A Screen Of Novel Apoptosis-inducers And Their Apoptosis Induction Mechanisms In Tumor Cells

Thirty-two 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile derivatives were designed and synthesized by State Key Laboratory of Fine Chemicals, Dalian University of Technology and we screened their apoptosis inducing abilities based on detection of apoptosis in cells by flow cytometer. Forteen derivatives had abilities of inducing apoptosis in MCF-7 cells. After MCF-7 cells were treated with 20μM S1 for 12h, the apoptosis ratio was 29.7%, MCF-7 cells were treated with 20μM E-10 for 72h, the apoptosis ratio was 29.3% , MCF-7 cells were treated with 20μM E-13 for 72h, the apoptosis ratio was 35.1%. The IC50 for S1 was 17μM. It was least than the IC50 for E-10 and E-13. S1 was applied a patent for an invention(2004100504495). So we studied the mechanism of S1 induced apoptosis in MCF-7 cells.After MCF-7 cells were treated with S1 for 12h, the morphological changes of cells were observed under the microphotography, apoptosis in MCF-7 cells were detected by flow cytometer, mitochondrial membrane depolarization was assayed by Rh123, Caspase activity were determined by absorption spectroscopy, Bcl-2 protein expression was determined by flow cytometer and Western Blotting, Bcl-2 gene were quantitative mensured by FQ-PCR. Results showed that caspase-9 and caspase-3 was activated, By contrast, not any change was found for caspase 8, neither was Bcl-2 mRNA. Bcl-2 protein expression and mitochondrial membrane potential decreased. S1/Bcl-2 binding were assayed by CD spectra and the docking simulations of S1 and Bcl-2 was analyzed by Insight II, results showed that S1 docked into the surface pocket of Bcl-2 and destroyed the a-helix of Bcl-2. The mechanism of S1 induced apoptosis was revealed that Bcl-2 protein was the first effector with which S1 interacts directly, S1 decreased Bcl-2 protein then caspase-9, caspase-3 were activtied then triggered the cascade of apoptosis in MCF-7 cells.This study screened a novel small apoptosis inducer. As an organic Bcl-2 protein inhibitor, S1 provides a new means for the study on the relationship of Bcl-2 protein family.