|Year : 2018 | Volume
| Issue : 10 | Page : 576-582
Trichostatin A induces apoptosis in oral squamous cell carcinoma cell lines independent of hyperacetylation of histones
Boonsil Jang1, Lee-Han Kim1, Seung-Youp Lee2, Kyung-Eun Lee3, Ji-Ae Shin1, Sung-Dae Cho1
1 Department of Oral Pathology, School of Dentistry, Institute of Oral Bioscience and Biodegradable Material, Chonbuk National University, Jeonju 561-756, Republic of Korea
2 Department of Orthodontics, School of Dentistry, Chonbuk National University, Jeonju 561-756, Republic of Korea
3 Department of Oral Medicine, School of Dentistry, Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute, Chonbuk National University Hospital, Jeonju 561-712, Republic of Korea
|Date of Web Publication||24-Sep-2018|
Department of Oral Pathology, School of Dentistry, Institute of Oral Bioscience and Biodegradable Material, Chonbuk National University, Jeonju 561.756
Republic of Korea
Source of Support: None, Conflict of Interest: None
Aim of Study: To investigate the apoptotic event of trichostatin A (TSA) and its associated mechanism in oral squamous cell carcinoma (OSCC) lines.
Materials and Methods: HSC-3 and Ca9.22 cell lines were evaluated using a trypan blue exclusion assay, histone isolation, soft agar assay, live/dead assay, 4%,6-diamidino-2-phenylindole staining, JC-1 mitochondrial membrane potential (MMP) assay, and Western blot analysis to demonstrate the anticancer activity of TSA.
Results: TSA decreased OSCC cell viability and proliferation without affecting the histone acetylation. TSA-induced caspase-dependent or -independent apoptosis according to cell types, TSA enhanced the expression levels of Bim protein by dephosphorylating ERK1/2 pathway in HSC-3 cells. TSA also damaged MMP and increased cytosolic apoptosis-inducing factor (AIF) in Ca9.22 cells.
Conclusion: The present study suggests that TSA may be a potential anticancer drug candidate for the treatment of OSCC through the induction of apoptosis.
Keywords: Apoptosis-inducing factor, Bim, oral squamous cell carcinoma, trichostatin A
|How to cite this article:|
Jang B, Kim LH, Lee SY, Lee KE, Shin JA, Cho SD. Trichostatin A induces apoptosis in oral squamous cell carcinoma cell lines independent of hyperacetylation of histones. J Can Res Ther 2018;14, Suppl S3:576-82
|How to cite this URL:|
Jang B, Kim LH, Lee SY, Lee KE, Shin JA, Cho SD. Trichostatin A induces apoptosis in oral squamous cell carcinoma cell lines independent of hyperacetylation of histones. J Can Res Ther [serial online] 2018 [cited 2020 Oct 24];14:576-82. Available from: https://www.cancerjournal.net/text.asp?2018/14/10/576/177220
Boonsil Jang, Lee.Han Kim and Seung.Youp Lee contributed equally to this work.
| > Introduction|| |
Histone deacetylases (HDACs) are important regulators of gene expression that remove the acetyl group from histones resulting in gene repression. Numerous studies have indicated that abnormal expression of HDACs in human tumors can be linked to key events of carcinogenesis.,, Recently, Chang et al. demonstrated that overexpression of HDAC in oral squamous cell carcinoma (OSCC) tissue was associated with advanced tumor size, metastasis, and shorter overall survival implying the possibility of being a good biomarker. In this concept, HDAC inhibitors were thought to be potentially effective anticancer drug candidates for the treatment of OSCC. Trichostatin A (TSA) originally identified as an antibiotic against fungi was a classic HDAC inhibitor. It was found that TSA can suppress the growth of several types of malignant tumors and promote tumor cell apoptosis., However, previous studies have not properly explained the antitumor activity of TSA in OSCC cell lines.
