Study On The Differentiation And Apoptosis Of Leukemia Cells Induced By Tributyrin And Its Mechanism

Objective: To study whether tributyrin (TB) can inhibit the proliferation and induce differentiation or apoptosis in human acute myeloid leukemia cell lines NB4, K562 and SHI-1 as well as primarily cultured cells from patients with leukemia. Elucidate the mechanism in the effects treated by TB in leukemia cells.Method: K562, NB4, SHI-1 cell lines and primarily cultured cells were used for in vitro culture study. Cell proliferation and viability was analyzed by trypan blue exclusion assay and MTT assay. Cell morphological analysis, NBT reduction, benzidine staining, the expression of CD11b, CD14, and CD71 , cell cycle analysis and binding of AnnexinV tested by flow cytometry, DNA electrophoresis were performed to evaluate whether TB could induce the differentiation and apoptosis in leukemia cells. The level of acetylated histone H3 was detected by Western blotting. p21WAFI/CIP1 expression was detected by semi-quantitative RT-PCR.Results: B inhibited K562, NB4, SHI-1 cells proliferation and viability in a time-dose dependent manner. Treated with TB 0.5mM for 72hs the viability of leukemia primarily cultured cells was lower than normal bone marrow cells apparently (p<0.01). Treated with TB for 16hs, K562, NB4, SHI-1 cells demonstrated accumulation of cells in Gl and/or G2 phase and a decrease of cells in S phase. The response was different between different doses and cell lines. 〦xposure to TB for 48hs with the dose of 0.5-1.0mM separately, the three cell lines and primarily cultured cells exhibited the morphological hallmarks of apoptosis. DNA ladder by gel electrophoresis, upregulation of apoptosis rate (marked by AnnexinV) were also observed. (3)The combined RA 0.05uM/TB 0.1 mM treatment induced partial differentiation observed by cell morphological analysis and NBT reduction in NB4 cells. Exposure of TB0.1mM can induce SHI-1 cells diffentiation partially. The NBT positivity was higher than control group. The expression of CD11b and CD14 are upregulated. TB (used only or in combination with ATRA) was unable to induce the differentiation in K562 cells. Treatment with ATRA/TB also induced differentiation of cells from a AML-M1 patient. (4)Histone H3 hyper-acetylation was detected in K562, NB4, SHI-1 cells treated with TB. The level of acetylated histone H3 increased 2hs after TB treatment and maintained through 24hs. (5) p21WAF1/CIP1 was increased in RNA level after 16hs treated by TB in all three cell types.Conclusion: (1)TB inhibits the proliferation and viability in K562, NB4, SHI-1 cell lines. Treatment with TB causes cells to arrest in Gl and /or G2 phase. (2)TB inhibits the proliferation of leukemia primarily cultured cells, while no effect seen on the proliferation of normal bone marrow cells. (3)TB used solely or combined with ATRA can induce differentiation and/or apoptosis in K562, NB4, SHI-1 cells and primarily cultured cells from patients with leukemia. The response was different between different doses and cell lines. (4)The effects of TB inhibiting proliferation and inducing differentiation and/or apoptosis in leukemia cells may associate with its up-regulation of acetylated histone and p21WAF1 mRNA level.