Open Access

ARTICLE

Metronomic Chemotherapy Response in MDA-MB-231 Triple-Negative Breast Cancer Cells under Nicotine Exposure

Alejandro Javier Español^1,2,*, Yamila Sanchez¹, Sofia Volpi²

1 Tumor Immunopharmacology Laboratory, Center of Pharmacological and Botanical Studies (CEFYBO)-National Council for Science and Technology (CONICET), University of Buenos Aires, Buenos Aires, C1121ABG, Argentina
2 Second Department of Pharmacology, School of Medicine, University of Buenos Aires, Buenos Aires, C1121ABG, Argentina

* Corresponding Author: Alejandro Javier Español. Email:

(This article belongs to the Special Issue: Exploring the Role of Cancer Stem Cells)

BIOCELL 2025, 49(8), 1449-1480. https://doi.org/10.32604/biocell.2025.068034

Received 20 May 2025; Accepted 08 August 2025; Issue published 29 August 2025

Abstract

Background: Triple-negative (TN) breast cancer, the most aggressive subtype of breast cancer, is usually treated with high doses of paclitaxel (PX), which induces resistance. To prevent this adverse effect, metronomic chemotherapy based on administering low doses of PX plus carbachol (Carb), a muscarinic acetylcholine receptor (mAChR) agonist, has emerged as an alternative. Other acetylcholine receptors also present in breast tissue are nicotinic ones. When activated by nicotine (Nic), these receptors can decrease the effectiveness of conventional chemotherapy. However, whether metronomic chemotherapy with PX and Carb is affected by Nic has not yet been described. This study aimed to determine the efficacy of metronomic chemotherapy with PX and Carb in human breast tumor MDA-MB-231 cells in the presence or absence of Nic and assess the intermediaries involved. Methods: Cell viability and proliferation were determined using colorimetric assays with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue. Nitrite levels in cell supernatant were determined using Griess reagent. The expression of proteins was determined by western blot assays. Apoptosis/necrosis and the proportion of cancer stem cells (CSC) were determined by flow cytometry. Mammosphere-forming units were determined with anchorage-free growth assays. Results: The metronomic chemotherapy combining PX and Carb effectively inhibited the viability of TN MDA-MB-231 cells in the presence and absence of Nic. These effects were mediated by the activation of mAChRs, triggering signaling pathways dependent on several kinases. These mediators induce increased expression of the inducible isoform of nitric oxide synthase (NOS). Only the inducible and endothelial isoforms were expressed in these cells, and their activity was increased by the metronomic chemotherapy with PX and Carb. Nitric oxide (NO), a product of NOS activity, may contribute to the observed increase in apoptosis. We also observed an increased sensitivity to PX in the residual cells after the metronomic chemotherapy, as well as a decrease in mammosphere-forming units and CSC proportion. We also determined a decrease in the expression of stemness proteins such as ATP-binding cassette super-family G member 2 (ABCG2), sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (OCT-4), and Nanog. Conclusions: Metronomic chemotherapy combining PX with Carb was selective for MDA-MB-231 cells and increased their sensitivity to conventional chemotherapy in the presence and absence of Nic, indicating that it could be a useful neoadjuvant strategy for the treatment of TN breast tumors.

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Supplementary Material

Supplementary Material File

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