# Curcumin Enhances Gemcitabine Sensitivity in Breast Cancer Cells Through ROS-Associated Mitochondrial Apoptosis and Transcriptional Reprogramming

## TL;DR

Curcumin boosts gemcitabine's effectiveness in breast cancer cells by increasing oxidative stress and triggering cell death, especially in aggressive triple-negative breast cancer.

## Contribution

The study reveals curcumin's ability to sensitize breast cancer cells to gemcitabine via ROS-mediated mitochondrial apoptosis and transcriptional changes.

## Key findings

- Combining curcumin with gemcitabine increases apoptosis and oxidative stress in breast cancer cells.
- Mitochondrial depolarization and ROS accumulation are selectively observed in cancer cells, not in normal breast epithelial cells.
- Transcriptomic analysis shows activation of pro-apoptotic pathways and suppression of survival signals in triple-negative breast cancer cells.

## Abstract

Breast cancer treatment is often limited by resistance to chemotherapy, particularly in aggressive subtypes such as triple-negative breast cancer. Gemcitabine is an effective chemotherapeutic drug, but its efficacy can be reduced by cellular survival mechanisms. Curcumin, a natural compound, has been suggested to enhance the activity of anticancer drugs. In this study, we show that curcumin significantly increases the sensitivity of breast cancer cells to gemcitabine. The combined treatment induces oxidative stress, disrupts mitochondrial function, activates programmed cell death, and suppresses angiogenesis-related signaling. Direct measurement of intracellular reactive oxygen species using the DCFH-DA assay demonstrated marked reactive oxygen species accumulation in breast cancer cells following combination treatment, whereas normal breast epithelial MCF-10A cells exhibited only minimal oxidative changes under identical conditions. Assessment of mitochondrial membrane potential using JC-1 staining further revealed substantial mitochondrial depolarization in malignant cells, while mitochondrial integrity was largely preserved in MCF-10A cells, supporting cancer-selective vulnerability. Blocking oxidative stress reverses these effects, demonstrating that reactive oxygen species are central to the synergistic interaction. Transcriptomic analysis further confirmed the activation of pro-apoptotic pathways and the inhibition of survival signals. These effects are particularly pronounced in triple-negative breast cancer cells. Overall, our findings provide a mechanistic basis for combining curcumin with gemcitabine to improve therapeutic efficacy in breast cancer.

Breast cancer is a leading cause of cancer-related mortality in women, necessitating new treatment strategies. Curcumin (Cur), a natural polyphenol, and gemcitabine (Gem), a standard chemotherapeutic, were investigated for their combined anticancer effects. We hypothesized that Cur sensitizes breast cancer cells to Gem via reactive oxygen species (ROS)-mediated apoptosis, and that this effect is associated with selective oxidative vulnerability in malignant cells compared to normal breast epithelial cells. MCF-7 (hormone receptor-positive) and MDA-MB-231 (triple-negative) cells were treated with Cur and Gem alone or in combination. Normal breast epithelial MCF-10A cells were included to evaluate therapeutic selectivity. Cell viability (MTT), apoptosis (Annexin V/PI), oxidative stress (TOS/TAS), intracellular ROS generation (DCFH-DA assay), mitochondrial membrane potential (ΔΨm) (JC-1 staining), caspase activation, synergy (Bliss/HSA/Chou-Talalay), VEGF secretion (ELISA), and transcriptomic changes (RNA-Seq) were assessed. Cur and Gem showed dose-dependent cytotoxicity. Combination treatment demonstrated strong synergistic activity, significantly enhancing apoptosis, oxidative stress, and caspase activation. Direct quantification of intracellular ROS revealed marked ROS accumulation in MCF-7 and MDA-MB-231 cells following combination treatment, whereas MCF-10A cells exhibited only modest oxidative changes. JC-1 analysis demonstrated substantial mitochondrial depolarization in breast cancer cells, which was largely reversible by ROS scavenging and minimal in MCF-10A cells. VEGF secretion was markedly suppressed. Transcriptomic analysis revealed profound alterations in apoptosis, cell cycle, and angiogenesis-related pathways, with more pronounced transcriptional reprogramming observed in the triple-negative subtype. Cur synergistically enhances Gem’s efficacy in breast cancer cells through ROS-mediated apoptosis and anti-angiogenic effects, characterized by cancer-selective ROS amplification and mitochondrial membrane depolarization, supporting its potential as a combination therapy, particularly for triple-negative breast cancer.