TRPM2 Mediates Histone Deacetylase Inhibition-Induced Apoptosis in Bladder Cancer Cells

Abstract

Purpose:

Inhibition of histone deacetylase (HDAC) activity results in growth arrest and apoptosis in multiple types of cancer cells. It has been well established that p21 is responsible for HDAC inhibitor (HDACi)-induced growth inhibition, while the mechanism underlying HDACi-elicited apoptosis in bladder cancer cells remains largely unknown.

Methods:

In this study, the apoptotic response to HDACi (trichostatin A and sodium butyrate) with different concentrations was determined by flow cytometry analysis and real-time polymerase chain reaction was conducted to examine the TRPM2 (Transient receptor potential cation channel, subfamily M, member 2) expression change on HDACi treatment. TRPM2 knockdown and overexpression were performed to investigate the role of TRPM2 in HDACi-induced apoptosis. The mechanism of HDACi-elicited upregulation of TRPM2 was studied by chromatin-immunoprecipitation.

Results:

HDACi efficiently induced cell apoptosis and TRPM2 upregulation in a time- and dose-dependent manner in T24 bladder cancer cells. Functional analysis revealed that TRPM2 overexpression promotes apoptosis of T24 cells. Conversely, TRPM2 depletion remarkably antagonized HDACi-induced apoptosis. Furthermore, HDAC inhibition-elicited TRPM2 upregulation is caused by the increase of acetylated H3K9 (H3K9Ac) enrichment in TRPM2 promoter.

Conclusions:

These data suggest that the HDACi-elicited upregulation of TRPM2 expression is required for HDACi-induced apoptosis in bladder cancer cells and that HDACi activated the enrichment of H3K9Ac-represented permissive chromatin in TRPM2 promoter.

Introduction

Bladder cancer is the most common malignancy of the urinary tract.¹ It is also a type of heterogeneous disease that makes treatment of bladder carcinogenesis problematic.^2,3 About 30% of bladder cancers are associated with a high risk of death from metastases.¹ Despite great advances in surgical and chemical therapies, the high incidence of metastasis and recurrence is the main cause of bladder cancer mortality. Patients with bladder cancer are usually treated with radical cystoprostatectomy to improve life quality,^4,5 and the chemical agents against the intrinsic therapeutic targets display promising prospects with controversy in bladder cancer treatment. Therefore, efforts in exploring the molecular mechanisms of the potential therapeutic agents against carcinogenesis are of particular significance.

Histone acetylation and deacetylation play an important role in the regulation of gene expression, and acetylated H3K9 (H3K9Ac) represents a permissive chromatin state.⁶ Epigenetic modulation of histone acetylation is recognized as an essential intrinsic mechanism contributing to the malignant phenotype.^7,8 Recently, histone deacetylases (HDACs) have been revealed to regulate cell proliferation and apoptosis in various solid malignancies.^9,10 Dysregulated HDAC activity is tightly associated with carcinogenesis by changing the expression pattern of HDAC target genes.^11,12 Inhibition of HDAC activity by HDAC inhibitors (HDACis, such as trichostatin A [TSA]/sodium butyrate [NaBu]), leads to growth arrest or apoptosis in cancer cells. Therefore, HDACi is considered a candidate drug in cancer therapy.^13

–16 It is widely accepted that induction of the cell cycle- or apoptosis-related genes is the main therapeutic pathway for HDACi. Numerous studies have revealed that p21 is the main factor responsible for HDACi-induced growth arrest.^17
–19 Although it has been documented that HDACi could induce apoptotic protein expression and downregulate survival signaling pathways,^20,21 the detailed mechanism underlying HDACi-induced apoptosis has not been fully understood.

