Effect Of Histone Deacetylase Inhibitor SAHA In Combination With ATO On NB4Cells, K562Cells And U266Cells

Objective：To investigate the effect of HDAC inhibitor Vorinostat(SAHA) incombination with ATO on NB4cells, K562cells and U266cells and explore possiblenew options of the treatment of hematological malignancies.Methods：Acute promyelocytic leukemia cell line (NB4cells), chronic myeloidleukemia cell line (K562cells) and multiple myeloma cell line (U266cells) wereemployed in the study.CCK-8assay was used to compare proliferation in differentgroups of three different cell lines.Annexin V and PI staining by flow cytometry andacridine orange and ethidium bromide stains (AO/EB stains) were used to detectapoptosis before and after treatment. Western blot was used to detect P53,P21, BCL-2,Akt, PML-RARa, Acetyl-Histone H3, Acetyl-Histone H4protein.Results：①T he results of CCK-8showed that SAHA and ATO presented significantanti-proliferation effects on both cells in a time-and dose-dependent manne（rp<0.01）.SAHA in combination with ATO treatment showed additive effect on NB4cells andK562cells. SAHA in combination with ATO treatment showed additive or synergisticeffect on U266cells.②A nnexin-V-FITC/PI double stains showed apoptosis ratesincreased in a dose-dependent manner. The NB4cells apoptosis rates were31.25±2.15％,19.25±1.35％,83.05±7.75％, respectively48hours after SAHA(0.5μmol/L),ATO (2μmol/L) and2μmol/L ATO combined with0.5μmol/L SAHA treatment. The K562cells apoptosis rates were10.85±0.65％,29.65±1.75％,84.00±0.80％, respectively48hours after SAHA (2μmol/L), ATO (8μmol/L) and8μmol/L ATO combined with2μmol/L SAHA treatment. The U266cells apoptosisrates were17.90±2.60％,26.45±4.95％,81.60±4.20％,respectively48hours afterSAHA (2μmol/L),ATO (16μmol/L),and16μmol/L ATO combined with2μmol/LSAHA treatment.③S ignificant changes in cell morphology were observed in AO/EB staining under a fluorescence microscope between treatment and control.SAHA-treatment group and ATO-treatment group showed obvious condensationnuclei, nuclear fragmentation, while SAHA in combination with ATO group showedmuch more changes.④On NB4cells, combination group showed lower level ofBCL-2and PML-RARa fusion protein than SAHA or ATO alone; SAHA group andcombination group showed increased level of Acetyl-Histone H3protein,Acetyl-Histone H4protein and Akt protein while ATO alone group showedincreased level of P53protein; SAHA alone treatment showed increased level ofp21WAF1/CIP1epression,while ATO alone and combination group showed nosignificant changes. On K562cells, both SAHA or ATO alone and combination groupshowed lower level of Bcr/abl fusion protein than control; SAHA group andcombination group showed increased level of Acetyl-Histone H3protein,Acetyl-Histone H4protein expression; SAHA alone showed increased level ofp21WAF1/CIP1epression, while ATO alone and combination group showed no significantchanges; Combination group showed lower level of Akt protein expression thanSAHA or ATO alone. On U266cells, SAHA group and combination group showedincreased level of Acetyl-Histone H3protein, Acetyl-Histone H4protein expression;Combination group showed increased level of P53protein while SAHA or ATO aloneand combination group showed no changes in the level of Akt protein.Conclusion：1. both SAHA and ATO inhibit proliferation and induce apoptosis onNB4cells, K562cells and U266cells in a time-and dose-dependent manner. SAHA incombination with ATO treatment showed additive or synergistic effect on NB4cells,K562cells and U266cells.2.Akt pathway possibly involved in the apoptosis process of NB4cells and K562cells induced by SAHA and ATO.