Repurposing Diphenhydramine against breast cancer: mechanistic insights into apoptosis, p53-independent cytotoxicity, and synergy with doxorubicin in MCF-7 cells
Authors: Syed Rashel Kabir 1*, Md Gausul Azam 1, Taufique Abdullah 1, Md. Abdul Alim Al-Bari 2, Mohammad Taufiq Alam 3
Institution: 1Department of Biochemistry and Molecular Biology, 2Department of Pharmacy, 3Department of Applied Chemistry and Chemical Engineering, University of Rajshahi, Rajshahi-6205
Introduction
Drug repurposing offers a potentially efficient strategy for identifying new anticancer applications for existing medicines with established pharmacological profiles. Diphenhydramine (DIP), a first-generation histamine H1-receptor antagonist, has pharmacological activities beyond its conventional antihistaminic effects, warranting investigation of its potential anticancer properties. However, its effects on breast cancer cells, the mechanisms associated with its cytotoxicity, and its interaction with conventional chemotherapy remain insufficiently characterized. This study investigated DIP-induced cytotoxicity in human breast adenocarcinoma MCF-7 cells, focusing on p53 dependence, apoptosis-associated mechanisms, reactive oxygen species (ROS), caspase signaling, NF-κB modulation, and its interaction with doxorubicin (Dox).
Methods
Cytotoxicity was evaluated in wild-type (WT) and TP53-knockout (KO) MCF-7 cells using the MTT assay following treatment with two-fold serial concentrations of DIP. Half-maximal inhibitory concentrations (IC₅₀) were estimated using four-parameter nonlinear regression. Apoptosis-associated nuclear changes were examined by Hoechst 33342 staining, and cellular morphology was assessed for characteristic alterations. Intracellular ROS generation was quantified to investigate oxidative stress. The involvement of apoptotic signaling pathways was evaluated using selective caspase-3, caspase-8, and caspase-9 inhibitors, while NF-κB-associated effects were investigated using pyrrolidine dithiocarbamate (PDTC). DIP–Dox interactions were assessed using the Chou–Talalay combination index (CI) method.
Results
DIP demonstrated dose-dependent cytotoxicity in both WT and TP53-KO MCF-7 cells, with comparable sensitivity supporting a largely p53-independent response under the tested conditions. Nuclear staining and morphological examination revealed changes consistent with apoptosis, accompanied by increased intracellular ROS generation. Pharmacological inhibition of caspases attenuated DIP-induced cytotoxicity, supporting the involvement of caspase-dependent cell-death signaling and both intrinsic and extrinsic apoptotic pathways. Modulation of NF-κB activity altered the cytotoxic response to DIP, suggesting a potential role for this pathway in cellular survival following treatment. Furthermore, combination experiments demonstrated synergistic cytotoxic interactions between DIP and Dox across the tested combinations, supporting further investigation of DIP as a potential adjunct to conventional chemotherapy.
Conclusion
Diphenhydramine exhibits p53-independent, ROS-associated, caspase-dependent cytotoxicity in MCF-7 breast cancer cells, with findings consistent with apoptotic cell death. Its synergistic interaction with doxorubicin and the observed modulation of cytotoxicity following NF-κB inhibition provide a rationale for further mechanistic and preclinical investigation. These findings support the exploration of DIP as a potential repurposed candidate for combination-based breast cancer research.