Abstract
Purpose
Oral cancer is the most common cancer among the Indian men and the second most common cancer among the Indian women. Such high incidence of oral cancer in India is due to consumption of tobacco in different form including smoking of cigarette. Smoke of tobacco contains different carcinogens which causes DNA damage. Such DNA damage if remain unrepaired due to faulty DNA repair system can cause mutation and eventual development of cancer.
Methodology
In the present study, we aimed to check the role of smoking as well as interaction of smoking and XPC polymorphism in risk modulation of oral cancer. Total of 372 subjects including 300 healthy controls and 72 patients of oral cancers been genotyped for the XPC PAT D/I, A/C and C/T polymorphisms with PCR based or PCR–RFLP based method. Genotype frequency was analyzed by chi-square test and strength of associations by odds ratio with 95% confidence intervals.
Results
The present study showed that compared to nonsmokers, smokers are at five times higher risk to develop oral cancer (p value= 0.001, OR= 5.03, 95% CI 2.91–8.69) and three times higher risk to develop node-positive (p value= 0.01, OR= 3.66, 95% CI 1.34–9.95) oral cancer. It has also been observed that individuals who were smokers and carrier of variant allele genotypes (AC and CC) for XPC A/C polymorphism were at threefold higher risk (p value= 0.01, OR=2.97, 95% CI 1.29–6.86) to develop oral cancer compared to individual who were smokers but do not carry the C allele (AA genotype). This observation indicates that C allele of XPC A/C polymorphism interacts with smoking and significantly increases the risk of oral cancer.
Conclusion
This study demonstrates a possible role of smoking and gene–smoking interaction in risk enhancement of oral cancer.
Keywords: Smoking, Oral cancer, XPC, Polymorphism
Introduction
Globally, oral cancer is the eighth most common cancer and it causes more than 128,000 deaths per year [1]. Estimated death due to oral cancer in 2018 is 9.6 million which makes it the second leading cause of death worldwide [2]. Important risk factors for oral cancer include occupational exposures to aromatic amines, polycyclic aromatic hydrocarbons, and polluted drinking water containing arsenic and chlorination by-products [3]. The presence of carcinogens in these exposures can induce DNA damage which if left unrepaired can induce cancer [4, 5]. Environmental factors like tobacco, alcohol, and viruses have also been found to alter the cellular epigenetic patterns and thereby affect changes in gene activation and cell phenotype [6].
In India, 57% of all men and 11% of women between the ages of 15–49 years use some form of tobacco which increases their risk of oral cancer. Betel quid (paan), gutka, zarda, kharra, mawa, and khainni also increase the risk of oral cancer [7]. Bidis, small hand-rolled cigarettes, produce three times more carbon monoxide than cigarettes and increase the risk of oral cancer by threefold compared to nonsmokers. It also increases the risk of lung, stomach, and esophageal cancer [8].
Genetic polymorphisms of various genes including those involved in the DNA repair pathway were found to alter the risk of oral cancer [9]. There are three major pathways involved in DNA damage and repair which play an important role in maintenance of genomic integrity such as base excision repair (BER), nucleotide excision repair (NER), and double-strand break repair (DSBR) [9]. XerodermaPigmentosum Complementation group C (XPC) plays an important role in the NER pathway and is present in the short arm of chromosome 3(3p25) which encodes for a 940 amino acids protein that in vivo form a supramolecular complex including HR23B, homolog to Rad23 in yeast, and centrin2 [10]. The binding of radiation repair HR23B protein to XPC forms a complex [11] that interacts with XPA and the presence complex RPA. It also interacts with TFIIH leading to the lesion site where this opens the double helix of DNA by acting as a helicase which allows for the excision, restoring of DNA [12]. Another function of XPC complex is to bind to the different types of lesions and recognition the DNA alteration [13]. Thus, the XPC acts as a potential carcinogenic abrasion for the NER pathway [14].
The present study is aimed to determine the role of smoking in the development of oral cancer. Simultaneously, interplay between smoking and XPC gene with respect to risk of cancer has also been evaluated.
Materials and Method
Materials
Study Subjects
The study was evaluated on a total of 72 patients with previously treated and pathologically confirmed oral cancer who were registered at the Department of Oral Pathology and Microbiology, King George’s Medical University, and 300 healthy controls. Data on risk habits (smoking, drinking, chewing) were obtained using a structured questionnaire via face-to-face interviews. Informed written consent was obtained from all subjects. Venous blood samples were collected in EDTA tubes and stored at − 80 °C, till DNA extraction. Genomic DNA extraction from blood samples was carried out by salting out method [15]. The study was evaluated, and ethical clearance was obtained from the Institutional Ethics Committee of King George’s Medical University, Lucknow.
