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Effect of EG00229 on Radiation Resistance of Lung Adenocarcinoma Cells.

ABSTRACT: Background: Neuropilin 1 (NRP1) is a pleiotropic receptor that interacts with multiple ligands and their receptors and plays a critical role in the process of tumor metastasis and radiation resistance in endothelial cells and tumor cells. In this study, we sought to investigate the mechanistic role of NRP1 in the radiation resistance of non-small cell lung cancer (NSCLC) cells and the role of EG00229 (an inhibitor of NRP1) on reversing radiation resistance. Materials and Methods: A549 and H1299 NSCLC cells were used to construct radiation resistance models. Western blot, ELISA, and qRT-PCR were used to detect protein and mRNA levels of NRP1, epithelial-mesenchymal transition (EMT) markers, and molecules in signaling pathways. Immunofluorescence was used to measure changes in co-expression of NRP1 and VEGF-165 in radiation-resistant model cells. An immunoprecipitation assay was used to detect the binding capacity of NRP1 and VEGF-165. Results: We successfully created two radiation resistant models (A549RR and H1299-RR). The expression levels of NRP1, EMT-related proteins, and proteins in metastasis-related pathways were increased in NSCLC cells with radiation resistance. After adding EG00229, the expression levels and binding capacity of NRP1 and VEGF-165 proteins were significantly reduced. The expression of EMT-related proteins and proteins in metastasis-related pathways were reduced in NSCLC cells with radiation resistance. Conclusion: Our data provide an insight into the molecular mechanisms of radiation resistance and suggest that EG00229 may contribute to reversing the radiation resistance of NSCLC cells by inhibiting the binding of NRP1 and VEGF-165. Our findings could provide a novel theoretical and experimental foundation for improving the efficacy of lung cancer radiotherapy.

SUBMITTER: Cong L

PROVIDER: S-EPMC8425201 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Lactic acidosis (3 to 40 mM, pH < 6.9) is a condition found in solid tumors because tumor cells have a high rate of glucose consumption and lactate production even in the presence of oxygen; nevertheless, the microenvironment might still provide a sufficient glucose supply. Lactic acidosis has been proposed to shift metabolism from aerobic glycolysis toward oxidative phosphorylation (OXPHOS). We tested if lung tumor cells cultured under lactic acidosis shift their metabolism from glycolysis to OXPHOS by consuming extracellular lactate, increasing growth rate. We analyzed lung adenocarcinoma (A-549, A-427) cell lines and non-transformed fibroblast cells (MRC-5), which were cultured using RPMI-1640 medium initially containing lactate (2 mM) and glucose (10 mM), at pH 7.2 or 6.2 and oxygen tension 21% O2 (normoxia) or 2% O2 (hypoxia). We obtained growth curves, as well as glucose consumption and lactate production rates (measured during exponential growth) for each cell line. HIF-1α (Hypoxia-inducible factor 1 α), CS (citrate synthase) and AMPK (AMP-activated protein kinase) transcript levels were analyzed using RT-qPCR. By flow cytometry, we determined: (a) expression of glucose transporters (GLUT)1 and 4; (b) lactate transporters (MCT)1 and 4; (c) cell cycle profile, and (d) protein levels of HIF-1α, total and phosphorylated AMPK (pAMPK). Mitochondrial functionality was evaluated by measuring O2 consumption in tumor cells using polarography and a Clark-type electrode. Tumor and non-transformed cells used both aerobic glycolysis and OXPHOS for obtaining energy. As of 48 h of culture, lactate levels ranged from (4.5-14 mM), thus forming a lactic environment. Lactic acidosis diminished GLUT1/GLUT4 expression and glucose consumption in A-549, but not in A-427 cells, and induced differential expression of HIF-1α, AMPK, and CS transcripts. A-427 cells increased pAMPK and HIF-1α levels and shifted their metabolism increasing OXPHOS; thus supporting cell growth. Conversely, A-549 cells increased HIF-1α protein levels, but did not activate AMPK and diminished OXPHOS. A-549 cells survived by arresting cells in G1-phase. Our findings show that lactic acidosis diminishes Warburg effect in tumor cells, but this change does not necessarily promote a shift to OXPHOS. Hence, lung adenocarcinomas show a differential metabolic response even when they are under the same microenvironmental conditions.

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Effect of EG00229 on Radiation Resistance of Lung Adenocarcinoma Cells.

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