Gallic acid-gold nanoparticles enhance radiation-induced cell death of human glioma U251 cells

Abstract

Glioblastoma multiforme (GBM) is among the most common adult brain tumors with invariably fatal character. Following the limited conventional therapies, almost all patients, however, presented with symptoms at the time of recurrence. It is dire to develop novel therapeutic strategies to improve the current treatment of GBM. Gallic acid is a well-established antioxidant, presenting a promising new selective anti-cancer drug, while gold nanoparticles (GNPs) can be developed as versatile nontoxic carriers for anti-cancer drug delivery. Here, we prepared gallic acid-GNPs (GA-GNPs) by loading gallic acid onto GNPs, reduction products of tetrachloroauric acid by sodium citrate, through physical and agitation adsorption. GA-GNPs, rather than GNPs alone, significantly inhibited the survival of U251 GBM cells, as well as enhanced radiation-induced cell death. Moreover, GA-GNPs plus radiation arrested the cell cycle of U251 at the S and G2/M phases and triggered apoptotic cell death, which is supported by increased BAX protein levels and decreased expression of BCL-2. Thus, GA-GNPs have great potential in the combination with radiation therapy in future studies for GBM treatment.

Keywords: gallic acid; glioma; gold nanoparticles; radiotherapy.

FIGURE 1

Characterization of gallic acid and gold nanoparticles. (a) Chemical structure of gallic acid. (b) pH values of gallic acid solutions with different concentrations (0, 100, 200, and 300 μg/ml). (c) The morphology of gold nanoparticles was detected by transmission electron microscope. Left panel: low‐magnification image (scale bar = 80 nm). Right panel: high‐magnification image (scale bar = 20 nm). (d) Particle size distribution of GNPs by dynamic light scattering instrument (DLS)

FIGURE 2

Generation of GA‐GNPs and definition of GA:GNP feed ratio. (a) The ultraviolet absorption peak of gallic acid solution detected by ultraviolet spectrophotometer analysis (~265 nm). (b) The linear equation of the relationship between the concentration of gallic acid solution and the absorbance at wavelength 265 nm. (c) Photographs showing the color change of GNP (pale red) reacted with gallic acid (colorless). The GA‐GNPs sample in the middle demonstrates pale purple. (d) The feed ratio of GA:GNPs was determined by adding different amounts of gallic acid to GNPs (1:1, 2:1, 3:1, and 4:1)

FIGURE 3

GA‐GNPs demonstrate cytotoxic effect on human glioma U251 cells and lead to sensitization to radiation. (a) U251 cells were treated with different concentrations of GNPs or GA‐GNPs (100, 150, 200 μg/ml) for indicated time points (days 1, 2, and 3). Cell survival was determined by MTT analysis. B. U251 cells were treated with combination of different concentrations of GA‐GNPs (100, 150, and 200 μg/ml) and multiple dose of radiation (0, 2, 4, 6, 8,10, and 12 Gy). After 48 hr, cell viability was determined by MTT analysis

FIGURE 4

GA‐GNPs enhance RT‐induced S and G2/M cell cycle arrest and cell death in U251 cells. (a) Cell cycle analysis of U251 cells treated with control, gallic acid, GNPs, and GA‐GNPs in the presence of radiation (8 Gy) by flow cytometry. (b) U251 cells were treated with control, gallic acid, GNPs, and GA‐GNPs in the presence of radiation (8 Gy), and the early apoptotic and dead cells were quantitated by annexin V and PI staining in flow cytometry analysis. (c) Immunoblotting analysis to determine the relative expression of BAX and BCL‐2 proteins in U251 cells treated with control, gallic acid, GNPs, and GA‐GNPs in the presence of radiation (8 Gy). (d) Western blots (n = 3) were quantitated for BAX (left panel) and BCL‐2 (right panel) protein levels relative to GPADH in the indicated treatment conditions