Genotyping
Genotyping for XPC PAT (D > I) polymorphisms was done by PCR method, whereas genotypes for XPC A > C and C > T polymorphisms were determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) technique. PCR products both undigested and digested were resolved on a 2% agarose gel and stained with ethidium bromide for visualization under UV light. PCR products were generated in 10 ul volume reactions containing 10 ng of genomic DNA, 2.0 mM MgCl2, 0.11mMeachdNTP, 0.3 mM each primer, and 0.5 U Taq DNA polymerase (Sigma-Aldrich, USA). PCR primers used have been reported previously [16].
Statistical Analysis
To determine the difference between the cases and controls with respect to the distribution of smokers and nonsmokers, Chi-square test (Yates corrected) was used. It was also used to find out difference in distribution of smokers and nonsmokers between different clinical parameters. The risk of oral cancer has been estimated among smokers and nonsmokers who were stratified within different genotypes of studied XPC polymorphisms. Estimated risk was expressed with odds ratio, and a P value less than 0.05 was considered as significant association. Odds ratios (OR), 95% confidence interval (CI) and p values for the assessment of associated risk were calculated by Epi-Info program (http://wwwn.cdc.gov/epiinfo/).
Results and Discussions
Distributions of smokers and nonsmokers among the subjects of oral cancer and healthy control are shown in Table 1. The frequency of smokers among the cases is significantly higher than healthy control. On the other side compared to the subjects of oral cancer, proportion of nonsmokers are significantly more among the healthy control (p value = 0.001). Our result shows that smoking increases the risk of oral cancer by five times (OR 5.03; 95% CI 2.91–8.69). Tobacco smoke is a mixture of chemicals most of which are carcinogen. Aromatic hydrocarbon benzopyrene and the tobacco-specific nitrosamines (TSNs), namely 4-(nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN), are the most important carcinogens in tobacco smoke. NNK and NNN in tobacco products have been found to cause tumors of the oral cavity, lung, esophagus, and pancreas in animal. These carcinogens of tobacco smoke and their metabolites covalently bind with DNA and form DNA adducts [17]. Such damaged DNA increase the mutational load of cells and eventually to cancer [18]. The role of smoking on oral cancer has been firmly established by epidemiological studies [19–22].
Table 1.
Effect of smoking on oral cancer development
| Status of smoking | Oral cancer | Control | p value | OR (95% CI) |
|---|---|---|---|---|
| No | 26 (36%) | 222 (74%) | Ref | 1 |
| Yes | 46 (64%) | 78 (26%) | 0.001* | 5.03 (2.91–8.69) |
*Significant association is considered when p value is less than 0.05 and both bondries of 95% confidence interval are more than 1 in risk association and less than 1 in protective association
We have evaluated the effect of smoking on different clinical parameters of oral cancer such as the size of tumor, nodal involvement, metastasis, stage, and grade documented in Table 2. Seventy-seven percent of smokers develop node-positive oral cancer while only 23% nonsmokers showed involvement of node. We observed that compared to nonsmokers, smokers are at four times higher risk in developing node-positive oral cancer (Table 2). The risk of larger tumor and nodal metastasis was found to be higher in smokers with laryngeal squamous cell carcinoma [23]. Wang C et al. [24] suggested that nicotine may promote tongue squamous carcinoma (TSCC) cell progression and may play a significant role in the progression and metastasis of smoking-related TSCC, a type of oral cancer.
Table 2.
