{"paper_id":"f6cbc69d-414b-48a3-8a63-51a35a6865ad","body_text":"Ovarian cancer (OC) is a leading cause of mortality among gynecological cancers, with five-year survival rates between 40% and 50%\n[ 1 ]. The poor prognosis associated with OC can be attributed to its aggressiveness, diverse\nhistological types and late-stage symptom manifestation [ 2 - 3 ].\nThe main therapeutic options for OC are chemotherapy, targeted treatments and radical cytoreductive surgery [ 4 ].\nHistologically, fallopian tubes, ovary and peritoneum are the primary sites for the development of OC [ 5 ].\nBased on the classification, there are 3 types of OC, including high-grade serous OC (HGSOC), which originates from the tubal epithelium\nand accounts for 75% of OC cases. Clear cell and endometrioid ovarian carcinoma, also known as endometriosis-related ovarian carcinoma\n(EROC), arise from endometriotic lesions and provide relatively superior clinical outcomes compared to high-grade serous ovarian\ncarcinoma (HGSOC) [ 6 ,  7 ]. Genetic factors play a\nsubstantial role in OC progression, including BRCA1 and BRCA2 alterations, but OC may develop from non-BRCA mutations\n[ 8 ,  9 ]. Single nucleotide polymorphisms (SNPs) represent\nprevalent genetic variations, including deletions, substitutions and insertions at specific loci within both coding and non-coding\nareas. More than 100 SNPs have been observed that significantly contribute to the development of OC [ 10 ].\nCytokines and chemokine's act as growth factors in cancer progression through paracrine and autocrine signaling. Their levels typically\nincrease in response to infection or injury and are often expressed by epithelial cells, which are frequent targets of infections\n[ 11 ]. Elevated levels of interleukin-8 (IL-8) and CRP have been detected in ovarian cancer (OC)\ntissues and ascites, with increased circulating IL-8 levels strongly associated with advanced tumor stages and poor outcomes. High IL-8\nconcentrations are consistently observed in OC patients [ 12 ]. Inflammatory chemokines, especially\nIL-8 and IL-6, play a significant part in tumor genesis across many malignancies, including OC [ 13 ,\n 14 ]. IL-8, a member of the CXC chemokine family initially recognized as a neutrophil\nchemo-attractant, has been shown to play a critical role in tumor growth, invasion, apoptosis and metastasis. It is a key contributor to\ncancer aggression, angiogenesis and metastatic progression [ 15 ,  16 ,\n 17 - 18 ]. Cancer cells secrete IL-8 in both paracrine and\nautocrine manners, as observed in ovarian cancer (OC). IL-8's interaction with CXCR1 and CXCR2, two cell-surface G protein-coupled\nreceptors, activates IL-8 signaling pathways [ 19 ]. The tumor microenvironment, comprising cancer\ncells, infiltrating neutrophils, cancer-associated macrophages and endothelial cells, often exhibits elevated IL-8 levels and receptor\nexpression. IL-8 activates angiogenesis in endothelial cells, promoting the survival of both cancerous and endothelial cells while\nfacilitating the recruitment of neutrophils to the tumor site [ 20 ]. Elevated IL-8 expression is\nobserved in cancerous ovarian tissues compared to normal tissues. Both OC and stromal cells within the tumor microenvironment are\nsignificant sources of IL-8 and concurrently express the CXCR1 and CXCR2 receptors [ 21 ,\n 22 ]. Eventually, IL-8 has been linked to the progression of chemo resistance through its role in\ninhibiting apoptosis and fostering the survival of OC cells [ 23 ]. IL-8 is located on chromosome\n4q12-q135, comprising four exons and three introns and encodes a protein of 99 amino acids. Its 5' promoter region contains multiple\nnuclear binding sites (NBDs) essential for transcriptional regulation. Nuclear factor κB (NF-κB) regulates IL-8\ntranscription via TNF receptor-associated factor 6 (TRAF6) and tumor necrosis factor (TNF) [ 24 ].\nThe regulation of IL-8 gene expression and structural variations can significantly impact its functionality. SNPs in the promoter region\nmay alter IL-8 expression, triggering a pro-inflammatory response associated with various tumor phenotypes. Additionally, structural\nmodifications can affect the receptor binding sites, potentially disrupting IL-8's interaction with its targets\n[ 20 ,  24 ]. Genetic variants in the IL-8 gene influence its\nexpression and protein structure, affecting IL-8-mediated signaling pathways. Notably, Five SNPs have been extensively studied 276A/T,\n+396G/T, +678T/C, +781C/T and +1633C/T [ 25 ]. However, the -251 A > T located in the promoter region\nand +781 C/T polymorphisms in intron 1, involved in gene regulation and transcription of IL-8, are extensively studied, with implications\nfor IL-8 levels and gene regulation [ 26 ]. Therefore, it is of interest to establish the\nassociation between IL-8 SNPs and OC risk.