A previous study has tried to identify HDACi targets in multiple cancer cell lines, and hundreds of the altered genes responding to HDACi treatment have been reported.²² Interestingly, a common set of genes, including TRPM2 (Transient receptor potential cation channel, subfamily M, member 2), were upregulated by multiple kinds of HDACi in T24 and MDA carcinoma cell lines,²² indicating that TRPM2 might participate in HDACi-induced cellular processes. TRPM2 is a nonselective calcium-permeable cation channel and is a part of the Transient Receptor Potential ion channel super family. TRPM2 is highly expressed in the brain and has been implicated in the genetic etiology of bipolar affective disorders.²³ The physiological role of TRPM2 has been suggested in the activation of NLRP3 inflammasome and toxicity of amyloid-β.^24,25 The TRPM2 ion channel is activated by free intracellular ADP-ribose, which is produced by PARP in response to oxidative stress and cell death signals.²⁶ Importantly, TRPM2 is tightly correlated with programmed cell death and apoptosis.^27

–30 Therefore, we hypothesized that the upregulation TRPM2 might be responsible for HDACi-induced apoptosis.

In this study, we find that HDACi induced cell apoptosis and TRPM2 upregulation in bladder cancer cells. TRPM2 is required for HDACi-induced apoptosis, and enrichment of H3K9Ac enrichment in TRPM2 promoter explains the induction of TRPM2 transcription by HDACi treatment. Collectively, our results demonstrate that TRPM2 is a key molecule involved in TRPM2-induced cell apoptosis and that TRPM2 is a potential therapeutic target for bladder carcinogenesis.

Materials and Methods

Cell culture and reagents

Human bladder cancer T24 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Invitrogen), 10 U/mL penicillin, and 10 mg/mL streptavidin (Sigma). The cultures were maintained at 37°C under a humidified 5% CO₂ atmosphere. TSA and NaBu were purchased from Sigma and used as indicated concentrations.

Real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from bladder cancer cell lines with TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. A260/A280 ratio of the total RNA was determined by NanoDrop Spectrophotometers for preliminary judgment of RNA quality. Then, the isolated RNA of each sample was electrophoresed on 1% nondenatured agarose/ethidium bromide gel in an RNAase-free electrophoresis tank and visualized with gel imaging system (Tanon). The density of 28S and 18S ribosomal RNA (rRNA) bands was used for RNA quality assessment. Five micrograms of total RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen). Real-time polymerase chain reaction (PCR) was conducted with SYBR Green real-time PCR master mix (Toyobo) to determine the relative gene expression, and the results were normalized to glyceraldehyde 3-phophate dehydrogenase (GAPDH) as an internal control. The primer sequences are listed next:

TRPM2 forward: 5′-TTCGTGGATTCCTGAAAACATCA-3′, reverse: 5′-CCAGCATCAGACAGTTTGGAAC-3′;

GAPDH forward: 5′-ATGACCACAGTCCATGCCAT-3′, reverse: GGTCTTACTCCTTGGAGGCCATGT-3′.

Western blotting

T24 cells were harvested in lysis buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1% NP-40, 0.25 mM phenylmethanesulfonyl fluoride, and protease inhibitors) at indicated time points. Lysates were separated by 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and blocked with 5% skim milk. Then, membranes were subjected to incubation with the primary against β-actin (1:10,000; Abcam), H3/H3K9Ac antibodies (1:1000; Millipore), Caspase-8 (1:300; Cell Signaling Technology), or TRPM2 (Bethyl Laboratories).

Plasmid constructs and transient transfection

The TRPM2 cDNA was obtained from Addgene (Cambridge) and constructed into pcDNA3 vector. A 19-nt short hairpin RNA (shRNA) sequence against human TRPM2 gene was designed, and the target sequence was AAAGCCTCAGTTCGTGGATTC. The control shRNA sequence was: 5′-GATTGTCCTGCCATCGCACTCTT-3′. The sequence was cloned into a self-constructed shRNA expression vector pSuper. Bladder cancer cells were transiently transfected using the X-treme GENE DNA transfection reagent (Roche) according to the manufacturer's instructions. After the indicated time intervals post transfection, the cells were harvested for further studies.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) was conducted using an EZ-ChIP chromatin immunoprecipitation kit (Millipore) according the manual instructions. Chromatin was immunoprecipitated using anti-H3K9Ac antibodies (Millipore). Human IgG was used as the negative control. The precipitated DNA was monitored by real-time PCR using specific primers for the TRPM2 or GAPDH promoters.

