The Effects Of Histone Deacetylase Inhibitor SAHA On Growth And Apoptosis In Jurkat T Cells And Mouse Lymphocytes

Aim：1. To analyze the effects of SAHA on the proliferation, cell cycle distribution,apoptosis and autophagy of Jurkat cells in order to elucidate its anti-canceractivity and the underlying mechanism.2. To investigate the effects of SAHA on the activation, proliferation, cytokinessecretion and apoptosis of mouse lymphocytes in order to elucidate itsanti-inflammatory activity and the underlying mechanism.Methods：1. Jurkat T cells were treated with various concentrations of SAHA. Theproliferation was measured by MTS assay. Cell cycle distribution was measuredby PI staining. Cell apoptosis was analyzed by AnnexinV-PE staining.Mitochondrial membrane potential was detected by JC-1staining. The expressionsof apoptosis-related proteins and acetylated histone were detected by westernblotting.2. Lymphocytes were isolated from lymph nodes of BALB/c mouse, andco-incubated with SAHA and polyclonal activators Con A or PDB plus ionomycin.The proliferation was analyzed by MTS assay. Activation and cytokines secretionwere evaluated with Immunofluorescence staining and flow cytometry. Cellapoptosis was analyzed by AnnexinV-PE staining. Mitochondrial membranepotential was detected by JC-1staining. The expressions of apoptosis-relatedproteins and acetylated histone were detected by western blotting.Results：1. SAHA inhibited the proliferation of Jurkat cells in a dose-dependent manner. PIstaining results shown that SAHA treated for24h arrested the Jurkat T cells at G₂/M phase (P <0.01). SAHA also induced apoptotic cell death as indicated byDNA fragmentation (sub-G0/G1peaks), loss of cell membrane integrity (annexinV staining), and decrease of mitochondrial membrane potential (JC-1staining)(P<0.01). Moreover, the results also demonstrated that SAHA induced theactivation of Caspase-3and poly (ADP-Ribose) polymerase cleavage. Autophagywas induced by SAHA treatment, since blockade of the autophagic flux bychloroquine (CQ) in SAHA-treated cells increased the level of LC3-II (P <0.01).However, blocking of autophagy by CQ did not significantly increase the ratios ofapoptotic cell death and expressions of Cleaved caspase-3and PARP. In addition,SAHA promoted the expressions of acetylated H3and phosphorylated H2A.X in adose-dependent manner (P <0.01).2. Our results demonstrated that SAHA inhibited the proliferation of ConA-activated lymphocytes in a dose-dependent manner (P <0.01). SAHA couldsignificantly downregulate the expression of early activation marker CD69andpro-inflammatory cytokines TNF-α, IL-6and IFN-γ in T lymphocytes (P <0.01).Furthermore, analysis of sub-G0/G1peaks and annexin V binding populationsrevealed that SAHA induced apoptotic cell death in a time-and dose-dependentmanner in Con A-activated lymphocytes (P <0.01). Consistent with these results,SAHA treatment also induced a decrease of mitochondrial membrane potentialand cleavage of caspase-3and PARP in these cells (P <0.01). Moreover, SAHAcaused an accumulation of phosphorylated histone H2A.X and acetylated H3.Conclusion：1. The effect that SAHA can markedly inhibit the proliferation of Jurkat T cells, mayresult from the cell cycle arrest and apoptosis induced by SAHA. SAHA-inducedapoptosis may be mitochondrial-and Caspase-3-dependent. SAHA also inducesautophagy of Jurkat T cells which has little effect on SAHA-induced apoptosis.2. Our results demonstrate that SAHA suppresses the proliferation, activation andpro-inflammatory cytokines secretion of lymphocytes in the dose-dependentmanner, and induces mitochondrial-and Caspase-3-dependent apoptosis of lymphocytes.