The role of mitochondrial dysfunction in the cytotoxic synergistic effect of gemcitabine and arsenic on breast cancer

Abstract

Breast cancer is the most common type of cancer in women worldwide. A common approach to cancer treatment in clinical practice is to use a combination of drugs to enhance the anticancer activity of drugs while reducing their side effects. In this regard, we evaluated the effectiveness of combined treatment with gemcitabine (GCB) and arsenic (ATO) and how they affect the cell death pathway in cancer cells. Cytotoxic activity of drugs individually or combined against MDA-MB-231 and MCF-7 was performed by MTT method and isobolographic analysis was used to determine the interaction between these factors. The combination of ATO and GCB showed synergistic anti-cancer activity (CI < 1) in both cancer cell lines. The combination of ATO and GCB induced sub-G1 phase arrest, apoptosis and death rates in MCF-7 and MDA-MB-231 cells. The apoptotic response induced by the combination of GCB and ATO was dependent on caspase 3/7. Combined treatment with mitochondrial membrane potential (MMP) reduction and increased reactive oxygen species (ROS) production caused mitochondrial dysfunction. Co-treatment significantly reduced catalase (CAT) activity in both cancer cells compared to the control group and cells treated with each monotherapy. A significant decrease in cellular GSH was observed in cancer cells treated with ATO and GCB. In addition, migration and invasion were significantly reduced in breast cancer cells treated with the combination of ATO and GCB compared to cells treated with ATO and GCB. In conclusion, the combined treatment of ATO and GCB synergistically increased the anti-cancer activity, and these findings provide an effective approach for the treatment of breast cancer. To the best of our knowledge, this is the first study showing promising results for combination therapy with ATO and GCB in breast cancer.

Fig 1

Cytotoxicity activity of (Aa) ATO on MCF-7 cells, (Ab) GCB on MCF-7 cells, (Ba) ATO on MDA-MB-231 cells and (Bb) GCB on MDA-MB-231 cells. Data are presented as mean ± SD (n = 3).

Fig 2. Rate of apoptosis in MCF-7 cells (ATO: 2.5 μM and GCB: 22.5 μM, 48 h, at 37°C).

Data are presented as mean ± SD. ****: Significant difference with control, ATO and GCB group (p < 0.0001). &: Significant difference with control group (p < 0.05).

Fig 3. Rate of apoptosis in MDA-MB-231 cells (ATO: 2.5 μM and GCB: 22.5 μM, 48 h, at 37°C).

Data are presented as mean ± SD (n = 3). ***: Significant difference with control group (p < 0.001). **: Significant difference with ATO, GCB group (p < 0.01). &&&: Significant difference with control group (p < 0.001).

Fig 4

Cell cycle arrest after treatment of A) MCF-7 and B) MDA-MB-231 cells with ATO (2.5 μM) and GCB (22.5) μM for 48 h at 37°C. Data are presented as mean ± SD (n = 3). ***: Significant difference with control group (p < 0.001). **: Significant difference with control, ATO, GCB group (p < 0.01). &: Significant difference with control group (p < 0.05). $: Significant difference with control group (p < 0.01).

Fig 5

Mean fluorescence intensity of (Aa) FDA, (Ab) PI, cell viability as determined by FDA-PI staining. (Ba) MCF-7 and (Bb) MDA-MB-231 cells stained with FDA/PI (alive cells stained green, whereas dead cells stained red).

Fig 6

Caspase 3/7 activity after treatment of A) MCF-7 and B) MDA-Mb-231 with ATO (2.5 μM) +GCB (22.5 μM) for 48 h at 37°C (n: 3). Data are presented as mean ± SD. ***: Significant difference with control group (p < 0.001). **: Significant difference with control group (p < 0.01). &: Significant difference with control group (p < 0.05). $: Significant difference with control group (p < 0.05).

Fig 7

Relative ROS production in A) MCF-7 and B) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB and measurement of MMP in C) MCF-7 and D) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB (ATO: 2.5 μM+GCB: 22.5 μM, 48 h at 37°C). Data are presented as mean ± SD. ***: Significant difference with control group (p < 0.001). **: Significant difference with control group (p < 0.01). &&: Significant difference with control group (p < 0.01). &: Significant difference with control group (p < 0.05). $: Significant difference with control group (p < 0.05). $ $: Significant difference with control group (p < 0.01).

Fig 8

The CAT activity in A) MCF-7 and B) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB and GSH content in C) MCF-7 and D) MDA-Mb-231 cells treated with ATO, GCB and ATO + GCB (ATO: 2.5 μM+GCB: 22.5 μM), for 48 h at 37°C. Data are presented as mean ± SD. ***: Significant difference with control and GCB group (p < 0.001). **: Significant difference with control, ATO, GCB group (p < 0.01). &: Significant difference with control group (p < 0.05). #: Significant difference between GCB and ATO group (p < 0.05). ##: Significant difference between GCB and ATO group (p < 0.01). $: Significant difference with control group (p < 0.05). $ $: Significant difference with control group (p < 0.01).

Fig 9

Inhibition of the invasion of A) MCF-7 and B) MDA-Mb-231 cells after treatment with ATO (2.5 μM) and GCB (22.5 μM) for 48 h at 37°C. Data are presented as mean ± SD. ****: Significant difference with control, ATO, GCB group (p < 0.0001). $ $ $: Significant difference with control group (p < 0.001). &: Significant difference with control group (p < 0.05). &&: Significant difference with control group (p < 0.01).

Fig 10

Inhibition of the migration of A) MCF-7 and B) MDA-Mb-231 cells after treatment with ATO (2.5 μM) and GCB (22.5 μM) for 48 h at 37°C. Data are presented as mean ± SD. ****: Significant difference with control, ATO, GCB group (p < 0.0001). $ $ $ $: Significant difference with control group (p < 0.0001). &&&&: Significant difference with control group (p < 0.0001).

Fig 11. Schematic presentation of mechanism of anti-cancer activity of ATO and GCB combination in breast cancer cells.