8-Methoxypsoralen is a competitive inhibitor of glutathione S-transferase P1-1

Abstract

The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy.

Keywords: 8-MOP; GST; glioblastoma.

Figure 1

(A) Concentration-dependent inhibition of GST-π activity by 8-MOP. Data did not present normal distribution and were analyzed by Kruskal-Wallis non-parametric test followed by Dunn's Multiple Comparison test; ^*p < 0.05 compared to the control group. (B) Michaelis-Mentem (R² = 0.9874 without 8-MOP and 0.9545 with 8-MOP) and (C) Lineweaver-Burk plots showing competitive inhibition of human GST-π toward CDNB by 8-MOP. The K_m and V_max of the enzyme for CDNB were 0.30 mM (SD = 0.05) and 11.58 μmol/min.mg (SD = 0.77), respectively, but these same values in the presence of 0.1 mM 8-MOP were 0.68 mM (SD = 0.06) and 11.71 μmol/min.mg (SD = 3.10). (D) Michaelis-Mentem (R² = 0.9733 without 8-MOP and 0.9736 with 8-MOP) and (E) Lineweaver-Burk plots showing an uncompetitive inhibition of human GST-π toward GSH by 8-MOP. The K_m and V_max for GSH were 0.25 mM (SD = 0.08) and 11.36 μmol/min.mg (SD = 1.31), respectively. These values in the presence of 0.1 mM 8-MOP were 0.09 mM (SD = 0.02) and 4.70 μmol/min.mg (SD = 0.21). The enzyme was used at 0.1 U/mL. The graphs are representative of five independent experiments.

Figure 2

(A) Absorption spectra of CDNB and 8-MOP in the presence of GSH. (B) Spectrophotometric evidences of DNP-SG, but not “8-MOP-SG,” in spectra after addition of GST-π. The maximum absorbance values for the solutions containing CDNB/GSH/enzyme (250, 340 nm) and 8-MOP/GSH/enzyme (248, 301 nm) were used for HPLC analysis. (C) Chromatographic detection of DNP-SG (RT = 4.6 min.), and (D) no evidences of “8-MOP-SG.”

Figure 3

(A) Interactions between 8-MOP (white) and GST after 2 ns simulation. The regions of interactions are in yellow. It is also showed details (interacting residues in yellow) and scheme of interactions between residues in the active site of GST and the 8-MOP. GSH is in Green. (B) The same for the inhibitor NBDHEX.

Figure 4

(A) GL-15 cells labeled positively to GST-π. (B) Conjugation reaction between CDNB and GSH under increasing concentrations catalyzed by GST in the cytosolic protein extract of GL-15 cells, followed until the maximum rate was attained. Line represents non-linear regression (R² = 0.9770) of the data. (C) Concentration-dependent inhibition of GST by 8-MOP under maximum rate conditions (1 mM CDNB). Data did not present normal distribution and were analyzed by Kruskal-Wallis non-parametric test (excluding the first group without DMSO) followed by Dunn's Multiple Comparison test ^*p < 0.05 and ^**p < 0.01 compared to the control group (1% DMSO, without 8-MOP). (D) Intracellular GSH assessed by MCB assay. GSH was present in control conditions and was depleted by CDNB after 30 min exposure, but not by 8-MOP.

Figure 5

(A) MTT assay data showing that treatment with vehicle and 8-MOP did not change cell viability. (B) GL-15 cells become more sensible to high concentrations of temozolomide (TMZ) after treatment with 8-MOP, that significantly reduced viability (MTT assay) of 0.6 and 1 mM TZM treated cells in 17.4% and 16%, respectively. (C) 8-MOP also acted as chemosensitizer for etoposide, what was statistically significant at 0.001 and 0.1 mM. DMSO was kept at 0.05% and 8-MOP at 0.05 mM in these experiments. ^*p < 0.05 and ^**p < 0.01. ^#p < 0.01 compared to group treated with 8-MOP alone in the experiment with TMZ (Two-way ANOVA and Bonferroni post-test). Values are means (±s.e.m.). (D) The pictures show reduction of cellularity and morphological changes promoted by the associations.

Figure 6

(A) Decrease of 21.5% (± 1.9), 29.4% (± 1.4) and 33.4% (± 1.8) on GL-15 viability after 48 h treatment with 8-MOP at increasing concentrations (gray bars). Normal cells (white bars) were less affected (decrease of 17.2% (± 5.3) only at maximal concentration). Data are from MTT assay. Values are means (±s.e.m.). ^*p < 0.01 (One-way ANOVA and Dunnett's Multiple Comparison post-test, data from each cell type were statistically analyzed separately). 0.05% DMSO was present in all groups. (B) Cell blebbing visualized by phase contrast microscopy and (C) condensed/fragmented Hoechst 33258 stained nuclei of GL-15. These findings are suggestive of apoptosis. No morphological evidences of apoptosis were found among treated astrocytes. (D) Reduced cell proliferation (evaluated by trypan blue exclusion assay) in GL-15 cells after long-term exposure to low dose of 8-MOP (10 days, 0.02 mM). The number of cells increased 380% (320,470) in control conditions against 270% (235,315) in 8-MOP treated group. Values are median (25% percentile, 75% percentile). Astrocytes were not affected at all. ^*p < 0.05 (Mann Whitney non-parametric test).