In vitro Investigation of Conessine-Mediated Apoptosis on Human Breast Cancer Cell Line (MCF-7) via Mitochondrial Pathway and APAF-1 Gene

Background

Breast cancer was the second most deadly cancer, especially in women. Recent data show that the incidence rate has been slowly rising over time and now it is becoming a global problem. This increasing problem shows how important it is to move away from traditional therapies and toward treatments that use bioactive compounds, which may be safer and more effective. The current study examines the antiproliferative effect of the steroidal alkaloid Conessine on the Human Breast Adenocarcinoma Cell Line (MCF-7).

Materials and Methods

The Tetrazolium Dye-Based Method (MTT) was used to test Conessine's cytotoxicity. Fluorescence microscopy was used to confirm apoptosis by using Acridine orange and Ethidium Bromide to stain the cells. The Muse™ Mito Potential kit by Muse™ cell analyzer was used to measure the potential of the mitochondrial membrane. Then, quantitative PCR was used to find out the gene expression status of apoptotic-related genes (Apaf-1, Caspase 3, and Caspase 9) and an Anti-Apoptotic Gene (BCLxL).

Results

The IC₅₀ value of Conessine against the MCF-7 cell line was 123.5981 µg/mL. Morphological analysis also demonstrated dose-dependent effects following treatment with Conessine. Dual staining with Acridine orange and Ethidium Bromide identified apoptotic cells in the treated groups, a finding corroborated by the MMP assay, which indicated an increased proportion of depolarized cells. At the molecular level observation denotes apoptotic genes (Apaf-1, Caspase 3, and Caspase 9) were upregulated and Anti-Apoptotic Genes (BCLxL) were down regulated.

Conclusion

The results of this study indicated that Conessine possesses anticancer efficacy against the MCF-7 cell line. The upregulation of apoptosis-related genes signifies the activation of the mitochondrial-mediated apoptotic pathway, primarily initiated by APAF-1 (apoptotic protease-activating factor), followed by sequential activation of caspase cascade components. In conclusion, Conessine exhibits significant potential as an anticancer agent, necessitating further exploration across additional cancer cell lines to validate its therapeutic efficacy.