Pharmacological Vitamin C-induced high H2O2 generation mediates apoptotic cell death by caspase 3/7 activation in breast cancer tumor spheroids

Sep 23, 2026

Uploaded by Logic

Pharmacological vitamin C (high-dose Vit-C) is explored as a potential cancer therapy using realistic three-dimensional breast cancer models. Two breast cancer tumor spheroid types are generated from MDA-MB-231 and MCF-7 cells and treated with escalating Vit-C concentrations. Vit-C causes dose-dependent loss of viability driven mainly by elevated H2O2 production and oxidative stress imbalance from GSH depletion. Increased H2O2 triggers apoptosis, with distinct susceptibility differences between spheroid subtypes.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages15

Reading Time38 min

AccessFree to download

Abstract

Introduction

Breast cancer burden and treatment challenges

Need for 3D models and tumor microenvironment complexity

Methods

Results

Conclusions

Mussa etal. Journal of Translational Medicine (2025) 23:31 [https://doi.org/10.1186/s12967-024-06016-7](https://doi.org/10.1186/s12967-024-06016-7) Journal of Translational Medicine RESEARCH Open Access Pharmacological Vitamin C-induced high H 2 O 2 generation mediates apoptotic cell death by caspase 3/7 activation in breast cancer tumor spheroids Ali Mussa 1,2, Mahasin Hamid3,4, Khalid Hajissa5,13, Ahmad Hafiz Murtadha6, Mohammad A. I. Al-Hatamleh7, Noor Fatmawati Mokhtar6,12, Vuk Uskoković8,9, Magdalena Plebanski 10, Rohimah Mohamud 11* and Rosline Hassan1* Abstract Background Pharmacological vitamin C (Vit-C), or high-dose Vit-C has recently gained attention as a potential cancer therapeutic. However, the anticancer activity ofVit-C has not been investigated in realistic 3D models of human cancers, especially with respect to breast cancer (BC), and its potential benefits remain under debate. Herein, we investigate the activity and mechanism of action of pharmacological Vit-C on two BC tumor spheroids. Methods We developed two distinct types of BC tumor spheroids from MDA-MB-231 and MCF-7 cells. The spheroids underwent treatment with a range of concentrations of pharmacological Vit-C (1, 5, 10, 15, and 20 mM) . Assessments were conducted to determine the cell viability, H2O2 levels, glutathione-to-glutathione disulfide (GSH/GSSG) ratios, and apoptosis. Both flow cytometry analyses of Annexin V/PI staining and caspase3/7 activity assay were used to check apoptosis. Results We showed that Vit-C induced dose-dependent cell death in both types of tumor spheroids, primarily driven by elevated H2O2 production and a concomitant oxidative stress imbalance induced by the GSH depletion. The high levels of H2O2 generated by Vit-C triggered the apoptosis of spheroids. In MCF-7 spheroids, Vit-C-induced H2O2 production was higher, with a more pronounced decrease in the GSH/GSSG ratio, indicating greater susceptibility to oxidative stress-induced cell death. However, MDA-MB-231 spheroids exhibited a more severe cytotoxic response. Conclusions This study reveals that Vit-C induces oxidative stress-mediated cell death in both non-aggressive and aggressive BC spheroids. Unlike traditional in vitro studies, this work provides novel insights into the response of two BC tumor subtypes to Vit-C, demonstrating its potential as a targeted common therapy for BC. Keywords Vitamin C, Pharmacology, Oncology, Spheroids, Breast cancer, Free radicals *Correspondence: Rohimah Mohamud [rohimahm@usm. my](rohimahm@usm. my)[ ](rohimahm@usm. my)Rosline Hassan [roslin@usm. my](roslin@usm. my) Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit [http://creativecommons.org/licenses/by-nc-nd/4.0/](http://creativecommons.org/licenses/by-nc-nd/4.0/.)[.](http://creativecommons.org/licenses/by-nc-nd/4.0/.) Mussa etal. Journal of Translational Medicine (2025) 23:31 Introduction Despite the advancements in treatments, breast cancer (BC) remain

FAQs

How was the breast cancer tumor spheroid model prepared for this study?

Two distinct breast cancer tumor spheroid types were developed from MDA-MB-231 and MCF-7 cells, then treated with pharmacological vitamin C across multiple concentrations.

What mechanism mediates Vit-C induced apoptotic cell death in these spheroids?

The study shows Vit-C elevates H2O2 production, creating an oxidative stress imbalance due to GSH depletion. The resulting H2O2 triggers apoptosis linked to caspase 3/7 activation.

Do the two breast cancer spheroid types respond to Vit-C differently?

Yes. MCF-7 spheroids show higher Vit-C induced H2O2 production and a stronger decrease in the GSH/GSSG ratio, indicating greater sensitivity to oxidative stress, while MDA-MB-231 spheroids display a more severe cytotoxic response.

Sep 23, 2026

Uploaded by Logic