Demethylzeylasteral induces G1 arrest and mitochondrial apoptosis via functional modulation of the MDM2-p21 module in murine and canine mammary tumor cells

Abstract

Canine mammary tumors are challenging to treat and share multiple molecular and pathological features with human breast cancer, particularly triple-negative breast cancer (TNBC). Demethylzeylasteral (T-96) exhibits significant antitumor activity; however, its mechanism of action against triple-negative breast cancer (TNBC) remains poorly understood. Moreover, pharmacokinetic information regarding intraperitoneal administration remains limited in BALB/c mice. This study aimed to investigate the therapeutic efficacy and underlying mechanisms of T-96 against TNBC and to characterize its pharmacokinetic profile. Transcriptomic analysis revealed that T-96 modulates pathways related to the cell cycle and apoptosis. Consistently, protein-level validation and MDM2 perturbation experiments support the functional involvement of the MDM2–p21 signaling module in T-96-associated phenotypes. At the molecular level, T-96 upregulated the expression of p21, BAX, and cleaved caspase 9 but downregulated the expression of MDM2 and Bcl-2. Pharmacokinetic analysis revealed that T-96 reached its peak plasma concentration (Tmax) at 2.0 h postadministration and had a half-life (t_1/2) of 23.5 h. In a mouse breast tumor model, T-96 significantly inhibited TNBC growth, increased the number of necrotic lesions in the tumor center, and reduced liver and lung metastases. Functional validation using transient MDM2 overexpression and inhibitor cotreatment supported a functional contribution of MDM2 to several T-96-associated phenotypes. To our knowledge, this study provides the first evidence that T-96 treatment is associated with reduced MDM2 levels and increased p21 expression, accompanied by G1 phase accumulation and activation of mitochondrial apoptosis markers in TNBC models, collectively contributing to the suppression of TNBC cell growth. Here, MDM2 was identified as a key functional mediator rather than a direct molecular target of T-96. Our findings indicate that the MDM2-p21 signaling module may partially functionally mediate T-96-associated cell cycle arrest and apoptosis, providing preclinical, comparative oncology–relevant evidence to support further evaluation of T-96 in additional models, including those of spontaneously occurring canine mammary tumors.

Graphical Abstract

Keywords

Demethylzeylasteral / MDM2-p21 axis / Cell cycle arrest / Apoptosis / Pharmacokinetics / Canine mammary tumor

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Funding

National Natural Science Foundation of China(32172925)

RIGHTS & PERMISSIONS

The Author(s)