Impact of oxygen status on 10B-BPA uptake into human glioblastoma cells, referring to significance in boron neutron capture therapy

Abstract

Boron neutron capture therapy (BNCT) can potentially deliver high linear energy transfer particles to tumor cells without causing severe damage to surrounding normal tissue, and may thus be beneficial for cases with characteristics of infiltrative growth, which need a wider irradiation field, such as glioblastoma multiforme. Hypoxia is an important factor contributing to resistance to anticancer therapies such as radiotherapy and chemotherapy. In this study, we investigated the impact of oxygen status on 10B uptake in glioblastoma cells in vitro in order to evaluate the potential impact of local hypoxia on BNCT. T98G and A172 glioblastoma cells were used in the present study, and we examined the influence of oxygen concentration on cell viability, mRNA expression of L-amino acid transporter 1 (LAT1), and the uptake amount of 10B-BPA. T98G and A172 glioblastoma cells became quiescent after 72 h under 1% hypoxia but remained viable. Uptake of 10B-BPA, which is one of the agents for BNCT in clinical use, decreased linearly as oxygen levels were reduced from 20% through to 10%, 3% and 1%. Hypoxia with <10% O2 significantly decreased mRNA expression of LAT1 in both cell lines, indicating that reduced uptake of 10B-BPA in glioblastoma in hypoxic conditions may be due to reduced expression of this important transporter protein. Hypoxia inhibits 10B-BPA uptake in glioblastoma cells in a linear fashion, meaning that approaches to overcoming local tumor hypoxia may be an effective method of improving the success of BNCT treatment.

Fig. 1.

The influence of 1% hypoxia on survival and cell growth in the T98G glioblastoma cell line. (A) A significant difference was observed between normoxia and hypoxia cell growth; however, viable cells could be identified even after incubation under hypoxia for 72 h. (B) Cells were incubated under hypoxia for 0 h or 72 h, and the cell cycle was analyzed by flow cytometry. Hypoxic conditions increased the percentage of T98G cells in G0/G1 phase and decreased the percentage of cells in S phase. No significant difference was found for G2/M phases. Values are expressed as the mean ± standard error; two asterisks indicate P < 0.01 compared with control cells at 0 h; NS = no significant difference.

Fig. 2.

Reduced oxygen concentration results in lower ¹⁰B-BPA uptake in glioblastoma cell lines. Cells were incubated under several oxygen concentrations—20.8% (normoxia), 10%, 3% or 1% oxygen (hypoxia)—for 72 h, exposed to ¹⁰B-BPA with a ¹⁰B concentration of 10, 20 or 30 ppm for 2 h under normoxic conditions, and analyzed by ICP-AES. (A) In T98G cells, ¹⁰B-BPA uptake gradually decreased in parallel with the decrease in oxygen. There was a significant correlation between ¹⁰B-BPA uptake and the ¹⁰B-BPA concentration of the culture medium. (B) In A172 cells the same trend was observed (i.e. a gradual decrease in ¹⁰B-BPA uptake with decrease in oxygen concentration), although this did not reach statistical significance. Values are expressed as the mean ± standard error; one asterisk indicates P < 0.05 and two asterisks indicate P < 0.01, compared with 10 ppm of ¹⁰B, at each oxygen concentration.

Fig. 3.

Reduced oxygen conditions decrease mRNA expression levels of LAT1. Cells were incubated at oxygen concentrations of 20.8% (normoxia), 10% or 1% oxygen (hypoxia) for 72 h, followed by total RNA extraction and qRT-PCR. (A) In T98G, relative expression of LAT1 was significantly lower than in cells incubated under normoxia. (B) In A172, similar results were obtained as for the T98G cell line. Values are expressed as the mean ± standard error.