Abstract
Background/Aim: Cathepsin G (CTSG) has been identified as an inhibitor of breast, bladder, and colorectal cancers. The G allele of the N125S (A/G, rs45567233) functional polymorphism of the CTSG gene confers increased serum CTSG activity and has been associated with cardiovascular and neurovascular diseases. This study examined the possible correlation between the pathogenesis of basal cell carcinoma (BCC) and the functional polymorphism CTSG N125S. Patients and Methods: A total of 197 DNA samples were examined, comprising 98 BCC patients and 99 control samples of Greek origin. The CTSG N125S polymorphism was molecularly genotyped using PCR amplification, followed by enzyme digestion, and agarose gel electrophoresis of the amplified DNA fragments. Results: There was no statistically significant difference in the genotypic and allelic frequencies between the patient and the control groups. Conclusion: There is no association between the CTSG N125S polymorphism and pathogenesis of BCC.
Basal cell carcinoma (BCC), previously known as basal cell epithelioma, is the most common skin cancer worldwide, with increasing incidence rates annually and represents the 80% of skin cancers other than melanoma. BCC mortality is rare and mostly affects immunocompromised patients (1, 2). Although, it has a high incidence rate. Tumors with aggressive histopathologic features (morphea-form, metatypical, basosquamous, infiltrating) are more likely to result in metastatic BCC, which concerns 1% of patients with BCC (3).
Multiple factors, including exposure to certain environmental conditions, underlying physical traits, and genetic predisposition variants, contribute to the development of BCC. The main factor contributing to BCCs and most skin malignancies is exposure to ultraviolet (UV) radiation from the sun, especially in early life and adolescence. History of skin cancer, such as melanoma or squamous cell carcinoma (SCC), increases the risk for BCC, and people with fair skin are more vulnerable. The majority of BCCs affect adults over 50 years old, while males are more likely to develop the disease than females (4-6). In recent years, our group has revealed that increased risk for BCC is associated with some functional DNA polymorphisms that increase gene expression and lead to elevated levels of the hormone angiotensin, which plays a key role in blood pressure regulation and electrolyte balance in the renin–angiotensin system (7).
With the aim to uncover additional associations of BCC risk with functional DNA polymorphisms, we investigated the involvement of the cancer inhibitor gene CTSG that encodes cathepsin G (CTSG), a 26-kDa serine protease that is expressed in azurophil granules of neutrophilic polymorphonuclear leukocytes during the promyelocyte stage of development (8-10). CTSG has a specificity similar to that of chymotrypsin C, and may aid in the destruction and elimination of ingested pathogens as well as the remodeling of connective tissue at inflammatory locations (8). It is released during neutrophil activation by several cytokines and platelet-activating factors and subsequently promotes platelet aggregation. CTSG provides antimicrobial action against a variety of bacterial species. Furthermore, by the activation of several extracellular matrix metalloproteinases, it significantly contributes to the degradation of extracellular matrix components. Additionally, through activating osteoclast precursors, CTSG promotes the synthesis of receptor activator for nuclear factor 
B ligand (RANKL), which is essential for bone remodeling (8). Several inflammatory disorders, such as rheumatoid arthritis, coronary artery disease, ischemia reperfusion injury, and a reaction to bone metastases, have been associated with CTSG. Also, it is related to a variety of viral and inflammatory disorders, such as cystic fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, and periodontitis (8).
The CTSG gene (14q11.2) spans 2.7 kb, and, like other genes encoding serine proteases, consists of 5 exons and 4 introns. A missense variant in exon 4 of the CTSG gene (CTSG: c.374A>G, p.Asn125Ser) has functional consequences (8). Individuals homozygous for the wild type (normal) variant A allele (AA genotype) have normal CTSG activity, while carriers of the variant G allele (heterozygous GA and homozygous GG) have increased serum CTSG activity and lactoferrin levels. Increased CTSG activity is linked to higher platelet activation and plasma fibrinogen levels, which may exacerbate cardiovascular and cerebrovascular disease (8).
Recent research suggests that cathepsin G plays a significant role in the pathophysiology of certain autoimmune diseases, in inflammation and immunological response (9). Most importantly, CTSG has also been identified as a cancer inhibitor gene in breast, bladder, and colorectal cancers (10-12). This study examined the potential association between the pathophysiology of BCC and the functional polymorphism CTSG-N125S.
Patients and Methods
Ethics statement. The protocol used was approved by the Ethics Committee of the University Department of Oral and Maxillofacial Surgery (27022019) in accordance with the standards of the 1964 Declaration of Helsinki. The individuals under study gave their informed consent to be included in the study.