Bcl-2 family proteins modulate mitochondrial membrane potential (MMP) to control the fate of cells to die, and thus it is necessary to target these proteins for inducing cancer cell death. Bim, a proapoptotic BH3-only protein of the Bcl-2 family members, can be upregulated to trigger cytochrome c release from mitochondria causing apoptotic cell death. The Bim-deficient mice increased the number of lymphoid and myeloid cells. Bim deficiency also can result in the formation and growth of the tumors in nude mice. These findings suggest that Bim is possibly a critical molecule of apoptosis in chemotherapy. Apoptosis-inducing factor (AIF) was known to be a novel apoptotic effector that induces chromatin condensation and DNA fragmentation. AIF is located in the mitochondria, which is critical for providing energy to support cellular functions. It may be released from mitochondria into cytosol causing caspase-independent cell death when cells got certain stress such as the treatment with genotoxic agents., Thus, AIF can be a very important target for caspase-independent apoptosis against cancer treatment.
The goal of our study was to investigate how TSA exerts its anticancer effect in human OSCC cell lines. To this end, we used two OSCC cell lines, HSC-3 and Ca9.22. With this study, we are the first to demonstrate that TSA can induce caspase-dependent or caspase-independent apoptosis according to cell type.
| > Materials and Methods|| |
Chemicals and antibodies
TSA and 4',6-diamidino-2-phenylindole (DAPI) were supplied by Sigma–Aldrich (Sigma–Aldrich, Louis, MI, USA). Z-VAD-FMK was obtained from R & D Systems (Minneapolis, MN, USA). Antibodies against AIF, actin and α-tubulin, were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA). Antibodies against acetyl-histone H3 and H2A (Lys5), cleaved caspase 3, cleaved PARP, Bim, Bax, Bak, Bcl-xL, Bcl-2, phospho-ERK, and total ERK were bought from Cell Signaling Technology, Inc., (Cell Signaling Technology, Charlottesville, VA, USA). COX4 antibody was obtained from Abcam (Abcam, Cambridge, UK).
Cell culture and chemical treatment
HSC-3 and Ca9.22 human OSCC cell lines were kindly provided from Prof. Shindo (Hokkaido, Japan). Both cell lines were cultured in Dulbecco's modified essential medium (DMEM; Welgene, Dae-gu, Korea) supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator.
Trypan blue exclusion assay
HSC-3 and Ca9.22 cells were treated with different concentrations of TSA (62.5, 125, 250, and 500 nM) for 24 h, and the number of viable cells was counted using a hemocytometer with trypan blue (0.4%). Each experiment was carried out in triplicate, and the results were expressed as means ± standard deviation for each treatment group.
To verify an HDAC inhibiting activity of TSA, acid-soluble proteins were extracted as follows. Isolated nuclei were mixed with 0.4 N sulfuric acid and incubated for 3 h on ice. After centrifugation at 15,000 rpm for 5 min at 4°C, the dissolved histones in the supernatant were precipitated with 20% trichloroacetic acid overnight at −80°C. The tubes were centrifuged and washed with acetone. The pellets were collected by centrifugation at 15,000 rpm for 5 min at 4°C and then dissolved in distilled water.
Cell death was further investigated using the live/dead viability/cytotoxicity kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, HSC-3 and Ca9.22 cells were seeded in 60 mm2 dishes, treated with different concentrations of TSA for 24 h, and harvested by trypsinization. Cell pellets were resuspended with calcein acetoxymethyl ester (2 μM) and ethidium homodimer (4 μM) at room temperature (RT) for 30 min, followed by washing with ice-cold PBS. Dead cells were finally observed with a fluorescence microscope (×200).
Nuclear condensation and fragmentation were determined by DAPI staining. Both cell lines were treated with TSA for 24 h and fixed in 100% ethanol overnight at −20°C. The next day, the cells were washed with ice-cold PBS (3X), fixed with 100% methanol at RT for 10 min, and then stained with DAPI solution (2 μg/ml). DAPI-stained cells were observed using a fluorescence microscope (×400).
Western blot analysis
Whole cell lysates were extracted with lysis buffer, and protein samples were quantified using a DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to immunoblot PVDF membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in tris-buffered saline and Tween 20 for 1 h 30 min at RT and maintained overnight at 4°C with designated primary antibodies, followed by incubation with HRP-conjugated secondary antibody at RT for 1 h 30 min. Antibody-bound proteins were detected using an ECL Western blotting luminol reagent (Santa Cruz, CA, USA).
Alterations in MMP were measured using the MMP detection kit (Stratagene, La Jolla, CA, USA). Ca9.22 cells were treated with TSA, incubated with 1X JC-1 Reagent for 15 min at 37°C in 5% CO2 incubator, and washed with PBS (3X). JC-1 fluorescence was measured using a microplate reader (Plate Chameleon, HIDEX).
