Effect of glucose level on chemical hypoxia- and hydrogen peroxide-induced chemokine expression in human glioblastoma cell lines

Abstract

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. The GBM-specific tumor microenvironment (TME) plays a crucial role in tumor progression, immune escape, local invasion, and metastasis of GBM. Here, we demonstrate that hypoxia, reactive oxygen species (ROS), and differential concentration of glucose influence the expression of cytokines and chemokines, such as IL-6, IL-8, and IP-10, in human glial cell lines. Treatment with cobalt chloride (CoCl₂) and hydrogen peroxide (H₂O₂) significantly increased the expression levels of IL-6, IL-8, and IP-10 in a dose-dependent manner in CRT-MG and U251-MG astroglioma cells, but not in microglia cells. However, we found strikingly different patterns of expression of cytokines and chemokines between H₂O₂-treated CRT-MG cells cultured in low- and high-glucose medium. These results suggest that astroglioma and microglia cells exhibit distinct patterns of cytokine and chemokine expression in response to CoCl₂ and H₂O₂ treatment, and different concentrations of glucose influence this expression under either hypoxic or oxidant-enriched conditions.

Keywords: Chemokines; Cytokines; Glioblastoma; Tumor microenvironment.

Fig. 1. Effect of CoCl₂ on IL-6, IL-8, and IP-10 expression.

CRT-MG and U251-MG astroglioma cell lines and HMO6 microglia cells were either treated with the indicated concentrations of CoCl₂ or not treated for 24 h. After incubation, supernatants were collected, and the protein levels of chemokines from the culture supernatants were analyzed via ELISA for IL-6 (A, D, G), IL-8 (B, E, H), and IP-10 (C, F, I). Absorbance was measured at 450 nm. (Inset) RT-PCR of CRT-MG, U251-MG and HMO6 cells treated with or without 0.5 mM CoCl₂. Data shown are representative of at least three independent experiments. ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. untreated control.

Fig. 2. Effect of H₂O₂ on IL-6, IL-8, and IP-10 expression.

CRT-MG, U251 MG, and HMO6 cells were either treated with the indicated concentrations of H₂O₂ or not treated for 24 h. After incubation, supernatants were collected, and the protein levels of chemokines from the culture supernatants were analyzed via ELISA for IL-6 (A, D, G), IL-8 (B, E, H), and IP-10 (C, F, I). Absorbance was measured at 450 nm. (Inset) RT-PCR of CRT-MG, U251-MG and HMO6 cells treated with or without 0.5 mM H₂O₂. Data shown are representative of at least three independent experiments. ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. untreated control.

Fig. 3. Effect of glucose concentration on CoCl₂-induced chemokine expression.

CRT-MG astroglioma cells were cultured in either low (1,000 mg/L) or high (4,500 mg/L) glucose medium with the indicated concentration of CoCl₂ for 24 h. After incubation, supernatants were collected and the level of IL-6 (A, D), IL-8 (B, E), and IP-10 (C, F) protein was measured via ELISA. Absorbance was measured at 450 nm. Data shown are representative of at least three experiments. ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. untreated control.

Fig. 4. Effect of glucose concentration on H₂O₂-induced chemokine expression.

CRT-MG cells were cultured in either low or high glucose medium with the indicated concentration of H₂O₂ for 24 h. After incubation, supernatants were collected, and the level of IL-6 (A, B), IL-8 (C, D), and IP-10 (E, F) protein was measured using ELISA. Absorbance was measured at 450 nm. (G) Indirect co-culture assay. CRT-MG astroglioma cells were placed on the 24 well-plate, and HMO6 microglia cells were seeded onto the microporous membranes of the transwell insert, and then cells were treated with either 0.5 mM H₂O₂ or 0.5 mM CoCl₂ for 24 h under low and high glucose conditions. After incubation, total RNA from CRT-MG and HMO6 cells were extracted and analyzed by RT-PCR for IL-8. Data shown are representative of at least three experiments. ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. untreated control.

Fig. 5. Effect of H₂O₂ and glucose concentration on phosphorylation of protein kinases.

CRT-MG cells cultured in low (1,000 mg/L) and high (4,500 mg/L) glucose medium were either treated with 0.5 mM of H₂O₂ or not treated for 24 h. After incubation, cells were lysed on ice for 30 min in lysis buffer, and 430 mg of cell lysate was analyzed for the phosphokinase assay as described in the Methods. (A) The duplicate spots corresponding to reduced phosphorylation of protein kinase in response to H₂O₂ plus low glucose compared to H₂O₂ plus high glucose or low glucose alone are highlighted in the solid box (A~G). Increased phosphorylation in response to H₂O₂ plus high glucose compared to high glucose alone, low glucose alone, or H₂O₂ plus low glucose is highlighted in the dashed box (a~e). The gray box indicates reference spots. (B) The intensity of spots corresponding to solid and dashed boxes is shown in A. The positive control was quantified using Image J software and subtracted from the background, then expressed as a ratio to the positive control. The ratio of each spot from untreated control cells, cultured in high glucose medium was assigned a value of one.