ABSTRACT
Objective To evaluate the effect of chidamide combined with matrine on proliferation and apoptosis of cutaneous T-cell lymphoma (CTCL) cell lines HH and Hut78,and to explore their apoptotic mechanisms.Methods Both HH and Hut78 cells were treated with 0.4 μmol/L chidamide and 0.6 g/L matrine alone or in combination for 24,48 and 72 hours,with those treated with dimethyl sulfoxide (DMSO) serving as control groups.MTS assay was performed to deteet cellular proliferation rates of HH and Hut78 cells at each time point.After 48-hour treatment,flow cytometry was conducted to detect cell apoptosis,and Western blot analysis to determine expression of apoptosis-related proteins in these cells.Statistical analysis was carried out by using repeated measures analysis of variance,one-way analysis of variance,and least significant difference (LSD)-t test for multiple comparisons.Results Compared with DMSO,chidamide and matrine alone or in combination could inhibit the proliferation of HH and Hut78cells to different extents (F =15.88,558.26,P ＜ 0.05,＜ 0.001,respectively).At 48 hours,the apoptosis rate in Hut78 cells was significantly higher in the matrine group (20.98％ ± 1.53％),chidamide group (22.44％ ± 7.74％) and combination group (44.53％ ± 1.85％) than in the control group (8.42％ ± 4.23％;LSD-t =4.76,5.31,13.69 respectively,all P ＜ 0.05),as well as in the combination group than in the matrine group and chidamide group (LSD-t =8.93,8.37 respectively,both P ＜ 0.01);no significant differences were observed in the apoptosis rate of HH cells between the matrine group (13.98％ ± 3.86％)or chidamide group (13.61％ ± 1.62％) and control group (11.44％ ± 1.43％,both P ＞ 0.05),while the combination group (20.94％ ± 0.64％) showed a significantly higher apoptosis rate compared with the control group,matrine group and chidamide group (LSD-t =7.37,5.40,5.69 respectively,all P ＜ 0.05).In the case of HH cells,the combination group showed significantly higher cleaved caspase-3 expression (all P ＜ 0.05),but significantly lower protein expression of E-cadherin,nuclear factor (NF)-κB,phosphorylated-Bad (p-Bad) and Bcl-2 compared with the other 3 groups (all P ＜ 0.05).In the case of Hut78 cells,the expression of E-cadherin,NF-κB,p-Bad and Bcl-2 was significantly lower in the matrine group,chidamide group and combination group than in the control group (all P ＜ 0.05),while cleaved caspase-3 expression was significantly higher in the chidamide group and combination group than in the control group (both P ＜0.05).No matter in HH cells or in Hut78 cells,there were no significant differences in Bad protein expression between the 4 groups (all P ＞ 0.05).Conclusion Chidamide in combination with matrine can inhibit proliferation and induce apoptosis of HH and Hut78 cells,likely by regulating the expression of apoptosis-related proteins E-cadherin,NF-κB,p-Bad,Bcl-2 and cleaved caspase-3.
ABSTRACT
Objective To evaluate effects of acitretin and interferon?α(INF?α)alone or in combination on the proliferative activity of and interleukin?15 expression in human cutaneous T?cell lymphoma Hut78 cells. Methods Cultured Hut78 cells were divided into several groups, including blank control group, negative control group, dimethyl sulphoxide (DMSO) group and experimental groups. Cells in experimental groups were additionally classified into several subgroups to be treated with acitretin(0.1-10μmol/L, acitretin groups)or INF?α(5 000-20 000 IU/ml, INF?αgroups) alone, or the combination of 1.0 μmol/L acitretin and IFN?α at concentrations of 5 000- 20 000 IU/ml (combination groups), for 24, 48 and 72 hours. Subsequently, cell counting kit 8(CCK8)assay was performed to assess the proliferative activity of Hut78 cells, and enzyme?linked immunosorbent assay(ELISA)to measure the expression of IL?15 in these cells. Results The proliferative activity of and IL?15 expression in Hut78 cells were both obviously suppressed in the acitretin groups and combination groups compared with the DMSO group, as well as in the INF?αgroups compared with the negative control group, and the inhibitory effects gradually increased with the increase in acitretin or INF?αconcentrations and treatment durations. As repeated measures analysis of variance revealed, there was a significant difference in both proliferation inhibition rates and IL?15 expression among different treatment durations and among different concentrations of acitretin or INF?α(all P<0.05), and there was an interaction effect between treatment durations and drug concentrations(all P<0.05). A significant difference was observed in both proliferation inhibition rates and IL?15 expression at 24, 48 and 72 hours when the 1.0?μmol/L acitretin+ 10 000/20 000?IU/ml IFN?αgroup was compared with the 1.0?μmol/L acitretin group and 10 000/20 000 IU/ml IFN?αgroup(all P<0.05). There was also a significant difference in IL?15 expression at 24, 48 and 72 hours between the 1.0?μmol/L acitretin+50 000?IU/ml IFN?αgroup and 5 000?IU/ml IFN?αgroup(all P<0.05). Conclusions Acitretin and IFN?αboth can inhibit the proliferation of and IL?15 expression in Hut78 cells, the inhibitory effects are enhanced with the increase in drug concentrations and treatment durations, and the combination of acitretin and IFN?α appears to have stronger inhibitory effects than acitretin or IFN?αalone.