E2F-1–Akt1 Interaction as Precursor to Cisplatin-induced Apoptosis in Triple-negative Breast Cancer Cells

Abstract

Objectives: We aimed to investigate the expression levels and interaction between E2F-1 and Akt1 in triple-negative breast cancer (TNBC) cells, and whether cis-diamminedichloroplatinum(II) (cisplatin) could influence such an interaction. Methods: A batch of MDA-MB-321 breast cancer cells were treated with increasing concentrations of cisplatin (2.5–40 μM) for 24 hours. Additional cells from the same source were used for control experiments. Cisplatin-induced apoptosis was confirmed biochemically using cleaved polymerase and flow cytometry analysis, and morphologically using hematoxylin and eosin staining, Hoechst staining, and scanning electron microscopy. Caspase-3 cleavage, an indicator of apoptotic induction, was measured by immunofluorescence. A western blot test was used to investigate the effects of cisplatin on E2F-1 and Akt1 expressions, while their co-localization and interaction were detected using immunofluorescence and immunoprecipitation, respectively. Results: A western blot analysis revealed an increase in E2F-1 and a decrease in Akt1 expression with increasing concentration of cisplatin, compared to untreated cells. Merged E2F-1 and Akt1 immunosignals observed by immunofluorescence demonstrated that cisplatin-treated cells exhibited co-localization of immunosignals in clusters and with increased intensity in the cytoplasm. Immunoprecipitation and western blot analysis results further confirmed the association between E2F-1 and Akt1, indicating a potential interaction between the two proteins in MDA-MB-231 cells. Conclusions: Our findings suggest a potential interaction between E2F-1 and Akt1, which in turn could be the precursor for the cisplatin-induced apoptosis in TNBC cells. Further studies are needed to determine whether this interaction occurs directly or via an intermediate protein.

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