Cell apoptosis assay

The cells were double stained with Annexin V-FITC and propidium iodide (PI) (Beyotime Institute of Biotechnology) and examined using a flow cytometer (FACS; BD Biosciences) to detect the percentage of late apoptotic (Annexin V⁺ and PI⁺) cells. The percentages of apoptotic cells were calculated.

Luciferase assay

The 5′-UTR region of TRPM2 (−2031 to+53 bp, including the core promoter region of TRPM2) was cloned from human MEF cells and ligated into pMD18-T vector. Then, this fragment was constructed into pGL3-basic vector (named pGL3-basic-TRPM2 promoter). Next, the vector or pGL3-basic-TRPM2 promoter plasmids were co-transfected with pRLTK (10:1) into T24 cells and dual luciferase activity was determined according to the manufacturer's instructions (Promega).

Statistical analysis

All results were obtained from at least three independent experiments. Data are presented as the mean±standard deviation (SD) and were analyzed using the Graphpad Prism 5. Gel Pro software was used to quantify relative band densities in western blots. Student's t-tests were performed to identify significant differences between different groups. *p<0.05 was considered statistically significant.

Results

TSA and NaBu induce T24 cell apoptosis

Inhibition of HDAC activity leads to apoptosis in multiple kinds of cancer cells.^13

–16 However, it remains unknown whether HDACi results in similar effects in bladder cancer cells. Human T24 bladder cancer cells were used in this study to investigate the pro-apoptotic effects of HDACi. T24 cells were treated with 100 nM TSA or 5 mM NaBu for 24–72 hours, and the cell survival rate was determined. Both HDACis (TSA and NaBu) efficiently induced cell apoptosis in a time-dependent manner (Fig. 1A). To explore the detailed HDACi effects, NaBu and TSA were added to the T24 culture medium with increasing concentrations for 48 hours. Then, the cell survival was examined by flow cytometry analysis. Consistently, both TSA and NaBu promoted cell apoptosis in a dose-dependent manner (Fig. 1B). Moreover, Procaspase-8 was downregulated and cleaved Caspase-8 was increased along with increasing concentrations of TSA/NaBu treatment (Fig. 1B), further confirming the observed HDAC inhibition effects in T24 cells. These data suggest that inhibition of HDAC activity results in apoptosis in bladder cancer cells.

FIG. 1.

TSA and NaBu induce T24 cell apoptosis. (A) T24 cells were co-cultured with DMSO, TSA (100 nM), or NaBu (5 mM) and the percentages of late apoptotic cells (Annexin V⁺/PI⁺) were examined at 24, 48, and 72 hours using flow cytometry analysis. (B) Cell apoptosis rate of T24 cells was determined by flow cytometry analysis under the treatment of TSA or NaBu at indicated concentrations at 48 hours. The TSA- or NaBu-treated cells were also subjected to western blot analysis of cleaved Caspase-8 protein levels. NaBu, sodium butyrate; PI, propidium iodide; TSA, trichostatin A.