Effect of smoking on different clinical parameters of oral cancer
| Tumor T status | Status of smoking | T3 + T4 | T1 + T2 | p value | Odds ratio |
95% CI |
|---|---|---|---|---|---|---|
| No | 10 (26%) | 16 (47%) | Ref | 1 | 1 | |
| Yes | 28 (74%) | 18 (53%) | 0.11 | 2.48 | 0.92–6.68 |
| Lymph node involvement | N1 + N2 + N3 | N0 | ||||
|---|---|---|---|---|---|---|
| No | 13 (23%) | 13 (52%) | Ref | 1 | 1 | |
| Yes | 44 (77%) | 12 (48%) | 0.01* | 3.66 | 1.34–9.95 |
| Metastasis | M1 | M0 | ||||
|---|---|---|---|---|---|---|
| No | 05 (28%) | 21 (40%) | Ref | 1 | 1 | |
| Yes | 13 (72%) | 32 (60%) | 0.53 | 1.70 | 0.53–5.49 |
| Tumor stage | III + 1 V | I + II | ||||
|---|---|---|---|---|---|---|
| No | 19 (37%) | 07 (37%) | Ref | 1 | 1 | |
| Yes | 33 (63%) | 1263 (%) | 0.79 | 1.01 | 0.34–3.01 |
| Cell differentiated grade | > Grade 1 | Grade 1 | ||||
|---|---|---|---|---|---|---|
| No | 16 (39%) | 10 (33%) | Ref | 1 | 1 | |
| Yes | 25 (61%) | 20 (67%) | 0.80 | 0.78 | 0.29–2.09 |
*Significant association is considered when p value is less than 0.05 and both bondries of 95% confidence interval are more than 1 in risk association and less than 1 in protective association
It is evident that not all smokers develop oral cancer and that is possibly due to different genetic makeup of individuals. It is possible that some smokers effectively repair their damaged DNA produced by carcinogens present in cigarette smoke due to better DNA repair capacity. XPC is a DNA repair protein and reported to harbor SNPs which have some functional impact. To check the interference of different genotypes from XPC PATD/I, C/T, and A/C polymorphisms with smoking in risk modulation of oral cancer, we have calculated the proportion of smokers and nonsmokers among the different genotypes of aforesaid XPC polymorphisms as given in Table 3. The risk of developing oral cancer with different genotypes of XPC polymorphisms among smokers and nonsmokers has been calculated separately and represented by odds ratio with 95% confidence interval. None of the genotypes of studied XPC polymorphisms were found to impact the risk of oral cancer in individuals who do not smoke. However, individuals who were smokers and carriers of AC and CC genotypes, i.e., carrier of C allele for XPC A/C polymorphism, were at threefold higher risk to develop oral cancer than those who were carrier of AA genotype. ShenH et al. [25] had also observed a borderline risk modulation of oral cancer in smokers with XPC PAT D/I polymorphism. The II genotype of XPC PAT D/I polymorphism was also found to increase the risk of breast cancer among smokers. In our previous study, we did observe modulation of risk of head and neck cancer and oral cancer [16] with different polymorphisms of XPC genes.
Table 3.
Interaction of XPC polymorphisms and smoking on development of oral cancer
| XPC A > C, C > T and D/I genotypes | |||||
|---|---|---|---|---|---|
| XPC genotypes | Oral cancer | Control | p value | Odds ratio |
95% CI |
| No habit | |||||
| D/I polymorphism | |||||
| DD | 15 (58%) | 99 (45%) | Ref | 1 | 1 |
| DI + II | 11 (42%) | 123 (55%) | 0.28 | 0.59 | 0.25–1.34 |
| C/T polymorphism | |||||
| CC | 09 (35%) | 51 (23%) | Ref | 1 | 1 |
| CT + TT | 17 (65%) | 169 (77%) | 0.29 | 0.57 | 0.23–1.35 |
| A/C polymorphism | |||||
| AA | 13 (54%) | 104 (46%) | Ref | 1 | 1 |
| AC + CC | 11(46%) | 120 (54%) | 0.61 | 0.73 | 0.31–1.70 |
| Smoking | |||||
| D/I polymorphism | |||||
| DD | 25 (57%) | 39 (50%) | Ref | 1 | 1 |
| DI + II | 19 (43%) | 39 (50%) | 0.59 | 0.76 | 0.36–1.59 |
| C/T polymorphism | |||||
| CC | 13 (28%) | 18 (23%) | Ref | 1 | 1 |
| CT + TT | 33 (72%) | 59 (77%) | 0.69 | 0.77 | 0.33–1.77 |
| A/C polymorphism | |||||
| AA | 10 (23%) | 37 (47%) | Ref | 1 | 1 |
| AC + CC | 33 (77%) | 41 (53%) | 0.01* | 2.97 | 1.29–6.86 |
*Significant association is considered when p value is less than 0.05 and both bondries of 95% confidence interval are more than 1 in risk association and less than 1 in protective association
Conclusion
In conclusion, our study indicates possible role of smoking both on development and on aggression of oral cancer. We have also found that the effect of smoking on development of oral cancer is further modified by A/C polymorphism of XPC gene. However due to fewer samples in the oral cancer group, our study is far from suggestive conclusions and warrant future studies with larger sample size.
Funding
The author, Kumud Nigam, is very grateful to Indian Council of Medical Research (ICMR), New Delhi, India (Project Id-3/2/2/54/2018/Online OncoFship/NCD-III), for providing the fellowship grant to conduct her Ph.D. work.
Compliance with Ethical Standards
Conflict of interest
All the authors declared no conflict of interest in this research work for the publication.
Footnotes
Shadab Mohammad and Somali Sanyal are sharing equal correspondence for the manuscript.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Shadab Mohammad, Email: shadab31aug@yahoo.com.
Somali Sanyal, Email: ssanyal@lko.amity.edu.
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