145 cells (Fig. 7A). Treated with 2 µM SAHA for 6 or 12 h did not have significant inhibition effect, while treated with 2 µM SAHA for 24 h significantly inhibited cell viability in DU145 cells (Fig. 7B). In colony formation assay, treated with low-dose SAHA for 1 d also significantly inhibited cell colony formation compared to control group (Fig. 7C), suggested no promoting effect of low dose SAHA on DU145 cells.

Fig. 7. The Effect of SAHA on Cell Viability and Colony Formation in DU145 Cells

(A) Cell viability of DU145 cells treated with 0–10 µM SAHA for 24 h. (B) Cell viability of DU145 cells treated with 2 µM SAHA for 6, 12, and 24 h. (C) Colony formation of DU145 cells treated with SAHA for 1 or 7 d.

DISCUSSION

CRPC is a lethal phenotype of advanced PC which results in poor prognosis,⁸⁾ and the 5-year survival rate for men diagnosed with metastatic PC between 2011 and 2017 was only 31%.⁴⁾ CRPC is resistant to a variety of comprehensive therapies based on androgen deprivation therapy, thus, development of novel therapies becomes a tough challenge.^27,28) HDACis were suggested to be a new potential drug to suppress tumor by modifying relative gene expression, but clinical trials have not yet met with success,¹⁴⁾ and the reason is not fully understood. In this study, we investigated the effect of SAHA, a representative of HDACis, on the growth of PC3 cells, and found that the inhibitory effect of SAHA on the proliferation of PC3 cells is both dependent on the effective dose and acting time, suggest that SAHA may promoting CRPC cells if not appropriately used in cancer treatment.

Though pre-clinical studies have showed HDACis have very high cancer inhibitory effect, disappointing results yielded in clinical trials.^23–25) The mechanisms under poor therapeutics of HDACis in clinical trials are not clear, which may relate to genes regulation and protein activation.²⁹⁾ However, the clinical situation is much more complex, such as, but not all, previous treatment, drug tolerance, and individual health status that may have an impact on HDACis treatment. Considering strong side effects indicated in clinical trials,^23,25,30) the actual dosage of HDACis used in clinical may often not reach the effective dose and acting time. As biphasic effect have been commonly appeared in anti-tumor drugs, that insufficient dose will stimulate cancer cell growth,³¹⁾ intermittent low doses HDACis treatment resulted in unsatisfactory and worse results in CRPC patients.²⁵⁾ In this study, the inhibitory effect of SAHA on PC3 cells was reversed when the effective dose is low, indicating maintaining a certain dosage is crucial for SAHA therapeutic effectiveness, and drug reduction caused by side effects maybe one of the reasons for unsatisfactory clinical treatment. However, we failure to repeat this phenomenon in another HDACi, TSA, or another CRPC cell line, DU145, suggested that the promoting effect of HDACis on CRPC cells are also dependent on both the specific HDACis and cell types.

The mechanism of how SAHA promotes PC3 cell growth is unknown. Inhibition of histone deacetylation is the main function of HDACis, and low dose SAHA (2 µM) have significant effect in inhibiting histone H3 deacetylation (Fig. 5A). However, histone H3 deacetylation would completely recover after SAHA was removed (Fig. 5B) which may be the reason that insufficient time treatment with SAHA have no significant effect in inhibiting PC3 cell growth. Autophagy may be evolved in, as SAHA have been reported to be effective in inhibiting acetylation of crucial autophagy genes, thereby deregulating autophagy and autophagic cell death (ADC) and facilitating cancer cell survival.¹⁹⁾ Autophagy is the key process to maintain intracellular homeostasis by degrading and recycling aberrant proteins and organelles that usually plays a protective role in cells, unless excessive disruption would lead to cell death.³²⁾ Therefore, slight autophagy induced by low dose or insufficient treatment with SAHA is the possible reason for promoting PC3 cell growth, however, this awaits further studies.

Although clinical trials got disappointed results, HDACis are still potential drugs for tumors that are resistant to conventional treatments, and new combination therapies were constantly being investigated, such as low dose SAHA with minor side effects have significant effect in enhancing the antitumor activity of chemotherapeutics^11,33,34) and radiotherapy.³⁵⁾ Developing HDACis based anti-tumor therapies still has the value. In this study, we found that the effective treatment time is important factor for SAHA exerting its inhibitory effect on PC3 cells even when the dose was low (Fig. 3B). Maintaining the drug concentration, especially at the tumor site, might greatly determine the clinical effectiveness of HDACis treatment, and developing new drug delivery and release techniques³⁶⁾ that may achieve good therapeutics.

In conclusion, we investigated the effects of SAHA on the proliferation, apoptosis, cell cycle progression, and colony formation of PC3 cells, and found dose-dependent biphasic effect of SAHA on cell proliferation, apoptosis, and time-dependent biphasic effect of SAHA on colony formation. These results indicate that treating with SAHA for insufficient time would promote PC3 cell growth, while no similar effects were observed in another HADCi, TSA, or another CRPC cell, DU145, more studies are required.

Acknowledgments

This study was supported by National Natural Science Foundation of China (82172679) and Medical Science and Technology Project of Zhejiang Province (2021KY010, 2023KY432).

Conflict of Interest

The authors declare no conflict of interest.

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