Preparation of cytosolic and mitochondrial fractions
Cell pellets were suspended for 1 min at RT in plasma membrane extraction buffer containing 0.05% digitonin. Following a centrifugation step at 15,000 g at 4°C for 5 min, the supernatant (cytosolic fraction) was separated from the pellet, which consisted of cellular debris (mitochondrial fraction). The pellets were resuspended in plasma membrane extraction buffer containing 0.5% Triton X-100 and centrifuged at 15,000 g at 4°C for 5 min. The supernatant from the last centrifugation contains mitochondrial proteins.
The data were analyzed for statistical significance using a Student's t-test, and P value compared with vehicle control was considered statistically significant.
| > Results|| |
Trichostatin A blocks viability of HSC-3 and Ca9.22 cells without affecting histone acetylation
We first examined a growth-inhibitory effect of TSA on HSC-3 and Ca9.22 cells using trypan blue exclusion assay. Both cell lines were treated with 62.5, 125, 250, and 500 nM of TSA for 24 h. TSA efficiently blocked the viability of HSC-3 and Ca9.22 cells in a concentration-dependent manner compared with DMSO-treated groups [Figure 1]a. To verify whether the antigrowth activity of TSA was mediated through the hyperacetylation of histones, we isolated histones and performed Western blot analysis using a specific antibody against acetyl-histones H2A and H3. HSC-3 and Ca9.22 cells treated with TSA for 24 h did not exhibit an increase in acetylation over the control except only HSC-3 cells treated with 500 nM of TSA whereas SAHA (4 μM)-treated HSC-3 cells as a positive control clearly caused hyperacetylation of histones [Figure 1]b. Overall, these results suggest that TSA can exert the antiproliferative activity oral cancer cell lines regardless of histone acetylation.
|Figure 1: Effect of trichostatin A on the viability and histone acetylation and of oral squamous cell carcinoma cells. (a) HSC-3 and Ca9.22 cells were treated with DMSO or different concentrations of trichostatin A (62.5, 125, 250, and 500 nM) for 24 h. The effects of trichostatin A on cell viability were analyzed by trypan blue exclusion assay (0.4%). The graph is mean ± standard deviation. *P < 0.05 compared with the DMSO treatment group; **P < 0.01 compared with the DMSO treatment group; ***P < 0.001 compared with the DMSO treatment group. (b) Histones were extracted using acid extraction and then analyzed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis to detect acetylhistones. Equal amount of proteins were measured by Coomassie blue staining. The respective positions of all core histones are indicated on the left. SAHA-treated HSC-3 cells were used as a positive control. The data represent two independent experiments|
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Trichostatin A augments caspase-dependent or -independent apoptosis in oral cancer cell lines
To characterize the apoptotic response induced by TSA, a live/dead assay was carried out. As shown in [Figure 2]a, TSA clearly increased red fluorescence-positive cells implying the increasing number of dead cells in HSC-3 and Ca9.22 cells. Next, we tested whether the nuclear morphological change was being affected by TSA. Both cell lines treated with TSA displayed nuclear condensation and fragmentation, which are typical apoptotic features while DMSO-treated cells had an intact nucleus [Figure 2]b. To examine the effect of TSA on the expression of apoptosis-regulating proteins in both cell lines, Western blot analysis using antibodies against cleaved PARP and cleaved caspase 3 was implemented. The results demonstrated that higher concentrations of TSA dramatically induced cleaved PARP and cleaved caspase 3 [Figure 3]a. To evaluate whether caspase 3 is associated in TSA-induced apoptosis, we then confirmed the dependency of caspase 3 in TSA-mediated PARP cleavage using a pan-caspase inhibitor, z-VAD-FMK. We found that z-VAD-FMK partly prevented caspase-related PARP cleavage in HSC-3 cells, but the apoptotic event triggered by TSA was not blocked by the z-VAD-FMK in Ca9.22 cells [Figure 3]b. These findings suggest that TSA-induced apoptotic cell death is mediated by either a caspase-dependent or -independent pathway dependent on cell types.
