Abstract
Background:
Canine mammary carcinoma is a common malignancy in female dogs, and CF41.Mg cells serve as a valuable translational model for this disease. Moringa oleifera possesses known anticancer properties; however, its effects on canine mammary carcinoma cells remain poorly understood.

Aim:
To evaluate the cytotoxic and pro-apoptotic effects of ethanolic M.oleifera leaf extract on CF41.Mg canine mammary carcinoma cells.

Methods:
CF41.Mg cells were treated with extract concentrations (100–550 µg/mL) for 24, 48, and 72 h. Cell viability was assessed using the MTT assay. Caspase-3 mRNA expression was quantified by RT-qPCR using the 2^(-ΔΔCt) method, normalized to GAPDH.

Results:
The extract significantly reduced cell viability in a time- and dose-dependent manner. At 72 h, 550 µg/mL induced the highest cytotoxicity, surpassing cisplatin. Caspase-3 expression showed a non-monotonic dose-response at 72 h: the lowest concentration (100 µg/mL) exhibited the highest fold change (~2142-fold), followed by 550 µg/mL (~880-fold), 400 µg/mL (~413-fold), and 250 µg/mL (~36-fold). Interestingly, this response was non-monotonic, with the lowest dose inducing the highest caspase-3 expression. All treated groups differed significantly from the control group (P < 0.05).

Conclusion:
M. oleifera ethanolic leaf extract induces cytotoxicity and upregulates Caspase-3 expression in CF41.Mg cells. The non-monotonic dose-response at 72 h suggests complex apoptotic regulatory mechanisms, including caspase-independent pathways at higher concentrations. Apoptosis was evidenced by the upregulation of Caspase-3 mRNA expression. The extract shows potential as a natural anticancer candidate for veterinary oncology.

Key words: Apoptosis; Canine mammary carcinoma; Caspase-3; CF41.Mg; Moringa oleifera.