AIP Conference Proceedings 2193, 040001 (2019); https://doi.org/10.1063/1.5139363 2193, 040001
© 2019 Author(s).
Effect of P21(C98A) polymorphism on the risk of head and neck squamous cell carcinoma in an Indonesian population
Cite as: AIP Conference Proceedings 2193, 040001 (2019); https://doi.org/10.1063/1.5139363 Published Online: 10 December 2019
Robi Sinambela, Bambang Tri Hartomo, Yurnadi Hanafi Midoen, et al.
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Effect of P21(C98A) Polymorphism on the Risk of Head and Neck Squamous Cell Carcinoma in an Indonesian
Population
Robi Sinambela
1, Bambang Tri Hartomo
1,2, Yurnadi Hanafi Midoen
3, Nurtami Soedarsono
1, Ferry Pergamus Gultom
1, and Elza Ibrahim Auerkari
1,a)1Department of Oral Biology, Faculty of Dentistry, Universitas Indonesia, Jl. Salemba Raya No. 4, Central Jakarta 10430 Indonesia
2Dental Medicine Program, Faculty of Medicine, Jenderal Soedirman University, Purwokerto, Indonesia
3Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
a)Corresponding author: elza.ibrahim@ui.ac.id
Abstract. The high prevalence of head and neck cancer in Indonesia could imply possible population-specific causative factors. The polymorphisms of the p21 gene may modify important cellular defenses to carcinogenesis through the involvement of p21 in the cell cycle. It is known that p21 has a role as a mediator of p53 tumor suppressor that is involved in DNA repair and apoptosis. This study aimed to investigate the possible association of p21 (C98A) polymorphism with the risk of head and neck squamous cell carcinoma (HNSCC) in Indonesia. The PCR-RFLP method was used to genotype stored DNA samples from 50 HNSCC patients and 50 healthy control subjects. The CA genotype was the most common variant in both case and control groups. Conclusion: There was no significant association between the genetic polymorphisms of p21 (C98A) with HNSCC in the tested Indonesian population.
Keywords: p21, p53 polymorphism, cancer, Indonesia
INTRODUCTION
Head and neck cancer (HNC) is the sixth most common cancer occurring worldwide, with more than 550,000 cases in the world with a mortality rate of about 300,000 annually. The incidence of cancer in men is higher than in women, with a ratio of 2:1 to 4:1. The most common form of HNC (90%) is head and neck squamous cell carcinoma (HNSCC). Most HNSCCs arise in the epithelial lining of the oral cavity, oropharynx, larynx, and hypopharynx [1].
There were 14.1 million new cancer cases, 8.2 million cancer deaths, and 32.6 million people living with cancer diagnosed within 5 years in 2012 throughout the world [2]. The Indonesian national study Riset Kesehatan Dasar (Riskesdas) in 2007 indicated that HNC prevalence in Indonesia is quite high with a prevalence of 4.7/100,000 population, occupying the fourth place of all malignancies in the period of 2001-2005 [3].
In cancer, cells can divide without control and invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph system. HNSCC begins from the squamous cells that line the mucosal surfaces in the head and neck (in the mouth, nose, and throat). Head and neck cancers are categorized by the area where they begin, such as salivary gland, paranasal sinuses, etc. Regional HNC may refer to locations of the paranasal sinuses, nasal cavity, oral cavity, tongue, salivary glands, larynx, or pharynx (including nasopharynx, oropharynx, and hypopharynx) [4-6]. Oral cancer can evolve through various stages that are influenced by environmental factors, such as chewing tobacco, smoking, alcohol consumption, and changes in genes [7].
Cancer is caused by changes in the genes that control the cellular functions, especially how the cells grow and divide. The genetic changes that lead to cancer can be inherited from parents or arise during the life of a person as a result of errors that occur when cells divide or due to DNA damage caused by exposure to certain environments.
Genetic alterations that contribute to cancer tend to affect the proto-oncogenes, tumor suppressor genes, and DNA repair genes. Proto-oncogenes are involved in normal cell growth and division. However, when the gene is altered in a certain way or more active than usual, the normal gene can become cancer-causing (oncogene). Tumor suppressor genes are also involved in controlling cell growth and division. Cells with specific changes in tumor suppressor genes can divide uncontrollably. DNA repair genes are involved in repairing damaged DNA. Cells with mutations in these genes are likely to develop additional mutations in other genes. Together, these mutations can cause cells to become cancerous [8-9].