|Figure 2: Effects of trichostatin A on the number of dead cells and morphological changes of oral squamous cell carcinoma cells. (a) Live (green) and dead (red) cells were determined by live/dead assay kit as mentioned in materials and methods (×200). (b) Fluorescence microscopy images of the 4',6-diamidino-2-phenylindole-stained in HSC-3 and Ca9.22 cells (×400). Dead cells or 4',6-diamidino-2-phenylindole-stained cells were counted, and the data shown in the graphs expressed the means ± standard deviation of triplicate experiments. *P < 0.05 compared with the DMSO treatment group; **P < 0.01 compared with the DMSO treatment group; ***P < 0.001 compared with the DMSO treatment group|
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|Figure 3: Effects of trichostatin A on caspase-dependent or independent apoptosis in oral squamous cell carcinoma cells. (a) Western blot analysis was performed to detect cleaved PARP and caspase 3. Actin was used to normalize the protein loading from each treatment. (b) HSC-3 and Ca9.22 cells were treated with trichostatin A (500 nM) for 24 h after 1 h pretreatment with Z-VAD (a pan caspase inhibitor, 2.5 μ M), followed by performing Western blot analysis|
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Trichostatin A may induce apoptosis in HSC-3 cells via ERK/Bim signaling
Several studies have reported that modulation of Bcl-2 family proteins by HDAC inhibitors in cancer cells plays a critical role in cancer development and progression.,, Thus, we determined whether TSA can regulate Bcl-2 family members. The results showed that HSC-3 cells treated with TSA significantly increased the expression level of Bim protein [Figure 4]a, whereas other Bcl-2 family members such as Bax, Bak, Bcl-xL, and Bcl-2 were not altered by TSA (data not shown). Because ERK1/2 pathway is necessary and sufficient to regulate Bim protein, we here evaluated whether TSA could affect ERK1/2 signaling. As shown in [Figure 4]b, TSA significantly dephosphorylated ERK1/2 but did not change total ERK protein. To confirm the relationship between ERK1/2 pathway and Bim protein during TSA-induced apoptosis, we used AZD6244, an MEK/ERK inhibitor. The results showed that AZD6244 dramatically potentiated TSA-induced PARP cleavages through upregulation of Bim protein compared to each single treatment [Figure 4]c.
|Figure 4: Effect of trichostatin A on Bim and ERK1/2 pathway in HSC-3 cells. (a) HSC-3 cells were treated with DMSO or various concentrations of trichostatin A for 24 h. Proteins were extracted to analyze the expression levels of Bim by Western blot analysis. Protein levels were normalized to actin. The graph was the mean ± standard deviation of three independent experiments and significance (P < 0.05) compared with the control group was indicated (*). (b) Phospho-ERK and total-ERK expression were detected by Western blot analysis. (c) HSC-3 cells were co-treated with trichostatin A and AZD6244 for 24 h, and cell viability was evaluated by trypan blue exclusion assay (0.4%). The expression levels of phospho-ERK, Bim and cleaved PARP were analyzed by Western blot analysis|
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Apoptosis-inducing factor may be related to caspase independency of trichostatin A-induced apoptosis in Ca9.22 cells
Next, in order to investigate whether modulation of MMP was involved in TSA-induced apoptosis in Ca9.22 cells, JC-1 assay was performed. The results found that TSA significantly disrupted MMP in a concentration-dependent manner [Figure 5]a. Recently, it has been reported that AIF leads to caspase-independent apoptosis by its translocation into cytosolic from mitochondria. Based on our results that TSA could mediate caspase-independent apoptosis [Figure 3]b, we determined the effect of TSA on cytosolic translocation of AIF in Ca9.22 cells. As shown in [Figure 5]b and [Figure 5]c, cytosolic AIF was increased in a concentration-dependent manner. These suggest that TSA recruits AIF to mediate caspase independency during apoptosis.