\n\nThis study was conducted according to a control and case design. Blood samples of control/healthy females were collected. Tissue\nsamples of OC patients were collected from King Abdulaziz University. All the samples were stored at -80°C before use.\n\nDNA of both OC tissue and blood samples of healthy was isolated using a Genomic DNA Purification Kit (Promega, United States,\nCat#A1125) following the manufacturer's protocol. The concentration and purity of DNA were measured using Nano Drop (ND-2000, Thermo\nScientific). Isolated DNA samples were amplified using conventional PCR (Applied Bio system Verity) with an exon-specific primer\ndesigned and manufactured from Macrogen Inc, South Korea (Forward-5'-AATCCTTAATCACTTTTTCCCCCAA-3', Reverse-5'-ACTACTGTAATCCTAACACCTGGAA-3',\nannealing temperature 56°C and amplicon size 265 bp). DreamTaq PCR Master Mix (Thermo Scientific, Cat# K1071) was used to amplify\nDNA. The master mix is a ready-to-use solution containing bacterially derived Taq DNA polymerase, dNTPs, MgCl 2  and reaction\nbuffers at optimal concentrations to ensure efficient DNA template amplification during PCR. For each run, the thermal cycle conditions\nwere as follows: at the denaturation step, initial denaturation was set at 95°C for 2 min, followed by 35 cycles of denaturation at\n95°C for 30 sec, annealing at (50-56°C) for 30 sec, extension at 72°C for 5 min. A final extension was set at 72°C for 5\nmin and the PCR product was kept at 4°C for an infinite time. Gel electrophoresis was performed to validate the amplification of\nDNA. SNPs of IL8 were determined by automated DNA sequencing (3500XL genetic analyzer-applied Biosystem). The cycle sequencing PCR\nreaction was performed using the forward primer according to the manufacturer's protocol (Big Dye Terminator Reaction Kit v3.1, Applied\nBio systems). The resulting chromatograms were assessed for quality, with peak analysis compared against reference DNA sequences. SNPs\nwere identified using sequence evaluation software and subsequently confirmed through reverse-strand sequencing.\n\nThe results were statistically analyzed using JASP statistical software v.0.18.3.0 for Windows [ 34 ]\nand the Vassar Stats Web-based tool for Statistical Computation [ 35 ]. Fisher's exact test was\nused to examine differences in genotype frequencies between patient and control groups, while age distribution across study groups was\nassessed with Student's t-test. A p-value of ≤ 0.05 was considered statistically significant.\n\nThe mean age of cases and controls was 57.60±14.88 years and 47.30±11.4 years, respectively. High-grade serous\ncarcinoma (HGSC) was the most prevalent histological type, representing 60% of cases. This was followed by low-grade serous carcinoma\n(LGSC) at 20%, endometrioid carcinoma at 10% and germ cell tumors at 5%. Histological data was unavailable for the remaining 5% of\npatients. The majority of OC cases (90%) were diagnosed at an advanced stage, with only 5% identified at an early stage. Tumor staging\ndata was missing for 5% of the patients.\n\nGenotyping of 20 OC cases and 10 healthy control women was performed using automated DNA sequencing in IL-8 exons 2. We identified a\nnon-synonymous SNP (nsSNP) in exon 2 of the IL-8 gene at position c.193 to 193 G>A, present in 25% of ovarian cancer patients. This\nnsSNP was found in 5 OC patients diagnosed with advanced-stage OC. The genotyping results are summarized in  Table 1 .\nThis c.193 G >A nsSNP resulted in the amino acid (AA) change (Glu65Leu).  Figure 1  represents the\nelectropherogram of nsSNP identified in automated DNA sequencing.\n\nWe identified deleterious analysis using four SNP prediction algorithms: Polyphen, PROVEAN, PhD-SNP and SNPs & GO. All the web\ntools predicted that nsSNP (Glu65Leu) may have a deleterious effect, as shown in  Table 2 .\n\nThe damaging impact of mutation Glu65Leu was predicted by the missense3D, as presented in  Table 2 .\nIn addition, Glu65Leu substitution also predicated the structural damage by buried H-bond breakage by disrupting all side\nchain/side-chain H-bond(s) and/or side-chain/main-chain bond(s) H-bonds formed by buried Glu residue. The structural change in the wild\ntype and mutant is shown in  Figure 2 .