| Objective: The aim of this study is to estimate the impacts of a novel chrysin derivative, 8-bromo-7-methoxychrysin(BrMC),upon the cell growth and apoptosis of human small cell lung cancer NCI-H446 cell line in vitro, and provide a laboratory and theoretic evidences to search for a new potent and promising new chemical entity for lung cancer chemotherapy.Methods: Human small cell lung cancer NCI-H446 cell line and Chinese Hamsters pulmonary epithelial CHL cell line were cultured in vitro. The cell viability inhibitory effect and the selection index of BrMC on NCI-H446 cells and CHL cells were measured by MTT assay. Plate colony-formation method was used to test inhibitory effect of BrMC on colony formation of NCI-H446 cells. BrMC-induced apoptosis rate and cell cycle of NCI-H446 cells were observed by Flow cytomtry(FCM) with PI staining. DNA ladder bands were observed by DNA agarosegel electrophoresis. The influence of BrMC on protein expression level and activity of Noxa, Mcl-1and Caspase-3 were analyzed by indirect immunofluorescence technique using flow cytometry.Results: (1) MTT assays showed that BrMC inhibited the cell viability of NCI-H446 cells in a concentration-dependent,the relative inhibitory rate of cell viability was 13.87%,22.14%,39.05%,51.97%,61.14% and 48.54% respectively,after NCI-H446 cells treated with various concentration at 0.3,1.0,3.0,10.0,30.0μM of BrMC and Chrysin at 30.0μM for 48h. The inhibition rate (61.14%) of BrMC at 30.0μM concentration is higher than that (48.54%) of the lead compound Chrysin at corresponding concentration. While there was no significant effect in CHL cells.The selective index to NCI-H446 cells was 46.19 (455.94/9.87). (2) Plate colony formation assay showed that the inhibitory rate of colony formation was 9.9%,25.34%,39.63%,47.92%,61.4% and 39.86% after NCI-H446 cells treated with various concentration at 0.3,1.0,3.0,10.0,30.0μM of BrMC and Chrysin at 30.0μM for 8d respectively, the colony inhibitory rate (61.4%) of BrMC at 30.0μM concentration is higher than that(39.86%) of the lead compound Chrysin at corresponding concentration. The BrMC caused colony formation inhibition of NCI-H446 cells in a dose-dependent manner. (3) FCM analysis showed that the apoptosis rate of NCI-H446 cells treated with 0.3, 3.0 and 30.0μmol/L BrMChR for 48h were 8.83%,23.7%,34.9% respectively and significantly higher when treated with 3.0 and 30.0μmol/L BrMC than that with 30.0μmol/L ChR (19.7%),and the cell cycle was arrested at G1 phase。(4) Indirect immunofluorescence flow cytometry demonstrated that the mean flourscence indensity (MFI) of Noxa was (1.18±0.15)%,(1.64±0.19)%,(1.95±0.26)% respectively, MFI of Mcl-1 was (0.22±0.05)%,(0.16±0.05)% , (0.15±0.09)% respectively , the ratio of Noxa to Mcl-1 was (5.6±2.2)%,(12.2±3.7)%,(19.5±4.5)% respectively, and the MFI of Caspase-3 was (1.78±0.1)%,(2.61±0.2)%,(3.89±0.7)% respectively after exposure to BrMC at 0.3,3.0,30.0μmol/L for 48h, indicating expression of Noxa ,Caspase-3 and the ratio of Noxa to Mcl-1 increased, while Mcl-1 down-regulated.Conclusion:1. BrMC possess significant inhibitory effect on the cell growth of human small cell lung cancer NCI-H446 cell line in vitro, in dose-dependent manner.2. BrMC can significantly induce apoptosis of human small cell lung cancer NCI-H446 cell line.3. BrMC induces apoptosis of NCI-H446 cells, which seems to be associated with increasing the ratio of Noxa/Mcl-1 protein and activating of Caspase-3.
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