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. 2020 Apr 7;10(1):6032.
doi: 10.1038/s41598-020-63034-3.

Glutathione S-transferasesP1 AA (105Ile) Allele Increases Oral Cancer Risk, Interacts Strongly With c-Jun Kinase and Weakly Detoxifies Areca-Nut Metabolites

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Free PMC article

Glutathione S-transferasesP1 AA (105Ile) Allele Increases Oral Cancer Risk, Interacts Strongly With c-Jun Kinase and Weakly Detoxifies Areca-Nut Metabolites

Pallavi Yadav et al. Sci Rep. .
Free PMC article

Abstract

The Glutathione S-transferases (GSTs) protects cellular DNA against oxidative damage. The role of GSTP1 polymorphism (A313G; Ile105Val) as a susceptibility factor in oral cancer was evaluated in a hospital-based case-control study in North-East India, because the habit of chewing raw areca-nut (RAN) with/without tobacco is common in this region. Genetic polymorphism was investigated by genotyping 445 cases and 444 controls. Individuals with the GSTP1 AA-genotype showed association with the oral cancer (OR = 3.1, 95% CI = 2.4-4.2, p = 0.0002). Even after adjusting for age, sex and habit the AA-genotype is found to be significantly associated with oral cancer (OR = 2.4, 95% CI = 1.7-3.2, p = 0.0001). A protein-protein docking analysis demonstrated that in the GG-genotype the binding geometry between c-Jun Kinase and GSTP1 was disrupted. It was validated by immunohistochemistry in human samples, showing lower c-Jun-phosphorylation and down-regulation of pro-apoptotic genes in normal oral epithelial cells with the AA-genotype. In silico docking revealed that AA-genotype weakly detoxifies the RAN/tobacco metabolites. In addition, experiments revealed a higher level of 8-Oxo-2'-deoxyguanosine induction in tumor samples with the AA-genotype. Thus, habit of using RAN/tobacco and GSTP1 AA-genotype together play a significant role in predisposition to oral cancer risk by showing higher DNA-lesions and lower c-Jun phosphorylation that may inhibit apoptosis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Analysis of SNP of GSTP1 A313G and its 3D monomer structure. (A) A complete gel picture of the PCR-product of GSTP1 from six samples; M-marker. (B) A complete gel picture shows the result of PCR-RFLP analysis for GSTP1 SNP. The presence of Alw26I restriction site yielded 328 and 105 bp fragments for the A allele and 222, 105 and 106 bp fragments for the G allele. (C) Partial sequence chromatograms of GSTP1 A313G polymorphism (arrow marked) are shown from three subjects whose RFLP data are depicted in B. (D) 3D structure of wild GSTP1 I105 (Camel colour) and mutant GSTP1 V105 (Carolina blue colour). Altered sites were marked by arrows and showed the amino acids and its position.
Figure 2
Figure 2
Effect of GSTP1 I105V polymorphism on c-Jun phosphorylation and expression of proapoptotic genes in normal oral tissue in OSCC patients. (A) Representative images of an immunohistochemical analysis of adjacent normal oral tissues in OSCC patients done with anti-c-Jun phosphorylation antibody. Human samples were collected from the persons whose GSTP1 proteins having Ile/Ile or Ile/Val or Val/Val at 105 positions. (B) The level of c-Jun phosphorylation in normal oral tissues analyzed by H-score were shown. Data were analyzed by Student’s t-test. *Two-tailed p < 0.004, n = 20 for Ile/Ile and 12 for Ile/Val samples; **P < 0.0001, n = 10 for Ile/Ile and 6 for Val/Val samples. The magnification of all these images is 40X. (C,D) Effect of GSTP1 genotypes on quatitative expression of Bim and PUMA mRNA. Total RNA was isolated from tumor cells collected from samples having either GSTP1 Ile/Ile or Ile/Val or Val/Val genotypes for real-time qPCR using either Bim primers (C) or PUMA primers (D) following the procedures mentioned in the Methods. Data are plotted as a histogram. Each bar is the mean ± SD derived from 20 Ile/Ile, 12 Ile/Val and 6 Val/Val samples. The value of p < 0.05 consider to be Significant in unpaired t-test.
Figure 3
Figure 3
An electrostatic interactions with toxic substances at active site cavity of GSTP1 and its influence on the individual sensitivity to genotoxic effects. (A) Comparative electrostatic interactions of reduced GSH and toxic metabolites like, GA, arecoline N-oxide and arecaidenylglycine derived from raw areca-nut, and NNAL derived from tobacco, with dimeric GSTP1 proteins having Ile/Ile, Ile/Val and Val/Val at 105 positions. Red colour indicates negative charge and blue positive charge. The distance of GSH from the active pocket indicates its relative affinity for the active site residues. (B) Quantitation of 8-OHdG (ng/ml) in DNA digests was performed by ELISA-kit in both blood and tumor DNA from oral cancer patients having either RAN-chewing or RAN + tobacco-using habit. Data are plotted as a histogram. Each bar is the mean ± SD derived from 12 Ile/Ile, 12 Ile/Val and 6 Val/Val samples. The value of p < 0.05 consider to be Significant in unpaired t-test.

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