The Study Of Sodium Butyrate-induced Apoptosis In Eca-109 Cells

Background and ObjectivesHistones are small basic proteins that organize and package DNA into the nucleosome core. The acetylation and deacetylation of core histones are alerted to transcription activity of genes.Inhibition of deacetylases (HDACi) represents a new therapentic approach in human cancer since these enzymes play a fundamemal role in regulating gene expression and chromatin assembly. Recently, Histone deacetylase (HDAC) inhibitors have been shown to exert various antitumor effects by modulating the expression of a number of genes, and currently are being tested for their efficacy in a variety clinical trials. Sodium butyrate (SB),a short no poisonous fatty acid, is the most common and physiologic member of HDAC inhibitors because it is formed in the colon by breakdown of dietary fibers and plays an important protective role in Colorectal carcinogenesis. Previous studies have demonstrated that SB suppresses cell proliferation and induces cellular differentiation and promotes apoptosis in a variety of tumor cells in vitro.However, the identity of the mechanisms that are involved in Sodium butyrate-induced apoptosis and the downstream effectors that confer the apoptotic response are unknown.The available evidence indicates that at least two well characterized pathways, caspase-dependent and caspase-independent pathway, commit a cell into the execution phase of apoptosis in the cellular response to other stimili. One pathway is predominantly initiated by engagement of the Fas receptors and thereby activation of caspase-8.On the other hand,activation of pro-appoptotic Bcl-2 family members such as Bax can trigger a sequence of events that leads to alterations in mitochondrial permeability transition, mitochondrial swell, mitochondrial electric potential (Ψm)descend, and release of cytochrome C and Smac/Diablo into the cytosol from mitochondrial. binding of cytosolic cytochrome C to Apaf-1 results in apoptosome-mediated caspase-9 activiation. Once activated, both caspase-8 and caspase-9 participate in a cascade that culminates in the activation of caspase-3,which cleaves several substrates including PARP, resulting in chromosomal DNA fragmentation and cellular morphologic changes characteristic of apoptosis. However, caspases are not the only effctors causing nuclear apoptosis. Some other mitochondrial pro-apoptotic proteins such as AIF, mediating cell apoptosis in a caspase-independent manner while are released from mitochondrial inter-membrane space. AIF translocates to the nucleus in a caspase-independent manner where it is involved in initial stages of chromatin condensation and large-scale DNA fragmentation.Recently, the role of caspases in Sodium butyrate (SB)-induced apoptosis had been reported. For example, SB enhances the susceptibility of hepatoma cells to anti-Fas-mediated cytotoxicity by altering the activation status of proteins that could be involved in the Fas signaling pathway. SB has been shown to require caspase-3 activity to induce apoptosis in lung, prostate carcinoma cells and Hela cells. The treatment of human myeloid leukemia cells and medulloblastoma cells with sodium butyrate have been demonstrated to associate with the activation of caspase-8 in undergoing apoptosis.Caspase-9 and cytochrome C has been found to be critical for apoptosis in response to sodium butyrate in colon carcinoma cells.At present, there is no published information on whether AIF might play a role in mediating cell apoptosis in tumour cells, especially in esophageal cancer Eca-109.In the current study, Eca-109 cells apoptosis was observed by agarose electrophoresis and MTT, to further elucidate the mechanism of SB-induced apoptosis in Eca-109 cells, we investigated caspases associated apoptotic events, translocation of cytochrome C from the mitochondria to the cytosol, and release of AIF from intermembrane space by western blotting.Methods1. Inhibition ratio of the extract from Sodium butyrate on Eca-109 cells was tested by MTT assay. The experiment was divided into 3 groups:①experimental group: Sodium butyrate at the different concentrations were administered to culture tumor cells with 1mmol/L, 2.5mmol/L, 5mmol/L;②negative control group: Sodium butyrate were not administered to culture tumor cells;③vacancy control group: the same volume of saline was administered to culture tumor cells .Every concentration of the extract from Sodium butyrate which was administered to Eca-109 cells, respectively, was set for 4 parallel holes, after culture of 24 hours ,48 hours ,72hours, the ratio of inhibition of cell growth was tested. The above experiment was repeated 3 times. The average value was figured out.2. Eca-109 cells apoptosis was observed by agarose electrophoresis; the caspase-8 activities from 2.5mmol/L Sodium butyrate-induced Eca-109 cells were examined by western blotting and showed by varying of Sodium butyrate-induced duration, the expression of apoptosis related-protein cytochrome C and AIF from mitochondrial and cytosolic were measured by western blotting and the quantitative amalysis was made with figure analysis system.Results1. The effect of growth inhibition of the extract from Sodium butyrate-induced of Eca-109 cells.Sodium butyrate can remarkably inhibit the growth of Eca-109 cells. After dealing with the concentrations of 1 mmol/L, 2.5 mmol/L, 5mmol/L after 24 hours, 48 hours, 72hours, respectively, the ratio of inhibition of Eca-109 cells growth was 13.38%, 33.40%, 39.04% after 24 hours; 14.06%, 34.25%, 46.50% after 48 hours; 32.10%, 40.65%, 48.23% after 72 hours. The ratio of inhibition of cells have remarkable duration-dose dependence which is increased by extendding time and raising does, according to concentrations of 1 mmol/L: concentrations of 2.5 mmol/L and 5mmol/L are significant after 24 hours, 48 hours, 72hours(P<0.01); according to concentrations of 2.5 mmol/L: concentrations of 5mmol/L are significant after 24 hours, 72hours(P <0.05) and 48 hours(P <0.01); according to 24 hours: 48 hours are significant at concentrations of 5mmol/L(P <0.01) and all of 72hours are significant(P <0.05); according to 48 hours: concentrations of 2.5 mmol/L is significant after 72 hours (P <0.05), concentrations of 1mmol/L is significant after 72 hours(P <0.01).2. Sodium butyrate(SB) inducing Eca-109 cells apoptosis2.1 After processed by Sodium butyrate (2.5mmol/L) for 48 hours and 72 hours, The DNA agarose electrophoresis showed obvious DNA ladder phenomenon in experimental group, however, there were no the ladder phenomenon in negative control group.2.2 The caspase-8 activities on the Eca-109 cells from 2.5mmol/L Sodium butyrate-induced after 0 hours, 12 hours, 24 hours, 48 hours, 72 hours were examined by the western blotting, the shift was accompanied by changing duration and appear at 12 hours until 48 - 72 hours of exposure. treatment of cells with SB caused a duration-dependent. The result showed that Sodium butyrate-induced apoptotic in Eca-109 cells may via caspase-independent pathway.2.3 We performed western blotting by determining the distribution of cytochrome C and AIF in Cytosolic and mitochondrial before and after Sodium butyrate inducted 2.5mmol/L Eca-109 cells after 48h. The result showed that cytochrome C and AIF in mitochondrial decreased singnificantly, while in cytosol, high levels of cytochrome C and AIF were detected.Conclusion1. Sodium butyrate can remarkably restrain the proliferation of Eca-109 cells, and the effect of anti- proliferation has the relation of duration-dose dependence.2. It is possible that Sodium butyrate restrains Eca-109 cells proliferation by inducing apoptosis, the available evidence indicates two well characterized pathways, caspase-dependent and caspase-independent pathway,indues apoptosis in Eca-109 cells.3. The molecular mechanism of Sodium butyrate inducing apoptosis of Eca-109 may be alerted to the expression of cytochrome C and AIF which are released from the mitochondria to the cytosol.