\n\nOC is one of the most common causes of death in women due to asymptomatic progression and delay in diagnosis as there is a lack of\nappropriate diagnostic tools for early detection of tumor [ 36 ,  37 ].\nOC is characterized by its strong interaction with the immune system, often initiating a systemic acute inflammatory response pathway\n[ 38 ]. Interleukins are signaling molecules that regulate immune and inflammatory responses.\nInterleukins play a central role in driving systemic changes, such as inflammation and immune modulation, which contribute to tumor\nprogression and metastasis [ 39 ]. Interleukin-8 (IL-8) initially recognized as a neutrophil chemo\nattractant and a key mediator of leukocyte-driven inflammation, plays a critical role in cancer progression in various cancers including\nOC [ 40 ,  41 - 42 ].\nElevated IL-8 levels in serum, ascetic fluid and tumor tissues, along with its overexpression, are strongly associated with poor\nprognosis and decreased survival in OC patients [ 43 ]. Furthermore, IL-8 polymorphisms have been\nlinked to an increased risk of developing OC [ 44 ]. Previous studies have primarily focused on two\nSNPs: -251 A/T in the promoter region and +781 C/T in intron 1, both SNPs play a role in the susceptibility to OC risk\n[ 44 ,  45 - 46 ].\nHowever, there is a lack of studies focusing on IL-8 polymorphisms within the coding region of IL-8 in OC. Hence, in this study, we\naim to investigate a possible polymorphism in the IL-8 coding region. Therefore, this study aimed to identify potential polymorphisms in\nthe IL-8 coding region and assess their impact. DNA was extracted from tumor tissues of OC patients and blood samples from healthy\ncontrols, followed by PCR amplification and automated DNA sequencing. Additionally, bioinformatics analysis was performed to predict the\neffects of identified SNPs on IL-8 protein structure and stability.\nIn the current study, we have identified a novel nsSNP at c.193G > A in the IL-8 exons 2, as shown in  Figure 1 .\nThis mutation involves a nucleotide substitution at position 193 (G to A), leading to an amino acid change from glutamic acid to leucine\nat position 65, as shown in  Table 1 . However, statistical analysis showed no significant\nassociation between this nsSNP and OC, as shown in  Table 1 . Hence, studies with larger cohort\nsizes are warranted to further investigate the correlation between this nsSNP and OC risk. Furthermore, studies have demonstrated\nsignificant associations between IL-8 polymorphisms, particularly the +781 (T/T) and +2767 (T/T) genotypes and an increased risk of OC\n[ 1 ,  44 ]. In addition,  in-silico  analysis was conducted to\npredict the impact of the amino acid change. Our results indicated that the c.193G>A mutation is likely damaging or deleterious, as\ndetermined by bioinformatics tools such as PolyPhen, PhD-SNP, PROVEAN and SNPs & GO, as shown in  Table 2 .\nNotably, protein stability analysis using MUPro suggested that the Glu65Leu nsSNP may decrease protein stability. Studies have shown\nthat mutations can lead to protein dysfunction by reducing solubility and impairing function when they cause destabilization beyond a\ncritical ΔΔG threshold [ 47 ,  48 ,  49 -\n 50 ]. Structural analysis using Missense3D also predicted significant damage caused by the\nGlu65Leu mutation. This mutation may disrupt essential hydrogen bonds, potentially compromising protein stability.\nThus, our observation revealed that novel nsSNP c.193G>A (Glu65Leu) in the IL-8 exons 2 could have a damaging effect on protein\nstructure/ and function, which may directly impact the interaction with its receptor CXR1\\CXR2. Thus, the present in silico functional\nanalysis of this novel deleterious nsSNP c.193G > A, needs to be further validated by correlating the level of IL8 production\n in vitro  study before drawing any definite association of this mutation with OC risk.\n\nOur findings suggest that IL-8 c.193 G>A (Glu65Leu) polymorphism has potential effect on IL8 structure. However, further\ninvestigation of the functional role of IL-8 polymorphism and its impact on the susceptibility of tumor remains ascertained prior to\nestablishing the significance of IL-8 polymorphism as a potential biomarker for OC risk assessment.\n\nThe authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia, for\nfunding this research work through the project number (1171).\n\nInformed consent was obtained from all subjects involved in the study.","source_license":"CC-BY-4.0","license_restricted":false}