|Figure 5: Effect of trichostatin A on mitochondrial membrane potential and apoptosis-inducing factor protein in Ca9.22 cells. (a) Mitochondrial membrane potential was measured using JC-1 assay kit and the data shown in the graph represent the mean ± standard deviation of triplicate experiments. *P < 0.05 compared with the DMSO treatment group (apoptosis inducing factor). (b) Apoptosis inducing factor protein expression was detected using Western blot analysis. COX4 and α-tubulin were used to normalize the mitochondrial and cytosolic protein, respectively. (c) The graph for cytosolic apoptosis inducing factor protein represents mean ± standard deviation of triplicate experiments, *P < 0.05 compared to the vehicle control group|
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| > Discussion|| |
HDAC inhibitors potentially inhibit the proliferation of various cancer cell lines in vitro and tumor growth in animal models.,, Recently, our group published that new synthetic HDAC inhibitors (Ky2, A248, and A1659) that increased the histone acetylation induced apoptosis by suppressing Sp1 protein in human breast and oral cancer cell lines., These findings suggest that HDAC inhibitors can be possibly apoptotic inducers against various solid tumors including oral cancer. Several studies demonstrated that TSA-induced apoptotic cell death in human liver, ovarian, and lung cancer cells., In the present study, our group also found that TSA significantly suppressed cell viability and increased the number of apoptotic cells in human OSCC cell lines (HSC-3 and Ca9.22) evidenced by live/dead assay and DAPI staining. Similarly, Suzuki et al. demonstrated that TSA inhibited cell growth and induced apoptosis in OSCC cell lines (HSC-4, Ho-1-N-1, and Ho-1-U-1) supporting our data. These strongly suggest that TSA can have a growth-inhibitory activity for the treatment of OSCC through the enhancement of apoptotic cell death. Next, we investigated the intracellular level of acetyl-histones H2A and H3 in both cell lines treated with TSA and the results showed that TSA did not affect the acetylation of histones. Although HDAC inhibitors have direct impact on gene transcription via direct inhibition of HDAC function on histone tail, they may have an indirect mechanism., Thus, we cannot exclude the possibility that TSA may exert its anticancer activities regardless of direct inhibition of HDACs.
Since Kerr et al. described a unique morphology of dying cells called apoptosis, it has been developed as an attractive approach of cancer therapy. Caspases are kinds of proapoptotic players sufficient to activate either intrinsic or extrinsic apoptotic pathway. In this study, we have shown that the treatment of OSCC cells with TSA clearly activated caspase 3. However, TSA induced the caspase-dependent apoptosis only in HSC-3 cells. Several studies reported that caspases can play critical roles in the apoptotic responses triggered by TSA., On the other hand, TSA also can induce caspase-independent apoptosis in human gastric and pancreatic cancer cell lines., These findings provide the possibility of both caspase-dependent and -independent apoptotic pathway in TSA-induced cell death of OSCC cells.
HDAC inhibitors can modify the cellular ability of the response to favoring apoptosis. Gene expression profile studies showed that HDAC inhibitors can alter the expression levels of proapoptotic proteins such as Bim, Bax, and Bak., TSA has been shown to increase the transcription of BH3-only proteins to disrupt MMP, induce the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm and activate caspase-9 during apoptosis, meaning that BH3-only proteins can be associated with apoptotic activity of HDAC inhibitors. This study showed that TSA could induce caspase-dependent apoptosis in HSC-3 cells by up-regulation of Bim. In accordance with these data, our group recently published that Bim affects parthenolide-induced apoptosis in oral cancer. The works of Ley et al. demonstrated that ERK1/2 is the major pathway to promote Bim phosphorylation for protein degradation via the proteasome. Our data showed that TSA significantly dephosphorylated ERK1/2 and AZD6244 (an MEK inhibitor) potentiated up-regulation of Bim protein slightly induced by TSA. These data imply that ERK1/2 may mediate Bim protein in TSA-induced apoptosis.
In our study, we observed that z-VAD, a caspase inhibitor, did not prevent TSA-induced apoptosis in Ca9.22 cells suggesting that this phenomenon is mediated by caspase-independent apoptotic pathway. AIF has a unique property to induce apoptosis-like changes in a caspase-independent manner. Recently, our group published that AIF could play an important role in caspase-independent apoptosis in oral cancer cells. Thus, we investigated whether TSA affects the cytosolic expression level of AIF protein and the result showed that it significantly increased AIF expression. Similarly, others also demonstrated that HDAC inhibitors including TSA-induced caspase-independent apoptosis in human pancreatic and lung cancer cell lines, and AIF are involved in HDAC inhibitors-mediated apoptosis strongly supporting our data. These findings suggest that TSA can induce apoptosis in either caspase-dependent or -independent apoptosis in OSCC cells. Collectively, the findings presented here provide the possibility of chemotherapeutic activity of TSA against OSCC.
Financial support and sponsorship
This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2014R1A1A2055874), the Ministry of Science ICT and Future Planning (2014R1A4A1005309) and research funds of Chonbuk National University in 2011.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]