Association of Interleukin, Vitamin D receptor (VDR) gene polymorphism and oxidateive stress in cancer

Abstract

Cancer has become a global disease and its burden is estimated to raise approximately 9.6 million deaths. 18.1 million new cases of cancers, registered in 2018. Etiology of cancer is not yet completely understood, however various factors including chronic inflammation, Vitamin D (VD) deficiency, dysregulation in mineral levels and genetic aberrations like single nucleotide polymorphism (SNPs) are being considered. Oxidative stress is the resultant burden of inflammatory response which triggers the abrupt proinflammatory cytokine levels, proposed as one of the causal candidate for carcinogenesis. Interleukins (IL) one of the cytokines, regulates the inflammatory response. The SNPs in IL genes may affect their circulating levels. The prospective of the current study is to assess the association of IL SNPs, minerals, VD and oxidative stress biomarkers with cancer. METHODOLOGY Present study is comprised of 200 blood samples of cancer patients with 50 controls, followed by informed consent from two tertiary care hospitals of Karachi. The DNA samples were analyzed, after extracting through Salting out technique. Oxidative stress biomarkers including Catalase (CAT), Xanthine Oxidase (XO), Malondialdehyde (MDA), Nitric Oxide (NO), Glutathione Reductase (GR), and Superoxide Dismutase (SOD) were estimated spectrophotometrically in plasma samples. In addition, Interleukin (IL-6) and VD were determined by ELISA, while calcium and magnesium levels were estimated by ion selective electrode (ISE) and spectrophotometric analysis respectively. Genotyping of SNPs including Vitamin D receptor- caudal homeobox 2 (VDR-Cdx2) polymorphism was performed using Tetra primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method. However, the IL-17 gene SNPs including, IL-17A (rs2275913 G/A) and IL-17F (rs763780 T/G) and IL-10 SNPs were investigated by the help of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Whereas, IL-6 (174 G/C) SNP was analyzed using allele specific PCR amplification protocol. The amplification yields were resolved on agarose gel, stained with ethidium bromide and visualized under UV light. XXII RESULTS Significantly high levels of oxidative markers; XO, MDA and NO whereas, markedly low plasma levels of antioxidant enzymes; CAT, SOD and GR along with decreased Vitamin D and Magnesium status in cancer patients were observed. Moreover, Pearson’s correlation of Vitamin D and IL-6 with oxidative stress biomarkers in different cancers were analyzed. The significant inverse relation was observed between Vitamin D and IL-6 in Oral cancer (OC), Colorectal cancer (CRC) and Multiple myeloma (MM) patients. A strong positive correlation between IL-6 and MDA was observed in OC and CRC groups. Although a negative correlation was found between Vitamin D and MDA in all cancer groups. Furthermore a strong negative correlation between NO and SOD has been observed in CRC patients. On the other hand strong negative correlation of Mg+2 with Ca:Mg ratio and other oxidative stress biomarkers in all cancer types were found. The genotypic assortment/distribution in the control and disease groups were according to Hardy Weinberg’s Equilibrium. These outcomes revealed that the genotypes including CC genotype of IL – 6 (174 G/C), GG and AA genotype of IL-10 (1082), CA and CC genotype of IL-10 (529), TC and TT genotype of IL-10 (819), GA and GG genotype of IL - 17A (G/A), TC and TT genotype of IL - 17F (T/C) and the AG and GG genotypes of VDR Cdx2 were found to be associated with cancer (p<0.05). CONCLUSION The outcomes of current study in the light of abovementioned results revealed that antioxidant profile, dysregulation in mineral levels and VD status could be used as diagnostic tool for individuals who are at higher risk. These results also unveiled that the above IL and VDR- Cdx2 genotypes were significantly different in cancer and control groups and could consider a peril for the initiation and progression of cancer. Conclusively, with regard to Pakistani population, the significant association between discussed SNPs and biochemical variants collaborate synergistically to promote the neoplastic process.