ABSTRACT
Objective To evaluate the inhibitory effect of chidamide combined with curcumin on the proliferation of cutaneous T-cell lymphoma (CTCL) cell line Hut78,as well as their promotive effect on its apoptosis,and to explore their therapeutic mechanisms in CTCL.Methods Some Hut78 cells were treated with different concentrations of chidamide (0.3,0.6,1.2,2.4 μmol/L) and 10 μ mol/L curcumin alone or the combination of 1.2 μ mol/L chidamide and 10 μ mol/L curcumin for 24,48 and 72 hours separately.MTS assay was conducted to estimate cell viability at each time point.After selection of chidamide concentrations,some Hut78 cells were treated with chidamide (0.6 and 1.2 μmol/L)and curcumin (10 μmol/L) alone or in combination (1.2 μmol/L chidamide and 10 μmol/L curcumin) for 24 hours,then,flow cytometry was performed to detect cell apoptosis and analyze cell cycle,real-time (RT)-PCR and Western-blot analysis were conducted to quantify the mRNA and protein expressions of apoptosis-associated genes Fas,caspase 8,nuclear factor (NF)-κB p65 as well as cell cycle-associated genes P21,CDK2 and cyclin E respectively.Statistical analysis was carried out by repeated-measures analysis of variance,one-way analysis of variance and the least significant difference (LSD)-t test.Results Chidamide could significantly inhibit the proliferation of Hut78 cells in a dosedependent and time-dependent manner (F =266.558,564.966,respectively,both P ＜ 0.001).After 48-and 72-hour culture,the combination of 1.2 μmol/L chidamide and 10 μmol/L curcumin showed significantly stronger inhibitory effect on cell proliferation compared with 1.2 μmol/L chidamide or 10 μmol/L curcumin alone (all P ＜ 0.001).As flow cytometry showed,the percentage of apoptotic cells was significantly higher in the combined treatment group than in the 0.6-,1.2-μmol/L chidamide groups and 10-μmol/L curcumin group (all P ＜ 0.001).Compared with the 0.6-μmol/L chidamide group and 10-μmol/L curcumin group,the combined treatment group and 1.2-μmol/L ehidamide group both showed significantly increased proportion of cells at G0/G1 phase and mRNA expressions of Fas,caspase 8 and P21,but decreased proportion of cells at S phase or G2/M phase and mRNA expressions of NF-κB p65,CDK2 and cyclin E (P ＜0.05 for proportion of cells at different phases,P ＜ 0.001 for mRNA expressions of different genes).Furthermore,the mRNA expression of Fas was significantly higher in the combined treatment group than in the 1.2-μmol/L chidamide group (P ＜ 0.001),while no significant differences were observed in the mRNA expressions of caspase 8,P21,NF-κB p65,CDK2 and cyclin E or the proportion of cells at any phase between the combined treatment group and 1.2-μmol/L chidamide group (all P ＞ 0.05).Western-blot analysis showed that protein expressions of Fas,caspase 8 and P21 significantly increased,but those of NF-κB p65,CDK2 and cyclin E significantly decreased in the combined treatment group compared with the 0.6-,1.2-μmol/L chidamide groups and blank control group receiving no treatment,which were in accordance with the above changes in mRNA expressions of these genes.Conclusion Chidamide can inhibit the growth of the CTCL cell line Hut78 by directly decelerating cell proliferation and inducing cell apoptosis,and the combibation with curcumin can markedly enhance the inhibitory effect of chidamide on the growth of Hut78 cells.
ABSTRACT
To investigate the melatonin receptor(MR) gene and protein expression in human HUT78 cell line, total RNA of HUT78 cells was isolated by single-step method of acid guanidinium-thiocyanate-phenol-chloform, and mt 1 and MT 2 mRNA were determined by reverse transcription-polymerase chain reaction(RT-PCR). The Envision method was applied to immunohistochemistry to identify and localize mt 1 and MT 2 protein. The mt 1 cDNA fragment of the expected size of 370bp was determined, but the MT 2 cDNA fragment of the expected size of 320bp was not determinable by RT-PCR. Sequencing result indicated that the positive product coincided with the cDNA of human mt 1. The mt 1 protein was observed by immunohistochemistry. These buffy positive granules were scattered with some areas stronger than the others, and were primarily located in cytoplasm and membrane, with rare location in nucleus; meanwhile the MT 2 protein was not observed. These results demonstrated the mt 1 mRNA and protein expression in HUT78 cell line. It is indicated that melatonin has directly immune-regulative effects on T lymphocyte, The changes of MR in physiological and pathological stages need to be investigated.