Antitumor Effects of 5-Aminolevulinic Acid on Human Malignant Glioblastoma Cells

Abstract

5-Aminolevulinic acid (5-ALA) is a naturally occurring non-proteinogenic amino acid, which contributes to the diagnosis and therapeutic approaches of various cancers, including glioblastoma (GBM). In the present study, we aimed to investigate whether 5-ALA exerted cytotoxic effects on GBM cells. We assessed cell viability, apoptosis rate, mRNA expressions of various apoptosis-related genes, generation of reactive oxygen species (ROS), and migration ability of the human U-87 malignant GBM cell line (U87MG) treated with 5-ALA at different doses. The half-maximal inhibitory concentration of 5-ALA on U87MG cells was 500 μg/mL after 7 days; 5-ALA was not toxic for human optic cells and NIH-3T3 cells at this concentration. The application of 5-ALA led to a significant increase in apoptotic cells, enhancement of Bax and p53 expressions, reduction in Bcl-2 expression, and an increase in ROS generation. Furthermore, the application of 5-ALA increased the accumulation of U87MG cells in the SUB-G1 population, decreased the expression of cyclin D1, and reduced the migration ability of U87MG cells. Our data indicate the potential cytotoxic effects of 5-ALA on U87MG cells. Further studies are required to determine the spectrum of the antitumor activity of 5-ALA on GBM.

Keywords: apoptosis; brain tumor; cell death; protoporphyrin; tumor cell line.

Figure 1

Cytotoxic effects of 5-aminolevulinic acid (5-ALA) on the human U-87 MG malignant GBM cell line (U87MG). Representative phase-contrast micrographs of U87MG cells following 1, 2, and 7 days of treatment with 5-ALA (0–3750 µg/mL) are presented. Cytotoxicity of 5-ALA on the viability of U87MG cells was evaluated using the MTT method. To ascertain the half-maximal inhibitory concentration of 5-ALA, the percentage of live U87MG cells was assessed following 1, 2, and 7 days of treatment with various doses of 5-ALA. The results are presented as means ± standard deviation. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 2

The effect of 5-aminolevulinic acid (5-ALA) on the viability of the human primary optic cells and NIH/3T3 cells. Representative phase-contrast micrographs of primary optic cells obtained from the human optic nerves and NIH/3T3 cells following 7 days of treatment with 5-ALA (500 µg/mL) are shown. Cytotoxicity of 5-ALA (0–3750 µg/mL) on the viability of these non-tumoral cells was evaluated using the MTT assay. To evaluate the half-maximal inhibitory concentration of 5-ALA, the percentage of live cells was assessed following 7 days of treatment with various doses of 5-ALA. The results are presented as means ± standard deviation. *, **, *** Indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 3

The effects of 5-aminolevulinic acid (5-ALA, 250 µg/mL) on the expression of cyclin D1 and cell cycle of the human U-87 MG malignant GBM cells (U87MG). (a) The cell cycle was assessed in U87MG cells incubated with 5-ALA for 7 days and the control group. Note the increased accumulation of U87MG cells in the SUB-G1 population as compared with the control group; (b) the application of 5-ALA significantly reduced the expression of cyclin D in U87MG cells as compared with the control group. The results are presented as means ± standard deviation. *** Indicates p < 0.001.

Figure 4

The effects of 5-aminolevulinic acid (5-ALA) on apoptosis and the mRNA expression of various apoptotic biomarkers (p53, Bax, and Bcl-2) of the human U-87 MG malignant GBM cells (U87MG). (a) U87MG cells were stained with Annexin V/propidium iodide and evaluated using the flow cytometry technique. U87MG cells were treated with 5-ALA at 250 µg/mL for 7 days. Untreated cells were evaluated as the control group. Diagrams quarter 4 (Q4) to Q1 represent live cells, early apoptotic, late apoptotic, and necrotic cells, respectively. U87MG cells incubated with 5-ALA showed a higher amount of early and late apoptotic cells; (b) 5-ALA significantly enhanced the values of Bax and p53, as well as decreased Bcl-2 expression as compared with the control group. The results are presented as means ± standard deviation. **, *** Indicate p < 0.01 and p < 0.001, respectively.

Figure 5

The effects of 5-aminolevulinic acid (5-ALA) on the induction of reactive oxygen species (ROS) in the human U-87 MG malignant GBM cells (U87MG) after 7 days. (a) Photomicrographs of ROS generation of U87MG cells in the control, 5-ALA (250 and 500 μg/mL), and tert-butyl hydroperoxide (TBHP) groups were obtained using fluorescence microscopy; (b) ROS generation was evaluated by the measurement of fluorescent intensities. Note a significantly greater ROS production after the application of 5-ALA at 500 μg/mL as compared with the cells treated with 5-ALA at 250 μg/mL and the control group. The results are presented as means ± standard deviation. *, **, *** Indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 6

The effects of 5-aminolevulinic acid (5-ALA) on the migration of the human U-87 MG malignant GBM cells (U87MG). The migration of U87MG cells was evaluated by the wound healing approach. Using a micropipette tip, a scratch wound was created through the surface of a confluent monolayer of U87MG cells, and then 5-ALA at 250 µg/mL was applied. The microscopic image were captured after 2, 24, and 48 h of treatment. (a) The migration rate of U87MG cells was assessed by the number of cells within the wound area; (b) 5-ALA exerted a significant inhibitory effect on the migration of U87MG cells after 24 h as compared with the control group. The results are presented as means ± standard deviation. * Indicates p < 0.05.