METHODS: Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
bromide assay. Cellular morphological changes (apoptosis and
necrosis) were evaluated using an electron microscope and
Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential.
Protein expression was determined by Western blotting analysis.
RESULTS: Here, we describe the ability of iso-
suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-
suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations (9.09, 18.17, or 36.35 μmol/L) but promoted lymphocyte proliferation at a high concentration (72.70 μmol/L).
After treatment of different concentrations of iso-
suillin (6.82, 13.63, or 20.45 μmol/L), the apoptosis rate of H446 cells increased with increasing concentrations of iso-
suillin (16.70%, 35.54%, and 49.20%, respectively, all P < 0.05 compared with the control), and the expression of related apoptotic
proteins in the mitochondrial pathway including
cytochrome c and
caspase-9 were up-regulated compared with the control (all P < 0.05). On the contrary, Bcl-2/Bax ratio was down-regulated compared with the control. Besides, the expression of
pro-apoptotic proteins in the
death receptor apoptosis pathway, including Fas-associating
protein with a novel death domain and
caspase-8, and the expression of
caspase-3, a downstream regulatory
protein of apoptosis, were also increased compared with the control (all P < 0.05). Inhibitors of
caspase-9 and
caspase-8 reversed the apoptosis process in H446 cells to varying degrees.
CONCLUSIONS: These results suggest that iso-
suillin could induce H446 cell apoptosis through the mitochondrial pathway and the
death-receptor pathway. Therefore, iso-
suillin might have a potential application as a novel
drug for
lung cancer treatment.