LBH589Enhances TRAIL-induced Apoptosis And Its Mechanisms In Kasumi-1Cells In Vitro

Background:Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to TNF superfamily, which can activate apoptosis signal transduction pathways specifically by binding with its death receptors DR4and DR5in diverse tumor cells and transformed cell including human leukemia cells, but it can spare against normal host cells. However, the resistance of tumor cells to TRAIL seriously limits its clinical application. So, the efforts to explore new agents to enhance TRAIL-induced apoptosis would increase the potential clinical utility for leukemic therapy.The acetylation and deacetylation of histones play an important role in regulation of gene expression, and the activity of histone deacetylases (HDACs) and histone acetylases (HATs) is related to tumorigenesis. Histone deacetylase inhibitors (HDACIs) can restrain HDACs and facilitate acetylation of histone to resist proliferation, promote differentiation and induce apoptosis through the regulation of chromatin remodeling and gene transcription. Researches show that the synergistic effects of anti-tumor of the combination of HDACIs and various agents make it become one of the new attracting drugs to the therapy of acute myeloid leukemia(AML), acute lymphocytic leukemia(ALL), and myelodysplastic syndrome(MDS). Panobinostat (LBH589) is a kind of micromolecule HDACIs, which belongs to hydroxamic acid, can block a variety of pathological pathways associated with tumor and tumorigenesis to lower the survival and induce apoptosis of cancer cells. It is reported that LBH589can improve histone H3and H4acetylation level and non-histone acetylase level and it can attenuate phosphorylation of signal protein and induce apoptosis in a dose-dependent manner in leukemia cells.Acute myeloid leukemia (AML) is a kind of commom disease and brings great harm to social. AML with t (8;21) which approximately accounted for10%-15%of acute myeloid leukemia is generally considered as a good prognosis subtype, but recurrent and resistence to drugs are still a tough problem in clinical. And due to its young onset age, the harm is even more remarkable. Kasumi-1cell line which was cultured from a patient with t (8;21) positive acute myeloid leukemia is a good model for us to research this subtype leukemia. So, it is important to explore new reagents and methods to conquer this problem. The effects of TRAIL, LBH589and their combination in Kasumi-1cells in vitro were observed, and the possible mechanisms were investigated in the study.Part I LBH589enhanced rsTRAIL induced apoptosis in Kasumi-1cells in vitroAIMS:To investigate the effects of apoptosis of rsTRAIL alone, LBH589alone and their combination in Kasumi-1cells in vitro. METHODS:Kasumi-1cells were treated with rsTRAIL alone, LBH589alone and their combination at variant concentrations. The changes of morphology of Kasumi-1cells were observed under microscope after Wrighting staining, the proliferation of cells was measured by CCK-8assay and the apoptosis was analyzed independently through Annexin V/PI double staining by flow cytometry. RESULTS:Both rsTRAIL and LBH589could induce the apoptosis of Kasumi-1cells, and rsTRAIL-induced apopotosis of Kasumi-1cells could be enhanced by LBH589, which was a time-and dose-dependent manner(P<0.05). The ratios of annexin V positive Kasumi-1cells were (26.03±2.48)%,(24.99±4.11)%and (66.01±7.15)%in lOng/mL rsTRAIL group,50nM LBH589group and the combination of the two agents group respectively compared with the control group (7.34±7.15)%, and the combination group was significantly higher than the group of rsTRAIL or LBH589alone (P<0.001). CONCLUSIONS:LBH589can enhance TRAIL induced apoptosis of Kasumi-1cells in vitro.Part II Mechanisms of the enhancement effects of LBH589in the apoptosis of Kasumi-1cells induced by rsTRAIL in vitroAIMS:To investigate the mechenisms of rsTRAIL alone, LBH589alone and their combination in Kasumi-1cells in vitro. METHODS:The expression of DR4, DR5, DcRl and DcR2at mRNA level was detected by real-time PCR The expression of signal transduction proteins, such as DR4, caspase-3, caspasep-9, Bid, Bax and Bcl-2was detected by Western blotting. RESULTS:LBH589can enhance rsTRAIL induced apoptosis of Kasumi-1cells and the mechanisms include the upregulation of DR4, activation of caspase-3, caspase-9and the degradation of Bcl-2.