2ⁿᵈ Edition of the Cancer R&D World Conference 2026

Abstract

Resistance to chemotherapy remains a major obstacle in effective breast cancer treatment and is frequently associated with impaired activation of apoptotic signaling pathways. Among the molecular factors contributing to this phenomenon is prolidase (PEPD), a multifunctional cytosolic enzyme primarily involved in collagen metabolism but also implicated in the regulation of intracellular signaling. Accumulating evidence indicates that prolidase directly interacts with the tumor suppressor protein p53, leading to functional inhibition of its transcriptional activity and attenuation of p53-dependent apoptosis induced by anticancer agents. Importantly, oxidative stress has been shown to promote dissociation of the PEPD–p53 complex, potentially restoring apoptotic signaling in tumor cells characterized by elevated prolidase expression.
The aim of this study was to investigate the impact of oxidative stress on p53-mediated apoptosis in breast cancer models exhibiting different levels of prolidase expression. Experimental analyses were conducted using wild-type MCF7 breast cancer cells, MCF7 cells with stable prolidase overexpression (MCF7PL), and a zebrafish embryo xenograft model, providing both in vitro and in vivo perspectives. Apoptosis was induced using doxorubicin, a chemotherapeutic agent known to directly activate p53 signaling. Oxidative stress was generated with tert-butyl hydroperoxide (t-BHP), while ascorbic acid was applied as an antioxidant to evaluate the reversibility of redox-dependent effects. Apoptotic cell death and mitochondrial membrane potential were assessed using the NucleoCounter NC-3000 system, DNA biosynthesis was quantified by a radiometric method, and prolidase activity was measured colorimetrically. The expression, activation, and intracellular localization of key proteins involved in apoptosis were analyzed by fluorescence microscopy and Western immunoblotting.
Doxorubicin induced apoptosis in a dose-dependent manner in wild-type MCF7 cells and in the zebrafish xenograft model, whereas MCF7PL cells displayed markedly reduced sensitivity to treatment. Induction of oxidative stress significantly enhanced apoptotic cell death, particularly in prolidase-overexpressing cells. This effect was associated with increased nuclear translocation of p53, elevated expression of active forms of caspases 9 and 7, loss of mitochondrial membrane potential, and inhibition of DNA biosynthesis. Notably, antioxidant treatment effectively reversed the pro-apoptotic effects of oxidative stress.
In conclusion, prolidase overexpression contributes to resistance against p53-dependent apoptosis in breast cancer cells. However, modulation of the cellular redox state restores apoptotic responsiveness, highlighting the potential of combination therapeutic strategies integrating p53-activating chemotherapeutics with controlled induction of oxidative stress in tumors characterized by elevated prolidase expression.