[Nitrosative stress promotes the malignant biological behavior of breast cancer through the glycolytic pathway]

Breast cancer is one of the most common malignant tumors in women, and some patients face poor therapeutic efficacy and recurrence. Tumor cells are extremely dependent on the energy produced by the glycolytic pathway, which not only provides adenosine triphosphate (ATP) for rapid tumor cell proliferation, but also creates favorable conditions for tumor cell growth by regulating the acidity of the tumor microenvironment. Nitrosative stress refers to a pathophysiological state characterized by intracellular accumulation of reactive nitrogen species (RNS). Studies have found that nitrosative stress is closely associated with the occurrence and progression of cancer. This study aims to investigate the effects of nitrosative stress and tumor cell glycolysis, to provide new ideas for breast cancer treatment.

Postoperative tumor tissues from breast cancer patients at the First Affiliated Hospital of Shihezi University were collected. Immunohistochemistry (IHC) staining was used to analyze the expression levels of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT) in breast cancer tissues and adjacent normal breast tissues. Fluorescent probes were used to detect nitric oxide (NO) and reactive oxygen species (ROS) levels in normal breast cells (MCF-10A) and breast cancer cells (MDA-MB-231 and MCF-7). Western blotting was used to compare the expression levels of iNOS and 3-NT between normal and breast cancer cells. The optimal concentrations of the peroxynitrite (ONOO⁻) donor (SIN-1) and the scavenger (FeTMPyP) were determined using cell counting kit-8 (CCK-8). Glycolysis levels were measured using a glycolysis assay kit. CCK-8, Transwell, and wound-healing assays were further used to detect the proliferation, migration, and invasion abilities of MDA-MB-231 and MCF-7 cells under the effects of the two drugs. Western blotting was used to detect the expression levels of E-cadherin, N-cadherin, and vimentin, as well as glycolysis-related proteins hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), and enolase 1 (ENO1).

The expression levels of iNOS and 3-NT in breast cancer tissues were significantly higher than those in normal breast tissues (P<0.001), NO (P<0.01), ROS (P<0.001), iNOS (P<0.05), and 3-NT (P<0.01) levels in breast cancer cell lines (MDA-MB-231 and MCF-7) were all significantly higher than those in normal breast cells (MCF-10A). Breast cancer cells exhibited higher glycolysis levels (P<0.001). Treatment with the nitrosative-stress down-regulating drug FeTMPyP significantly inhibited glycolysis levels (P<0.001), and the expression of glycolytic enzymes HK2, PKM2, and ENO1 was significantly decreased (all P<0.05). The proliferation, migration, and invasion abilities of the two breast cancer cell lines were weakened, and the expression of EMT-related protein E-cadherin was increased, while the expressions of N-cadherin and vimentin were significantly decreased (all P<0.05). After treatment with the nitrosative-stress up-regulating drug SIN-1, glycolysis levels were significantly enhanced (P<0.001), and the expression of glycolytic enzymes HK2, PKM2, and ENO1 was significantly increased (all P<0.05). The proliferation, migration, and invasion abilities of the 2 breast cancer cell lines were enhanced, and the expression of EMT-related protein E-cadherin was reduced, while the expressions of N-cadherin and vimentin were significantly increased (all P<0.05).

Nitrosative stress can promote the malignant biological behavior of breast cancer cells through the glycolytic pathway.