Histone Deacetylase Inhibitor Trichostatin A (TSA) Induced Growth Inhibition And Caspase-independent Apoptosis In Human Gastric Cancer Cell

Backround: Gastric cancer is one of most common digestive malignant tumors, and it is the first leading cause of mortality in china [1]. Although the 5-year survival rate of gastric cancer has increased dramatically in the past years, the prognosis of advanced gastric cancer is still not ideal, especially in developing country. Meanwhile, the efficacy of traditional multi-model treatment strategies including surgery, chemotherapy and radiation are very limited. Thus, it is necessary to explore new therapeutic methods to improve the survival of advanced gastric cancer, and recently, a novel therapeutic agent, histone deacetylase inhibitors, have been extensively studied for the treatment of cancer.It has been reported that histone deacetylase inhibitors (HDACIs) can induce apoptosis, cell cycle arrest and differentiation in various tumor cells [2-7], including gastric cancer [3], but the detail mechanisms on gastric cancer cells, however, have not been well characterized. Objective: To determine the roles of TSA on human gastric cancer SGC-7901 cells. And then characterize the detail mechanisms of TSA on gastric cancer cells.Methods: To determine the roles of TSA on human gastric carcinoma cells SGC-7901, cells were treated with TSA at different time points and concentrations and then analyzed by cell proliferation assay, Western Blot, TUNEL assay, Flow cytometry by annexin V-FITC conjugated with PI staining, Cell Cycle Analysis, and Immunofluorescence analysis respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 21073 distinct cDNA of human genes and the data were confirmed with Quantitative Real time PCR.Results:I,TSA inhibited cell proliferation and induced cell cycle arrest in gastric cancer SGC-7901 cells.Fig. 1 is a photomicrograph composite of SGC-7901 cells treated with 400 nM TSA, or untreated control. The control cells appeared as undifferentiated small round clumps of cells. Examining these cells on successive hours indicated that they remained undifferentiated and were rapidly dividing, reaching confluence by the third day. The treated cells showed a dramatically different morphology after TSA treatment for 24 h, resulting in a stellate-like appearance. TSA treated cultures also had a significantly reduced cell density as compared with control group.As shown in Fig. 2A, treatment SGC-7901 with 400 nM TSA resulted in marked slowing of cell growth followed by cell death. Fig. 2B showed that as low as 100 nM TSA could inhibit the cell growth of SGC-7901 cell, and the growth inhibition mediated by TSA was concentration-dependent.To better define the basis of this proliferation, cell cycle was analyzed after the treatment of TSA. The results showed that TSA increased the cell number of G0/G1 phase of cell cycle, and decreased the cell number of S phase significantly (p<0.05) in the SGC-7901 cells. As is shown in Fig. 2C, the percentage of G0/G1 population of cells increased from 49.01% to 69.5%, and the percentage of S population of cells decreased from 39.1% to 17.6% after treatment of 400 nM TSA for 18 h. This result indicated that TSA could induce the cell cycle G1/S arrest in gastric cancer SGC-7901 cell line (Fig. 2C).The expressions of cyclin-dependent kinase inhibitor and its down stream genes were examined with Western blot analysis. As expected, TSA dramatically increased the expressions of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and p27KIP1, but decreased the expression of cyclin-dependent kinase 2 (CDK2) and cyclin D1 at the protein level (Fig. 2D). Moreover, TSA increased the expression of p53 in SGC-7901 cells (Fig. 2D) cancer SGC-7901 cells.Tunel assay showed significantly increased apoptotic nuclei in SGC-7901 cells after TSA treatment (Fig. 3A). Quantitatively, apoptotic cells and total number of cells were scored in three random fields, and the percentage of apoptotic cells increased significantly in the treated cells than it in the untreated control (Fig. 3A). FACS analysis confirmed the apoptotic nature of the cell death in SGC-7901 cells induced by TSA (Fig. 3B), the percentage of Annexin V positive cells was 36.7% in TSA treated group and 1.7% in control (p<0.01). To explore whether the caspase was involved in this process, the pan-caspase inhibitor z-VAD-fmk was applied, and