Effect of trichostatin A on histone deacetylase 1 (HDAC 1) and CIP/KIP (p21CIP1/WAF1, p27KIP1, and p57KIP2) gene expression, cell growth inhibition and apoptosis induction in lung cancer COR-L105 cell line

Lung cancer is one the leading cause of cancer-related death worldwide, with more than 1.2 million deaths each year. In addition to genetic mutations, epigenetic modifications may contribute to the induction and progression of cancer. The term epigenetic refers to several molecular mechanisms and pathways that regulate gene expression without changing the DNA sequence. These mechanisms include alterations in the histone modification, the methylation status of DNA, chromatin remodeling, and microRNAs. Cancer cells exhibit changes in histone modification patterns at individual genes. Epigenetic deregulation can affect several aspects of tumor cell biology comprising cell growth, cell differentiation, and cell death. Histones are highly conserved alkaline proteins that can become post-translationally modified at the amino acid residues located on their N and C- terminal tails. There are four core histones include histone 2 A, histone 2 B, histone 3, and histone 4, and one linker histone, histone 1. Histone modifying enzymes, such as histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), histone demethylases (HDMs), is often responsible for the aberrant histone modifications. HDACs and HATs are two counteracting enzyme families whose enzymatic activity controls the acetylation state of protein lysine residues. Aberrant histone acetylation is associated with several solid cancers. In mammals, HDACs form three groups based on their sequence homology and are classified as HDACs I, II, and III. These enzymes have a critical role in modulating the balance between pro- and anti-apoptotic proteins. Dysregulation of HDACs and aberrant deacetylation have been implicated in the pathogenesis of various cancers. Overexpression of these enzymes correlates with tumorigenesis. Two known mechanisms, methylation and histone deacetylation seem to be the best candidate mechanisms for inactivation of CIP/KIP (p21CIP1/WAF1, p27KIP1, and p57KIP2) and INK4 (p15INK4b, p16INK4a, p18INK4c, and p19INKd) families. Histone deacetylation is a general mechanism for the inactivation of the CIP/KIP family in various cancers such as gastric cancer, human colon cancer cell line HT -29, human breast cancer MDA231 cell line, human non-small cell lung cancer c ell line, A549 cells. HDAC inhibitors have been considered to be a novel class of cancer treatment agent. These compounds include trichostatin A (TSA), butyrate, trapoxin (TPX), MS-27-275 (a synthetic benzamide derivative) and apicidin. Previously, we reported the effect of trichostatin A on hepatocellular carcinoma (HCC). The present study was aimed to investigate the effect of TSA on histone deacetylase 1 (HDAC 1), CIP/KIP (p21CIP1/WAF1, p27KIP1, and p57KIP2), cell growth inhibition and apoptosis induction in lung cancer COR-L105 cell line.

First, the lung cancer COR-L105 cells were cultured in DMEM supplemented with sodium butyrate, sodium bicarbonate, sodium pyruvate, 10% FBS and antibiotics (penicillin G and streptomycin) at 37°C in 5% CO2 overnight and then seeded into 96-well plates (3× 105 cells per well). After 24 h, the medium was replaced with an experimental medium containing various concentrations of TSA (0, 1, 2.5, 5, 7.5, 10, and 20 μM). The control groups received DMSO only, at a concentration of 0.05 %. After a period of 24 and 48h, the cells were investigated by MTT assay according to Standard protocols to determine cell viability. Therefore, MTT solution was added to each well for 4 h at 37℃ and then the MTT solution was changed by DMSO and shaken for 10 min to dissolve all of the crystals. Finally, the optical density was detected by a microplate reader at a wavelength of 570 nM. Each experiment was repeated three times (triplicates). To determine apoptotic cells, the COR-L105 cells were cultured at a density of 3 × 105 cells/well and incubated overnight and then treated with TSA (2.5 μM) for different periods (24 and 48 h). Subsequently, the treated and untreated cells were harvested by trypsinization, washed with cold PBS, and resuspended in Binding buffer (1x). Finally, Annexin-V-(FITC) and PI were used according to the protocol to determine the apoptotic cells by FACScan flow cytometry (Becton Dickinson, Heidelberg, Germany). Real-time quantitative RT-PCR amplification and analysis were achieved to quantitatively estimate the expression of histone deacetylase 1(HDAC 1) and CIP/KIP (p21CIP1/WAF1, p27KIP1, and p57KIP2) in TSA (25 µM)-treated COR-L105 cells at different times. After treatment times, Total RNA was isolated by RNeasy mini kit (Qiagen) according to the manufacturer’s protocol and then treated by RNase free DNase (Qiagen) to eliminate the genomic DNA. The RNA concentration was determined using a Biophotometer (Eppendorf). Total RNA (100 ng) was reverse-transcribed to cDNA by using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s instructions. Real-time RT-PCR was performed by the Maxima SYBR Green RoxqPCR master mix kit (Fermentas). The primer sequences are shown in Table1. Real-time PCR reactions were performed using the Steponeplus (Applied Biosystem). Data were analyzed using the comparative Ct (ΔΔct) method, the relative expression level of the genes were calculated by determining a ratio between the amount of these genes and that of endogenous control. GAPDH was used as a reference gene for internal control.

To test the effect of TSA (0, 1, 2.5, 5, 7.5, 10, and 20 μM) on the lung cancer COR-L105 cell viability, MTT assay was utilized. Our findings showed that the rate of cell growth inhibition was significantly increased in than that of control groups after 24 and 48 h. Results showed that the number of viable cells decreased significantly, as the concentration of the compounds and duration increased; indicating a dose- and duration-dependent relationship (p<0.001). The IC50 values were determined with approximately 2.5 μM for TSA. Flow cytometric analysis was achieved to determine whether TSA (2.5 μM) can induce apoptosis in the lung cancer COR-L105 line. The percentage of treated and un-treated COR-L105 apoptotic cells was evaluated by staining with annexin V-FITC and PI after 24 and 48 h of treatment. After treatment with TSA, the apoptosis percentage increased significantly. The effect of TSA (2.5 μM) on the histone deacetylase 1(HDAC 1) and CIP/KIP (p21CIP1/WAF1, p27KIP1, and p57KIP2) gene expression was investigated by quantitative real-time RT-PCR analysis. The result indicated that the treatment of lung cancer COR-L105 cells with TSA (2.5 μM) for 24 and 48 h reactivated the p21CIP1/WAF1, p27KIP1, and p57KIP2 gene, down-regulated HDAC 1 significantly.

TSA can down-regulate HDAC 1 and up-regulate p21CIP1/WAF1, p27KIP1, and p57KIP2 gene expression and induce apoptosis in lung cancer COR-L105 cell line.