Inhibition of HDAC activity induces TRPM2 expression in T24 cells

Based on previous gene expression profiling data, TRPM2 expression was induced by multiple kinds of HDACi.²² However, the alterations of TRPM2 expression in response to HDACi have not been verified by further studies. Therefore, the TRPM2 transcriptional level was examined under the stimulation of NaBu or TSA. Here, we found that TRPM2 was upregulated by NaBu treatment at both mRNA and protein levels along with extended 5 mM NaBu exposure (Fig. 2A, B). Intriguingly, TRPM2 mRNA level was observed to be increased even at 2 hours, indicating that HDACi-elicited TRPM2 upregulation might directly occur at the transcriptional level. Moreover, the effects of NaBu treatment with distinct concentrations were also determined. Our results showed that mRNA and protein expression of TRPM2 was induced by NaBu in a dose-dependent manner (Fig. 2C, D). The effect of TSA stimulation was also determined, and it showed that TSA also elicited TRPM2 upregulation in a dose- and time-dependent manner at both mRNA and protein levels (Fig. 2E, F). To verify the hypothesis that TRPM2 is transcriptionally regulated by HDACi, we clone the TRPM2 promoter into the pGL3-basic and dual luciferase activity was performed. Consistently, the TRPM2 promoter activity was strongly induced on TSA or NaBu treatment (Fig. 2G). Taken together, these results demonstrate that inhibition of HDAC activity induces TRPM2 expression in bladder cancer cells.

FIG. 2.

Inhibition of HDAC activity induces TRPM2 expression in T24 cells. (A–B) Real-time PCR (A) or western blot (B) analysis of TRPM2 mRNA and protein levels under the stimulation of 5 mM NaBu at indicated time points. The relative protein level relative to β-actin was analyzed by Gel Pro software. (C–D) T24 cells were treated with NaBu (1, 5, 20 mM) for 12 hours, and mRNA as well as protein levels were determined by real-time PCR (A) or western blot (B), respectively. The relative protein level relative to β-actin was analyzed by Gel Pro software. (E) Real-time PCR analysis of TRPM2 expression responding to 100 nM TSA stimulation at different time points. (F) T24 cells were treated with 20, 100, and 300 nM TSA for 12 hours, and TRPM2 mRNA and protein levels were examined. (G) The relative luciferase activity of pGL3-basic or pGL3-basic-TRPM2 promoter was determined on indicated concentrations of TSA or NaBu treatment for 24 hours. HDAC, histone deacetylase; PCR, polymerase chain reaction; TRPM2, transient receptor potential cation channel, subfamily M, member 2. *p<0.05 was considered statistically significant.

HDACi-induced TRPM2 overexpression mediates apoptosis of bladder cancer cells

Given that HDACi induces apoptosis of bladder cancer cells, it is accompanied by the upregulation of TRPM2, which is tightly associated with cell death.^27

–30 Therefore, the role of TRPM2 in HDACi-elicited apoptosis was addressed. Based on these observations, we postulate that TRPM2 might be a positive regulator involved in this process. TRPM2 was overexpressed in T24 cells by transient transfection of pcDNA3-TRPM2 plasmid, which resulted in about 10-fold overexpression at the transcriptional and translational levels in T24 cells when compared with the control group (Fig. 3A). Then, the apoptotic analysis was conducted in TRPM2-overexpressed T24 cells. Notably, the percentage of apoptotic cells in TRPM2-overexpression group was significantly increased along with cell cultivation, while cell apoptosis in the control group was maintained at a low level (Fig. 3B). It suggests that the upregulated TRPM2 expression promotes cell apoptosis.

FIG. 3.

HDACi-induced TRPM2 overexpression mediates apoptosis of bladder cancer cells. (A) pcDNA3 vector or pcDNA3-ATF3 plasmids were transfected into T24 cells for 48 hours, and the cells were harvested for real-time PCR or western blot analysis. (B) The TRPM2 overexpression effect on cell apoptosis was determined in T24 cells at 0, 24, 48, and 72 hours. (C) TRPM2 was knocked down efficiently by TRPM2 shRNA. Control or TRPM2 shRNA plasmids were transfected into T24 cells, and the mRNA as well as protein levels were determined. (D) Control or TRPM2 shRNA-expressing T24 cells were subjected to flow cytometry analysis with or without TSA/NaBu treatment at indicated time points. These shRNA-expressing cells with or without TSA/NaBu treatment were subjected to western blot analysis of cleaved Caspase-8 proteins. HDACi, HDAC inhibitor; shRNA, short hairpin RNA. *p<0.05 was considered statistically significant.

