Effect of Trichostatin A on Histone deacetylases 1, 2 and 3, p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 Gene Expression in Breast Cancer SK-BR-3 Cell Line

DNA methylation, the covalent addition of a methyl group to cytosine, and histone modification play an important role in the establishment and maintenance of the program of gene expression. Hypermethylation of tumor suppressor genes (TSGs) are well documented in various cancers [1].The balance of histone acetylation and deactylation is determined by the activities of two groups of enzymes including histone acetyltransferases (HATs) and histone deacetylases (HDACs). Histone deacetylation are generally associated with silencing gene expression resulting in several solid tumours and haematological malignancies [2]. HDACs include a family of 18 genes, which are grouped into classes I–IV based on their homology to their respective yeast orthologues. Classes I, II, and IV consist of 11 family members, which are referred to as “classical” HDACs, whereas the 7 class III members are called sirtuins [3]. Abstract


Introduction
DNA methylation, the covalent addition of a methyl group to cytosine, and histone modification play an important role in the establishment and maintenance of the program of gene expression. Hypermethylation of tumor suppressor genes (TSGs) are well documented in various cancers [1].The balance of histone acetylation and deactylation is determined by the activities of two groups of enzymes including histone acetyltransferases (HATs) and histone deacetylases (HDACs). Histone deacetylation are generally associated with silencing gene expression resulting in several solid tumours and haematological malignancies [2]. HDACs include a family of 18 genes, which are grouped into classes I-IV based on their homology to their respective yeast orthologues. Classes I, II, and IV consist of 11 family members, which are referred to as "classical" HDACs, whereas the 7 class III members are called sirtuins [3].

Masumeh Sanaei, Fraidoon Kavoosi
Overexpression of HDAC1, 2 and 3 has been reported in breast cancer [4]. In addition to breast cancer, upregulation of HDACs has been reported in other cancers. Several studies have indicated that class I HDAC isoforms 1 and 2 are highly expressed in renal and urothelial carcinomas [5][6]. Semilar mechanism has been shown in prostate cancer [7]. Mammillary Cell cycle progression is a regulated process that involves several checkpoints that assess extracellular signals and DNA integrity. Cyclins and their partner, cyclin-dependent kinases (CDKs), are positive regulators of cell cycle progression; whereas CDK inhibitors (CDKIs) are important negative regulators. The abnormal expression of CDKs or CDKIs leads to tumorigenesis. There are two types of CDKIs, including the CIP/KIP family, p21Cip1/Waf1/Sdi1 (p21), p27Kip1 (p27), and p57Kip2 (p57), and the INK family, p16, p15, p18, and p19. HDACs activity can affect the expression of apjcb.waocp.com Masumeh Sanaei, et al: Effect of Trichostatin A on Histone deacetylases 1, 2 and 3, p21Cip1/Waf1/Sdi1, p27Kip1, and CDKIs by deacetylation of these genes resulting in genes silenced and cancer induction [8]. HDAC inhibitors (HDACIs) are the new class of potential anticancer compounds for the treatment of solid and hematological cancers. These agents vary in structure and exert various anticancer activities such as cell cycle arrest and cell apoptosis. They are classified into hydroxamic acids (such as trichostatin A [TSA]), short-chain fatty acids (such as valproic acid [VPA] and phenylbutyrate), cyclic peptides (such as depsipeptide), and synthetic benzamides (such as MS-275). HDACI TSA was one of the first natural compounds determined to inhibit HDACs [9]. It has been shown that TSA induces apoptosis in colon cancer HCT116 cells [10]. Previously, we reported that TSA and VPA, as HDACIs, can induce apoptosis in HCC and colon cancer, respectively [11][12]. The current study was designed to evaluate the effect of TSA on histone deacetylases 1, 2, and 3, p21, p27, and p57 gene expression in breast cancer SK-BR-3 cell line.

Materials
The human breast cancer SK-BR-3 cell line was obtained from the National Cell Bank of Iran-Pasteur Institute and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics and fetal bovine serum 10% in a humidified atmosphere of 5% CO 2 in air at 37℃. All materials and compounds including TSA, various necesary kits, and instruments used in cell culture, flow cytometry assay, 3 (4,5 dimethyl 2 thiazolyl) 2, 5 diphenyl 2H tetrazolium bromide (MTT) assay, RNA extraction, reverse transcription, and real-time PCR were provided as obtained previously [12-13-14].

Cell culture and cell viability
The breast cancer SK-BR-3 cell viability was determined using MTT assay. The cells were cultured and seeded in 96-well plates at a density of 3 × 10 5 cells per well and treated with TSA (0.5, 1, 5, and 10 μM) dissolved in DMSO for 24 and 48 h. The control groups received the solvent of the drug, DMSO. After incubation for different periods, the cells were exposed to MTT, MTT (0.5 mg/mL) was added to each well for 4 h. Finally, the MTT metabolite was dissolved in DMSO (200 mL) and the optical density was detected by a microplate reader at a wavelength of 570 nM. Each experiment was repeated three times (triplicates).

