(T0930-04-24) Urea-Based Compound UR-1-1-13 Affects the Mitochondrial Health of Triple-Negative Breast Cancer Cells

Purpose: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer that lacks the expression of three things: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) genes. That is why, hormonal therapy and HER2-based treatment are ineffective against it. Approximately 15% of breast cancers are TNBC, and 28% of TNBC patients suffer from brain metastases, leading to decreased survival rates compared to people suffering from other types of breast cancer. Due to the high heterogeneity of TNBC, six subtypes with distinct sensitivity to existing chemotherapeutic agents were identified. Our lab has synthesized a novel class of anticancer agent, UR-1-1-13, with cytotoxicity against TNBC cell lines, a high ability to cross the BBB in vitro and in vivo, and the ability to reduce tumor growth in vivo by > 95%. In this study, we investigate if our compound affects the mitochondrial health of cancer cells.

Methods: The chemicals we have used for this study are MitoView Green, JC-1, MitoSOX Red, a mitochondrial superoxide indicator, oxidizing agents like dimethoxy-naphthoquinone (DMNQ) and hydrogen peroxide (H₂O₂), and antioxidants like ascorbic acid (vitamin C), n-acetylcysteine (NAC), and glutathione (GSH). We measured mitochondrial ROS and membrane depolarization by our compound on TNBC cell line MDA-MB-231 by colorimetric assay, flow cytometry, and immunofluorescence with and without antioxidants.

Results: We used the MTT assay to determine that UR-1-1-13 has comparable cytotoxicity in five TNBC cell lines. Tested cell lines belong to the MSL, BL1and BL2 subtypes of TNBC. Our compound UR-1-1-13 showed good anti-cancer activity against all the cell lines. We have selected the MDA-MB-231 cell line for further experiments as it is commonly used to model late-stage breast cancer. Some experiments were also performed in the MDA-MB-468 cell line. A colorimetric assay using DMNQ and H₂O₂ as positive control and GSH and vitamin C as a negative control showed that the activity of our compound is mediated through increased ROS levels. These data were further confirmed by flow cytometry and immunofluorescence using MitoSOX Red dye. We also examined the effects of antioxidants on the cytotoxicity of our compound where vitamin C, but not NAC diminished the cytotoxicity of our compound. As vitamin C, but not NAC, can enter the mitochondria, we conclude that UR-1-1-13 induces ROS production in mitochondria.
Furthermore, our compound showed mitochondrial membrane depolarization in cancer cells but not in human breast epithelial cells (MCF10a). UR-1-1-24, a structural analog of UR-1-1-13 that lacks cytotoxicity in tested cell lines, showed no effect on mitochondrial membrane polarization and ROS level. Therefore, our compound exhibits its anticancer activity by generating ROS inside the mitochondria and causing mitochondrial membrane depolarization.

Conclusion: This study showed that UR-1-1-13 has comparable cytotoxic activity in several TNBC cell lines that belong to different subgroups, whereas MCF10a cells were the least affected. The observed anticancer activity correlates with the level of mitochondrial membrane depolarization and ROS production in the cancer cells.