Subsequently, the hypothesis that TRPM2 might be partially responsible for HDACi-induced apoptosis was tested in the following knockdown studies. TRPM2 was remarkably knocked down by TRPM2 shRNA (Fig. 3C). Then, control or TRPM2 shRNA-expressing cells were treated with TSA or NaBu for 24–72 hours and flow cytometry analysis was performed to determine cell apoptosis. As expected, both TSA- and NaBu-induced apoptosis was significantly decreased in TRPM2-depleted cells, but TRPM2 shRNA did not reduce the HDACi-induced apoptosis to the basal level (Fig. 3D). Furthermore, the naturally occurring cell death during culture without HDACi treatment was also inhibited by TRPM2 shRNA (Fig. 3D). To verify this result, the protein level of Caspase-8 was determined by western blot analysis. We found that HDACi-elicited upregulation of cleaved Caspase-8 was hindered by TRPM2 knockdown (Fig. 3D(c–f)), which is agreement with TRPM2 shRNA effects in HDACi-induced apoptosis. Collectively, these data indicate that HDACi-induced TRPM2 overexpression is partially required for HDACi-induced apoptosis.

HDAC inhibition-elicited TRPM2 upregulation is caused by increased acetylated H3K9 enrichment in TRPM2 promoter

In view of the essential role of TRPM2 in HDACi-induced apoptosis, the molecular mechanism underlying HDACi-elicited upregulation of TRPM2 expression appears to be important to understand the mechanism of the anticancer activity of HDACi. HDAC proteins act mainly through deacetylation of acetylated histones, which could be inhibited by HDACi. Moreover, the transcriptional activation or repression by histone acetylation or deacetylation is a rapid process. In Figure 2A and E, we found that TRPM2 transcription could be activated by HDACi within 2 hours, suggesting that the action of HDACi is most likely a direct process mediated by histone acetylation modifications. Therefore, the level of H3K9Ac, a permissive histone marker, was determined in HDACi response experiments. As expected, the protein level of H3K9Ac was enhanced even within 2 hours by 5 mM NaBu (Fig. 4A) or 100 nM TSA (Fig. 4B). It proposes that HDACi promotes the global histone acetylation level and transcriptional activity in bladder cancer cells.

FIG. 4.

HDAC inhibition-elicited TRPM2 upregulation is caused by increased acetylated H3K9 enrichment in TRPM2 promoter. (A, B) H3K9Ac level was determined by western blot with anti-H3K9Ac antibodies when T24 cells were treated with 5 mM NaBu (A) or 100 nM TSA (B) for 0, 2, 1, and 24 hours. H3 served as a loading control. (C, D) ChIP assay was performed with anti-H3K9Ac antibodies. The enrichment of H3K9Ac in TRPM2 and GAPDH promoters was determined by real-time PCR. IgG served as a negative control for TRPM2. ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde 3-phophate dehydrogenase.

However, it remains unclear whether the upregulation of histone H3K9Ac is correlated with TRPM2 expression. To test H3K9Ac enrichment in TRPM2 promoter, ChIP assay was conducted with H3K9Ac polyclonal antibodies. It showed that H3K9Ac was occupied at TRPM2 promoters and the H3K9Ac enrichment activity was increased with the increasing concentrations of HDACi treatment (Fig. 4C, D). However, the occupancy of H3K9Ac at the promoter region of housekeeping gene-GAPDH was not affected by HDACi stimulation (Fig. 4C, D). It demonstrates that the induction of TRPM2 expression by HDACi is indeed elicited by the HDACi-elevated H3K9Ac.