we found that z-VAD-fmk did not inhibit the apoptosis induced by TSA (Fig. 3B). The percentage of Annexin V positive cells in z-VAD-fmk + TSA group was 34.6%, that was no different with the percentage (36.7%) of apoptotic cells in the TSA treated only group (p>0.05). This indicated the apoptosis induced by TSA in SGC-7901 cells was through a caspase-independent pathway. Furtherly, immunofluorescence assay was applied to determine whether other apoptotic regulatory proteins such as AIF and EndoG were involved in this process. As expected, AIF and EndoG were detected to translocate from the mitochondria to the nucleus by immunoflurescence stain in treated SGC-7901 cells (Fig. 4A). Untreated SGC-7901 cell showed normal nuclear morphology by DAPI staining (blue fluorescence) and distinct punctuate pattern of II,TSA induced a caspase in-dependent apoptosis in gastric AIF and EndoG immunostaining (green fluorescence). AIF and EndoG positive were found in the peri-nuclear area, indicating its normal location in mitochondria. Cultures of SGC-7901 cells treated with TSA for 24 h demonstrated a more diffuse and nuclear immuostaining pattern indicating the translocation of AIF and EndoG into the nucleus. The subcellular fractionation analysis by western blot also confirmed that AIF and EndoG were released from mitochondria to cytosol (Fig. 4B). We could detectost of AIF and EndoG in untreated pellet and treated cytosol fraction, but very little amount of AIF and EndoG in treated pellet and untreated cytosol fraction. .To further test the mechanism of caspase-independent cell death observed in TSA treated SGC-7901 cell line; Western blotting was applied to assess the function of poly (ADP-ribose) polymerase (PARP) in the cell death process. We did not detect the 116 kd and 85 kd cleavage fragment of PARP in TSA treated SGC-7901 cell (Fig. 4C). The gastric cell line AGS were used as a positive control, which harbor a wild type p53 and were treated with indomethacin 0.4 mM for 48 h to induce the cleavage of PARP as previously reported [8].III,TSA induced the expression of Bax and decreased the expressions of Bcl-2 and Survivin in SGC-7901 cell.Western blotting was applied to examine the expression of pro-apoptotic gene Bax and anti-apoptotic gene Bcl-2 and Survivin. We clearly found the expression of pro-apoptotic Bax was increased, while the expressions of anti-apoptotic genes Bcl-2 and Survivin were decreased significantly in SGC-7901 cells after TSA treatment (Fig. 4D).â…£,Gene profiles in TSA treated SGC-7901 cells.We employed Agilent Human gene expression micorarray which included 21,073 human sequences to screen the genes, and found more than 4710 genes were altered (22.3%), including 905 up-regulated genes (ratio>2) and 3805 down-regulated genes (ratio<0.5). Table 1 showed parts of cell cycle-related and apoptosis-related genes that are significantly regulated by TSA. We confirmed a few of the data with quantitative real time PCR. Table 2 showed the results, and it was consistent with the microarray data. From the table1 we found that TSA increased the expressions of a couple of CDKIs, including CDKN1A (p21WAF1/CIP1), CDKN1B (p27KIP1), CDKN1C (p57), CDKN2D (p19), and decreased the expressions a number of CDKs and its down stream genes, such as CDK2, CDK4, CDK8, cyclin B1, cyclin B2, cyclin A2, cyclin D1. But the expressions of p15 (CDKN2B), p16 (CDKN2A), p18 (CDKN2C) were not increased significantly by TSA and the expressions of CDK6, cyclin E1 were not decreased significantly too (data not shown). This told us that TSA selectively regulated the cell cycle-related genes. The micorarray and real time PCR data also confirmed the expressions of p53, bax, p21WAF1/CIP1 and PTEN were up-regulated and the expression of caspase 3, caspase 1, caspase 4, caspase 9, bcl-2, survivin and BAG were down regulated. Conclusions:1. TSA can inhibit the cell proliferation of gastric cancer SCG-7901 cells in a time and dose dependent manner.2. TSA can induce the cell cycle arrest at G1 pahse, it also can induce the G2 arrest, but it was not significant.3. TSA and induce a caspase in-dependent apoptosis in gastric cancer cells4. TSA acts on the gastric cancer cell through the regulation of the expression of multiple tumor-related genes.5. TSA can be used as a therapeutic agent for the gastric cancer, and it will provide a novel therapeutic ways combined with the traditional methods.