Study On The Mechanism Of Apoptosis Through Activation Of The Death Receptor Pathway By HDAC Inhibitor In Leukemia Cells

Recently, attention has focused on the HDACI (histone deacetylase inhibitor) as a new anticancer agents for the less toxic and more effective treatment. HDACI might develop as a wide promising anti-cancer new drug. It has been reported that sodium butyrate (SB) could suppress the growth of tumor cells, induce cellular differentiation and apoptosis. It is a kind of regulator of genetic expression. We had done some research about HDACI. Such as SB can improve the expressoin of costimulatory molecules CD80, CD86 in acute leukemia cells. And We had proved that NFκB was an important transcription factor involved in the up-regulatoin of CD80 and CD86. The acetylation degree was accumulated in SB treated NB4, HL60 and U937 cells. SB made the cells chromatin remodeling. And it made transcriptional factor CREB activate and be combined with DNA. This improve the transcription of CD86 and made it express more than untreated cells. And we researched the proteomics of apoptotic NB4 cells induced by SB. It proved that SB can induce apoptosis and many functional protein changes in tumor cells. These results pave the way to further explore the anti—tumor mechanism of SB. This experiment is based on the previous results. We study on the mechanism of apoptosis induced by SB in acute leukemia cells NB4, U937 and Jurkat. It has been found recently that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is a member of TNF ligand super family, and death receptor 4 (DR4) and DR5 are the receptors of TRAIL. TRAIL binds two receptors, that could induce tumor cell apotosis through activation of the death receptor pathway. In the study,we have evaluated the effect of the leukemic cells induced by HDACI. And we will discussed the mechanism of the apoptosis. Does it activate the death receptor pathway? This will provide experimental evidence for clinical application.We selected 3 cell lines as the study objects which were NB4, U937 and Jurkat. The cells were treated with sodium butyrate (SB) at 1.0mM concentrations at 24h, 48h and 72h. We detected the change of apoptosis at 24h, 48h and 72h by Annexin V-FITC apoptosis detection kit. The results showed that SB could induce apoptosis of 3 leukemic cell lines. And it was time dependent. Then we detected the expression change of TRAIL and it receptor DR4/DR5 protein by westernblot at 24h 48h and 72h. Then we found that the TRAIL and DR5 protein were both increased. But DR4 was the same as untreated by SB. Then reverse transcriptase (RT)-PCR was used to examine the gene transcription of TRAIL and DR4/DR5 treated by SB at 24h 48h and 72h. The results showed that the TRAIL and DR5 mRNA were accumulated in all the cells. The expression of DR4 mRNA was not changed .These results verified that: 1,HDACI SB can induce apoptosis of the acute leukemic cells NB4, U937 and Jurkat. The apoptosis induced by SB expressed time dependent. And the apoptotic effect induced by SB was most obvious in the NB4 cell line at 72h. It is 68.5%. 2,SB up-regulated the expression of TRAIL and DR5 protein is an important mechanism of leukemic cells apoptosis. 3,DR4 may not participate in the apoptosis induced by SB in the acute leukemia cell NB4, U937 and Jurkat. 4,SB can promote the transcription of TRAIL and DR5, indicating that the up-regulation of TRAIL and DR5 happened at the transcriptional level, not at post-transcriptional level. We presumed SB raised the acetylation degree of core histones. That may relax the chromatin which will make it accessible for transcriptional factors and promote the transcription of TRAIL and DR5.This study showed that SB can induce apoptosis of the leukemic cells NB4, U937 and Jurkat. And we discussed the apoptosis mechanism on the cellular and molecular level. We confirmed that SB up-regulated the TRAIL and DR5 protein expression in acute leukemia cells, NB4, U937 and Jurkat,and proved the conclusion on the transcriptional level. The up-regulation of TRAIL and DR5 activated death receptor pathway. This is an important mechanism of inducing apoptosis. It is provided theory support and experimental evidence. The study on the apoptotic pathway and the regulation of apoptosis induced by HDACI will be our new research direction. Study on the anti-tumor effect of HDACI will contribute not only to explaining the pathogenesis of tumor, but also provided new clinical therapeutic regimen .