Effects of Glucose-6-Phosphate Dehydrogenase Deficiency on Cytotoxicity of Benzoquinone on K562 Cells

Objective To evaluate the effects of glucose-6-phosphate dehydrogenase (G6PD) deficiency on the cytotoxicity of benzoquinone (BQ) on K562 cells.

Methods After treatment with different concentrations of BQ (0, 10, 20, and 40 μmol/L), MTT assay was used to detect the relative growth rate of K562-WT cells, and Western blot assay was used to measure the protein expression level of G6PD. RNA interference lentivirus targeting G6PD gene was constructed and transfected into K562-WT cells, while negative control group was transfected with empty vector. Quantitative real-time polymerase chain reaction (real-time PCR) was applied to measure the mRNA expression of G6PD. MTT assay and colorimetric assay were used to detect the relative growth rate as well as reduced glutathione (GSH) and oxidized glutathione (GSSG) in G6PD defective K562-WT cells (K562-G6PD△) and negative control cells after treatment with different concentrations of BQ.

Results The results of MTT assay indicated that the relative growth rates of K562-WT cells remarkably decreased with higher BQ concentrations compared with 0μmol/L BQ group (P < 0.05) at 24, 48, and 72 h. The results of Western blot showed that the G6PD protein level was increased first (r=0.809, P=0.008) and then decreased when the cells were exposed to 40 μmol/L BQ, but was still higher than that of 0 μmol/L BQ group (P < 0.05). The results of real-time PCR showed the G6PD mRNA expression of K562-G6PD△ cells was decreased by 86.65% compared with the negative control cells. That suggested K562-G6PD△ cells were successfully constructed. The results of MTT assay indicated that the relative growth rate of K562-G6PD△ cells was remarkably decreased compared with the negative control cells at each concentration of BQ (P < 0.05). The results of colorimetric assay showed that the level of GSH in K562-G6PD△ cells was decreased when exposed to BQ concentrations of 20 and 40 μmol/L, while in the negative control cells the decrease occurred at 40 μmol/L. The level of GSSG in K562-G6PD△ cells increased significantly compared with the negative control cells when exposed to the BQ concentration of 10μmol/L (P < 0.05).

Conclusion  The results of this study suggested that the proliferation of K562-WT cells could be inhibited with exposure to BQ, while G6PD is activated to produce GSH to resist oxidative damage. So, G6PD couldn't be activated in G6PD defective cells, which might lead to GSH depletion and GSSG accumulation as well as increase the cytotoxicity when the cells are exposed to relatively low doses of BQ.