TP53-Associated Mechanisms of Loperamide-Enhanced Doxorubicin Cytotoxicity in MCF-7 Breast Cancer Cells
Authors: Syed Rashel Kabir1#*, Md Gausul Azam1#, Taufique Abdullah1, Mohammad Taufiq Alam2
Institution: 1Department of Biochemistry and Molecular Biology and 2Department of Applied Chemistry and Chemical Engineering, University of Rajshahi, Rajshahi-6205, Bangladesh
Introduction
Breast cancer remains a leading cause of cancer-related mortality among women, while treatment resistance and chemotherapy-associated toxicity remain major challenges. Drug repurposing offers an opportunity to identify anticancer applications of established drugs with known pharmacological profiles. Loperamide, an FDA-approved antidiarrheal drug, has demonstrated anticancer activity and doxorubicin-sensitizing effects in other breast cancer models. However, whether loperamide produces synergistic cytotoxicity with doxorubicin in parental, hormone receptor-positive and TP53-wild-type MCF-7 cells, independently of an MDR1-overexpressing phenotype, remains insufficiently defined. In particular, the functional contribution of TP53 and NF-κB-associated signaling to this interaction has not been established. Addressing these questions may help clarify whether loperamide-mediated chemosensitization extends beyond drug-efflux modulation and involves broader cellular mechanisms relevant to hormone receptor-positive breast cancer.
Methods
MCF-7 cells were treated with loperamide and doxorubicin individually or in combination. Cytotoxicity was assessed by MTT assay and the IC₅₀ was obtained by linear interpolation between the two concentrations that bracket 50% inhibition The drug interactions were quantified using the Chou–Talalay combination index (CI) and dose-reduction index. Apoptotic morphology, caspase activity, reactive oxygen species (ROS), and mitochondrial membrane potential were evaluated using Hoechst 33342, fluorescent caspase substrate, DCFH-DA, and JC-1 assays. Expression of TP53, CDKN1A, TNF-α, BAX, BCL2, PARP-1, MAPK, and NFKBIE was analyzed by real-time PCR. NF-κB-associated signaling was further examined using PDTC, and molecular docking was performed against NF-κB- and MAPK-associated targets. The functional role of TP53 was investigated by comparing wild-type and CRISPR-Cas9-mediated TP53-knockout MCF-7 cells.
Results
Loperamide and doxorubicin produced IC₅₀ values of 8.55 and 0.64 µg/mL, respectively. Across 16 concentration combinations, synergistic interactions were observed in 11, with the strongest synergy at 8 µg/mL loperamide plus 1 µg/mL doxorubicin (CI=0.288). The doxorubicin dose reduction index reached 10.46. Combination treatment enhanced apoptotic morphology, ROS generation, caspase activation, and mitochondrial membrane depolarization. Caspase inhibition demonstrated a prominent contribution of caspase-7, with additional involvement of caspase-8 and caspase-9. Treatment increased TP53, CDKN1A, and TNF-α expression and altered BAX, BCL2, PARP-1, MAPK, and NFKBIE expression. PDTC further increased cytotoxicity of loperamide alone and the loperamide-doxorubicin combination, supporting involvement of NF-κB-associated survival signaling. Molecular docking demonstrated favorable predicted interactions of loperamide and doxorubicin with NF-κB- and MAPK-associated targets. Importantly, TP53 knockout increased the loperamide and the doxorubicin IC₅₀ remarkably. Synergy remained detectable in all six tested combinations in TP53-knockout cells but was generally weaker; for 8 µg/mL loperamide plus 1 µg/mL doxorubicin, CI increased from 0.288 in wild-type cells to 0.417 after TP53 knockout. Maximum combination-induced inhibition decreased from 96.61% to 84.36%. These findings indicate that functional TP53 enhances loperamide sensitivity and contributes to the magnitude of drug interaction, while residual synergy in TP53-deficient cells suggests involvement of additional pathways.
Conclusion
Loperamide potentiates doxorubicin-induced cytotoxicity in parental MCF-7 cells through coordinated apoptotic, oxidative, mitochondrial, TP53-associated, and NF-κB/MAPK-associated mechanisms. TP53 is not absolutely required for loperamide activity, but functional TP53 contributes to drug sensitivity and the magnitude of loperamide-doxorubicin synergy. These findings extend previous observations from MDR1-overexpressing and triple-negative breast cancer models by demonstrating chemosensitization in parental MCF-7 cells and identifying a functional contribution of TP53. The substantial reduction in the effective doxorubicin dose observed in combination treatment provides a rationale for further investigation of loperamide as a potential repurposed chemosensitizing agent in breast cancer.