Effects Of Diallyl Disulfide On The Growth Inhibition And Apoptosis In Human Small Cell Lung Cancer Cell Line H446 In Vitro

Objective: To investigate the effects of different concentration of diallyl disulfide (DADS) on human small cell lung cancer H446 cells in vitro in order to provide experimental and theoretical basis for new medicine exploitment.Methods:H446 cell line was cultured in vitro.Cell proliferation inhibition was measured by growth curve analysis, average doubling time, vitality detection and MTT assay. Cell morphologic changes were observed by inversion microscope, optics microscope (HE staining) and fluorescence microscope (AO-EB staining and Hoechst 33258 staining). The apoptosis rate and phase distribution of cell cycle of H446 cell line induced by DADS was analyzed using flow cytometry (FCM) with PI staining. The change of Intracellular free calcium concentration was measured by laser scanning confocal microscope (LSCM) and Immunocytochemical staining detected the expression of PCNA and Bcl-2.Results:MTT assay showed that DADS from 4 to 60μg/ml significantly inhibited H446 cells and exhibited a dose-dependent and time-dependent model. After exposure to DADS,H446 cell average doubling time retarded from 25.40 hours in normal cultured cells to DADS experimented H446 cells 145.64 hours(P<0.05).Under inverted microscope, optics microscope and fluorescence microscope, H446 cellular heteromorphism diminished,nucleocytoplasmic proportion was reliable to reasonableness, cellular apparatus was abundant in plasm,nuclear and partial cell organs manifest retrograde alters and partial cells manifest apoptosis morphologic alters. All these showed above suggest that H446 cells malignancy and proliferation capacity is declined because of DADS treatment.Flow cytometry analysis revealed that the cell content of Go-phase declined ,however, G2-phase increased after exposure to DADS, which indicated that H446 cells arrested in G2-phase(P<0.05);Hhyodiplod peak was higher, which means cells apoptosis induced by DADS.CLSM revealed that intracellular Ca2+concentration in DADS groups was significantly higher than the control group.Immunocytochmical stain and morphometric quantitative analysis indicated that the expression of Bcl-2 and PCNA reduced (P<0.05).Conclusion:1. DADS could significantly inhibit the proliferation of human small cell lung cancer cell line H446 and this effect presents a scene of dose-dependent and time-dependent.2. DADS can induce apoptosis and G2/M-phase arrest in human H446 cells and its mechanism probably relate to down regulated expression of Bcl-2 and PCNA.3.The inducing apoptosis effect of DADS on H446 cells could be through the sequential Mechanism of Ca2+homeostasis disruption.