Discussion

In this study, we show that inhibition of HDAC activity induces apoptosis of T24 bladder cancer cells. Mechanistic studies reveal that HDACi promotes TRPM2 expression, and the upregulated TRPM2 mediates HDACi-induced apoptosis in bladder cancer cells. Furthermore, H3K9Ac occupancy in TRPM2 promoter is responsible for HDACi-elevated TRPM2 expression. Our data for the first time demonstrate an essential role of TRPM2 in HDACi-induced apoptosis in bladder cancer cells. As a kind of potential anticancer reagents, HDACi results in cell apoptosis by its cytoxity in both cancer cells and normal cells.³¹ Hence, suppression of TRPM2 activation might be an effective way to reduce the side-effect of HDACi in cancer treatment.

HDAC activity is required for various cellular processes as well as for carcinogenesis. In recent years, in view of the reversibility of epigenetic markings, inhibition of HDAC by chemical inhibitors is considered a promising method for cancer therapy. Accordingly, the effects of HDACi on cell growth and apoptosis were widely studied. Previous findings propose that HDACi induces cell growth arrest and apoptosis in various kinds of cancer cells.^12,32 It is widely accepted that HDACi achieves its growth inhibition or arrest by transcriptional upregulation of the cell cycle inhibitor-p21.¹⁷ HDACi treatment also leads to apoptosis in several types of cancers, such as breast, prostate, lung and thyroid carcinoma, leukemia, and multiple myeloma.^33
–35 This study extends the spectrum of HDACi-induced apoptosis to bladder cancer cells. Coinciding with previous findings, HDACi induced apoptosis of bladder cancer cells in a time- and dose-dependent manner (Fig. 1).

In consideration of the unclear mechanism of HDACi-induced apoptosis, this study tried to solve this problem in bladder cancer cells. Coincidently, TRPM2, an ion channel involved in programmed cell death, has been reported to be upregulated by HDACi in microarray analysis,²² while its role has not been understood. In agreement with their findings, TRPM2 expression was markedly induced by HDACi at both mRNA and protein levels in T24 cells (Fig. 2). With the use of TRPM2 overexpression and knockdown assays, we found that TRPM2 overexpression induced apoptosis of bladder cancer cells. On the contrary, TRPM2 depletion antagonized HDACi-induced apoptosis (Fig. 3). However, we note that the effect of TRPM2 overexpression is not as strong as the effect of HDACi on apoptosis and TRPM2-specific shRNA could only partially inhibit the HDACi-elicited apoptosis (Fig. 3). We reason that blocking HDAC activity results in the global transcriptional changes with multi-aspects of side-effects. Furthermore, there may exist some TRPM2-independent components, such as Caspase-3, Caspase-8, and Caspase-9,³⁶ which are involved in HDACi-triggered cell apoptosis. Thus, TRPM2 partially mediated HDACi-elicited apoptosis in bladder cancer cells.

It has been previously reported that TRPM2 mediates the H₂O₂-induced [Ca²⁺]_i rises and whole-cell currents, and it also modulates H₂O₂-induced apoptosis in vascular endothelial cells.³⁷ Consistently, here, we addressed the fact that TRPM2 mediates HDACi-elicited apoptosis in bladder cancer cells. Combining the mechanisms of TRPM2 functions in cell death as an ion channel,^27

–30 it is reasonable to postulate that the role of TRPM2 in HDACi-induced apoptosis might be also a result from [Ca²⁺]_i rises, which needs to be explored in future studies. Once TRPM2 activates Ca²⁺ influx, oxidative stress-induced cell death will be triggered through well-characterized apoptotic pathways, including Caspases cleavage and PARP inactivation.³⁸

In conclusion, our study provides strong evidence for the functional role of TRPM2 in HDACi-induced apoptotic cell death in bladder cancer cells. Based on our findings, we believe that TRPM2 channels may represent a potential target for the treatment of bladder cancers.

Footnotes

Disclosure Statement

The authors declare that they have no competing interests.

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