Cell apoptosis assay
To determine the apoptotic breast cancer SK-BR-3 cells, the cells were cultured and seeded at a density of 3 × 10 5 cells/well and treated with TSA (1 μM) for 24 and 48 h. Following treatment, all treated and untreated cells were harvested by trypsin and washed twice with PBS and then resuspended in Binding buffer (1x). Finally, the cells were stained with annexin V-FITC (5 μl) and propidium iodide (5 μl) in the dark at room temperature for 15 min and counted by FACScan flow cytometry (Becton Dickinson, Heidelberg, Germany).

Real-time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
To determine the relative expression level of histone deacetylases 1, 2, and 3, p21, p27, and p57 gene expression genes expression, the qRT-PCR was performed. The SK-BR-3 cells were cultured at a density of 3 × 10 5 cells/well and treated with TSA (1 μM) for 24 and 48 h, based on IC 50 values. After 24 and 48 h of treatment, the total RNA was harvested using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer protocol and treated by RNase-free DNase (Qiagen). The RNA was transcribed to complementary DNA (cDNA). QRT-PCR was done as described previously [15]. The program for the PCR was as we reported previously [16]. The primer sequences of the genes are indicated in Table1. GAPDH was used as an endogenous control. Data were analyzed using the comparative Ct (ΔΔct) method.

Statistical analysis
The database was setup with the SPSS 16.0 software package (SPSS Inc., Chicago, Illinois, USA) for analysis. The data were acquired from three tests and are shown as means ± standard deviations. Statistical comparisons between groups were performed with ANOVA (one-way ANOVA) and Turkey test. A significant difference was considered as P < 0.05.

Result of cell viability by the MTT assay
To evaluate the effect of TSA (0.5, 1, 5, and 10 μM) on the breast cancer SK-BR-3 cell viability, MTT assay was utilized. Our results indicated that the rate of cell growth inhibition was significantly increased in treated groups than that in control groups after 24 and 48 h. Results showed that the number of viable SK-BR-3 cells decreased significantly, as the concentration of the

Result of determination of genes expression
The effect of TSA (1 μM) on the histone deacetylase 1, 2, and 3, p21, p27, and p57 gene expression genes expression was investigated by quantitative real-time RT-PCR analysis. The result indicated that treatment of breast cancer SK-BR-3 cells with TSA (1 μM) for 24 and 48 h downregulated histone deacetylases 1, 2, and 3 and upregulated the p21, p27, and p57 genes significantly,

Discussion
Epigenetic inactivation of TSGs is a hallmark of cancer cells. These epigenetic molecular mechanisms include crosstalk between histone modification, DNA methylation, and other components of chromatin higher-order structure, and lead to the regulation of gene expression. Histone deacetylation plays an important role in the silencing of cancer-related genes, thereby affecting numerous processes, such as the cell cycle checkpoint, signal transduction, apoptosis, angiogenesis, and cell adhesion. Re-expression of TSGs epigenetically inactivated can result in the suppression of cancer cell growth [19]. In the current study, we report that TSA can down-regulate HDAC 1, 2, and 3 and up-regulate p21, p27, and p57 resulting in cell growth inhibition and apoptosis induction in breast cancer SK-BR-3 cell line.
Similarly, it has been reported that HDACs suppresses the growth of breast cancer cell MCF-7, induce apoptosis, and arrested G1 phase of MCF-7 cells by p21 up-regulation and cyclin D1 down-regulation [20]. Several in vitro study have demonstrated that DACIs increased p57Kip2 gene expression in HL-60 (from promyelocytic leukemia); K562 (from an erythroleukemia); HT-29 and CaCO 2 (from colon cancers); HeLa (from cervical cancer), Lan-5, SK-N-SH and SK-N-BE (from neuroblastomas); and HEK293 (from the human embryonic kidney) [21]. Meanwhile, other studies have confirmed that the HDACs are the main molecular target for anticancer effect of TSA in breast cancer [22]. In colon cancer, it has been demonstrated that Class I HDACs are involved in repressing p21 and suggest that the growth inhibitory  and apoptotic effects induced by HDAC inhibitors are mediated through the inhibition of these enzymes [23]. All reports mentioned above are inconsistent with our findings. Additionally, other researchers have reported various molecular pathways for HDACIs. It has been shown that induction of p21WAF1/Cip1 mediated by silencing of HDAC4 arrests cancer cell growth in human glioblastoma model [24]. In vitro study has been indicated that HDAC4 promotes gastric cancer cell progression mediated through the repression of p21 [25].
In urothelial cancer (HDAC4, HDAC5, and HDAC7) cell lines, treatment with the pan-HDAC inhibitor vorinostat down-regulates the HDAC2 and HDAC8 [26]. Besides, it has been shown that HDACIs can Down-regulate pro-survival proteins such as Bcl-1 and Bcl-2 which regulate mitochondrial integrity, and up-regulate pro-apoptotic proteins like Bim, Bak, and Bax, which function as sensors of cellular stress and initiate the intrinsic pathway [27]. In summary, HDACIs can play their roles through two molecular mechanisms including intrinsic and extrinsic pathways. In this study, we did not evaluate the intrinsic mechanism. Therefore, this evaluation is recommended.
In conclusion, our findings indicated that the TSA can reactivate the p21, p27, and p57 through down-regulation of deacetylase 1, 2, and 3 gene expression which can open a new window in breast cancer treatment.