Abstract:

Objective To investigate the inhibitory effect and killing mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma （NK/TCL）. Methods Human NK cell leukemia cell line YT and human NK/TCL cell line NK92 cells were treated with 0，0.05，0.10，0.20，0.30，0.40，0.50 μmol/L BDA-366. CCK-8 assay was used to calculate the half inhibitory concentration （IC₅₀） value of BDA-366 on these cells. The apoptosis levels of cells in control group and IC₅₀ BDA-366 treated group were detected by flow cytometry. Western blotting was used to detect the expression levels of apoptosis-related proteins in cells of control group and 1/2 IC₅₀，IC₅₀，2× IC₅₀ BDA-366 treated groups. TMRE and Fluo-3 fluorescent probe were used to detect mitochondrial membrane potential of control group and IC₅₀ BDA-366 treated group，and the intracellular Ca²⁺ concentration of control group，IC₅₀，2× IC₅₀ BDA-366 treated groups. NOD-SCID mice in control group and 10 mg/kg BDA-366 intraperitoneal injection group were weighed and HE staining was performed to evaluate the toxicity of BDA-366 in vivo. Results The IC₅₀ of BDA-366 for YT and NK92 cells were 0.065 and 0.086 μmol/L respectively. The apoptosis rates of YT cells in the control group and 0.065 μmol/L BDA-366 group were （6.62±1.59） % and （34.60±3.06） % respectively. The apoptosis rates of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were （5.57±0.88） % and （29.18±0.90） % respectively，both with statistically significant differences （t=14.05，P<0.001； t=32.58，P<0.001）. The relative expression of Bax in NK92 cells of the control group，0.043，0.086 and 0.172 μmol/L BDA-366 groups were 0.85±0.00，1.26±0.04，1.51±0.18，1.15±0.10 （F=20.70，P<0.001），the relative expression of Bax in BDA-366 groups were higher than that in the control group （all P<0.05）. The fluorescence intensity of TMRE of YT cells in the control group and 0.065 μmol/L BDA-366 group were 8 372.00±330.47 and 6 419.67±311.34，and that of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were 9 169.00±535.72 and 7 311.67±295.52 respectively，and there were statistically significant differences （t=7.45，P=0.002； t=5.26，P=0.006）. In YT cells，the intracellular Ca²⁺ concentrations of 0.065 and 0.130 μmol/L BDA-366 groups were significantly higher than that of the control group （5 791.67±220.45，6 729.33±585.39，4 874.67±112.61，F=19.16，P=0.003） （P=0.039； P=0.002）. In NK92 cells，the intracellular Ca²⁺ concentrations of 0.086 and 0.172 μmol/L BDA-366 groups were significantly higher than that of the control group （4 553.67±17.62，4 740.33±254.50，4 185.67±17.67，F=10.96，P=0.010） （P=0.039； P=0.007）. There was no statistically significant difference in body weight change on day 12 compared with day 0 of NOD-SCID mice between BDA-366 group and control group [（3.18±0.01） g vs. （2.73±0.58） g，t=0.60，P=0.570]，and HE staining showed no abnormal morphology of heart，liver，spleen，lung and kidney in BDA-366 group. Conclusion BDA-366 promotes NK/TCL cells apoptosis in vitro，but does not cause weight loss and morphological changes of organs by HE staining in vivo. The inhibitory effect of BDA-366 on NK/TCL cells may be achieved by increasing Bax expression，inducing Ca²⁺ release and reducing mitochondrial membrane potential.

Key words: Proto-oncogene proteins c-bcl-2, Apoptosis