Subjects. This study included a total of 197 individuals of Greek origin; 98 BCC patients aged between 28 and 96 years, and 99 controls in the same age range. The patients included 41 males (41.8%) with a mean age of 70.1 (±12.5) years and 57 females (58.2%) with a mean age of 70.2 (±10.2) years. The healthy control group consisted of 45 males (45.5%) with a mean age of 68.2 (±11.6) years and 54 females (54.5%) with a mean age of 71.0 (±11.8) years. The diagnosis of BCC for all patients was set by clinical examination and biopsy findings.
Genotyping. The genotyping of CTSG N125S polymorphism was carried out using PCR amplification based on the protocol developed by Pérez-Is et al. (8), using the following primers: Forward: 5’-GCT GAG CGG GAA CGC CTA CA-3’ and Reverse: 5’-CGT AGG AAC CGA AGA TGC GG-3’. The initial denaturation step at 95°C was followed by 31 cycles at 95°C for 1 min, 58°C for 1 min, 72°C for 1 min, and finally an elongation step at 72°C for 8 min. PCR products were incubated overnight at 37°C with the restriction enzyme Bsp1286i (New England Biolabs, Ipswich, MA, USA) followed by agarose gel electrophoresis.
Statistical analysis. Software SPSS v.21.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Allele and genotype frequencies of the group of patients were compared to the respective frequencies of the control group using the Fisher’s exact test. All observed genotype and allele frequencies were prior tested for compliance with Hardy–Weinberg equilibrium. A p-value of less than 0.05 was considered statistically significant. In case of significant value, the Mantel–Haenszel method would be used for the calculation of all odds ratios with a 95% confidence interval (CI).
Results
In the group of healthy controls, the observed and expected frequencies of CTSG-N125S genotypes were not significantly different (Table I). Therefore, the control population under study was in Hardy–Weinberg equilibrium for this polymorphic trait and further analysis was valid.
Results of the Hardy–Weinberg equilibrium comparison for the control group.
Table II displays the observed genotype frequencies of the CTSG N125S polymorphism that were found in the groups of patients with BCC and healthy controls. According to the genotyping results, there was no statistically significant difference in the allelic and genotypic frequencies between the control and patient groups. Consequently, no correlation was discovered between the CTSG gene’s N125S polymorphism and the pathophysiology of BCC or protection against it.
Genetic and allele frequency comparison between controls and patients in relation to the CTSG-N125S polymorphism.
Discussion
CTSG has been linked to a variety of inflammatory conditions, particularly those that affect the bones, like rheumatoid arthritis, periodontitis, and bone metastases. CTSG has also been reported as cancer inhibitor gene in breast (10), bladder (11) and colorectal (12) cancers. Following neutrophil stimulation by platelet-activating factor, tumor necrosis factor-alpha, and interleukin-8, CTSG is released. This causes calcium mobilization, fibrinogen’s surface expression and release, platelet secretion and aggregation, and a systemic release of the platelet thrombogenic products that cause intravascular thrombosis through the CTSG platelet receptor protease-activated receptor 4.
The CTSG N125S G allele has been correlated with elevated plasma fibrinogen levels in myocardial infarct patients, bacterial bone infection osteomyelitis, and certain neurological disorders (8). In this study, we examined the possible association of the CTSG N125S polymorphism with the pathophysiology of BCC.
The frequency of heterozygotes of the CTSG N125S polymorphism in our healthy control group (10.1%) was extremely similar to that seen in previous studies in other Caucasian populations. More specifically, it was 9.4% in Spaniards, 10% in Caucasian Americans, and 11.7% in French and British Caucasians (8, 13, 14). In addition, our Greek control population was in Hardy–Weinberg equilibrium for the polymorphic trait under study, therefore further analysis was valid. Nevertheless, the studied functional polymorphism of the CTSG gene was shown to be unrelated to an increased or decreased risk for BCC as evidenced by the lack of significant differences in genotypic and allelic frequencies between patients and healthy controls, matched for age and sex.
Although cathepsin G has been shown to play a cancer inhibitor role in some cancer types, CTSG levels do not appear to influence the occurrence of BCC. Further investigation is needed in order to identify potential genetic susceptibility factors for BCC, aiming eventually to develop preventive therapy of high-risk individuals.
Acknowledgements
The Authors wish to thank all individuals (BCC patients and healthy controls) who participated in this study.
Footnotes
Authors’ Contributions
Anna Douka performed the main work of molecular analysis, prepared the initial draft of the manuscript, and performed statistical analysis; Iphigenia Gintoni participated in the conception and design of the study, assisted in initial laboratory work and in the initial draft of the manuscript; Georgia Vairaktari assisted in research logistics and performed statistical analysis; Spyridoula Derka and Stavros Vassiliou collected patients and made corrections in the manuscript; Christos Yapijakis conceived the study, supervised molecular analysis and approved the final draft of the manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received March 6, 2024.
- Revision received March 26, 2024.
- Accepted March 27, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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