The Mechanism Of Apoptosis Induced By Lapatinib In Her2 Positive Breast Cancer Cells And Synergistic Effective Between ABT-737 And Lapatinib

Objective:(1) To explore the role of BH3-Only family proteins in the apoptosis induced by Lapatinib in Her2 positive breast cancer cells BT-474 and SK-BR3 cells and whether regulated by FOXO3a protein. (2) To study the synergy effect between BH3 mimitic compound, ABT-737 and Lapatinib in Her2 positive breast cancer cell SK-BR3 and explore the mechanism of the synergy effect.Methods:(1) The extent of apoptosis was evaluated by flow cytometric analysis utilizing annexin V-fluorescein isothiocyanate staining method. The Her2 pathway protein, BH3-Only family protein and protein FOXO3a were measured by Real-time PCR and Western blot methods. RNA interference method inhibiting the express of Bim and FOXO3a protein to ascertain the role of them in the apoptosis induced by lapatinib in Her2 positive breast cancer cells BT-474 and SK-BR3 and whether Bim expression regulated by FOXO3a protein. (2). Utilize CCK8 method to study the toxic effect of BH3 mimitic compound, ABT-737 and Lapatinib in the Her2 positive breast cancer cell SK-BR3. According median-effect principal (Chou-Talalay combination index method), utilize the Calcusyn software analysis the combination index (CI) to ascertain the synergistic effective between them in SK-BR3 cell. The difference of BCL-2 family proteins induced by Lapatinib alone or combined with ABT-737 in SK-BR3 cell was detected by Western blot method. The association of BCL-2 and Bim protein was detected by Coimmunoprecipitation method.Result:(1). The apoptosis induced by Lapatinib in Her2 positive breast cancer cells BT-474 and SK-BR3 cells accompanied by a pronounced increase of Caspase 3 cleavage and the conformational change of BAX. The expression of one of the BH3-only family protein Bim and FOXO3a increase after treatment of Lapatinib. RNA interference of Bim and FOXO3a inhibit the apoptosis induced by Lapatinib in SK-BR3 cell, and the increase of Bim protein induced by Lapatinib was inhibited by RNA interference of FOXO3a. (2) The BH3-mimitci compound ABT-737 synergizes the antitumor activity of Lapatinib in SK-BR3 cell. With the increase of Bim protein induced by Lapatinib the expression of MCL-1 protein was decreased. Lapatinib-induced Bim is primarily sequestered by Bcl-2 rather than Mcl-1 and Bcl-xL; these associations are disrupted by ABT-737.Conclusion:(1) Lapatinib induced apoptosis require Bim through mitochondrial pathway and regulated by FOXO3a in Her2 positive breast cancer cells. The AKT-FOXO3a-Bim axis may be the important role in lapatinib induced apoptosis. (2) The BH3 mimetic compound ABT-737 synergize the antitumor activity of Lapatinib for Her2 positive breast cancer cell SK-BR3. The mechanism may be the Lapatinib induced decrease of MCL-1 protein enhancing the activity of ABT-737, on the other hand ABT-737 disrupted the associations in Lapatinib-induced Bim sequestered by Bcl-2.