Not all changes in the body tissues are cancerous. Some changes can develop into cancer if not treated promptly.
As examples of changes that are not cancer tissue to become cancerous, hyperplasia occurs when cells in the tissues divide faster than normal cells. However, each cell and how the network is set looks normal under the microscope.
Hyperplasia may be caused by several factors or conditions, including chronic irritation. Dysplasia is a condition more serious than benign cells where there is an accumulation of excess cells. But the cells look normal, and there is a change in the way the network is set up. Cells and tissues display abnormal appearance, and the greater the abnormality, the higher the chance that cancer will form. Some types of dysplasia may need to be monitored or treated.
Examples of dysplastic nevus dysplasia are formed on the skin. A dysplastic nevus can turn into melanoma, although only in a minority of cases [10].
The p21 gene is part of CKI or CIP / KIP family of cyclin-dependent kinase inhibitors that can be attached to and inhibit cyclin-CDK. CKI is a negative regulator of the function of the proliferation, control checkpoint, and tumor suppressor. Various cyclin-CDK complexes are required to phosphorylate substrate proteins that are important for every stage of the cell cycle, initiation of DNA replication, and the onset of mitosis. A specific group of cyclin-CDK will regulate each cell cycle phase. For example in the G1 phase, the cell cycle is catalyzed by cyclin D-CDK4 and p21 cyclin E-CDK2. The p21 gene is located on chromosome 6 short arm (p) 21.2 and consists of three exons and two introns, located in exon 2. The p21 gene is present as a mediator of p21 suppressor activity and as an inhibitor of p53 tumor suppressor in the cell cycle progression due to its ability to inhibit the activity of cyclin E-CDK2. Also, p21 can bind and prevent kinase activity of cyclin E-CDK2 which can affect the growth at some stages in the cell cycle [11- 14].
Through cyclin-CDK, p21 acts as the mechanism involved in DNA repair and apoptosis. Furthermore, p21 can inhibit the activity of cyclin-CDK inhibitors for increasing cell levels. Therefore, p21 can function as inhibitors of CDK and plays an important role in the development of cancer as in the regulation of cell cycle control, DNA repair, and apoptosis. Several studies have shown that p21 polymorphisms may affect the expression, protein activity, and susceptibility to various types of cancer, including cancers of the head and neck [15-16].
Despite the clear involvement of p21 in cancer formation, mutations in p21 are rare. However, some reports have suggested that polymorphic variants of p21 are associated with a higher risk of many types of cancer. Polymorphisms in the gene p21 are Ser31Arg (rs1801270C > A), with the basic change from C to A to produce non-identical serine to arginine substitution in the protein. Many molecular epidemiology studies have been conducted to evaluate the effect of p21 polymorphisms Ser31Arg on cancer risk. The important position of p21 in control of cell cycle and DNA damage indicating that p21 is required for some or all effects of p53 [17-18].
MATERIALS AND METHODS
This study was conducted using a cross-sectional analysis in the laboratory. Samples of this research using biological materials stored in the Laboratory of the Department of Oral Biology Faculty of Dentistry, Universitas Indonesia in a refrigerator at -20°C. The material was in the form of DNA that was previously extracted. DNA used for the study consisted of 50 DNA samples of HNSCC patients and 50 DNA samples of the control subjects.
Polymorphism detection was conducted using the PCR-RFLP method. This method begins with DNA amplification using forward primer 5'-GTC TGG AGA GGA GGC ACC TG-3' and reverse primer 5'-CCA CTC CTC ACT CAT GG CCC-3'. Amplification was using 25 uL PCR mix consisting of 12.5 mL PCR master mix (KAPA), 0.75 mL of forwarding primer, reverse primer 0.75 mL, 10.5 ddH2O, and 0.5 mL of the DNA sample. After that, the sample tubes were inserted into the PCR thermocycler to start a cycle of amplification using the two-step technique.
The process of amplification included an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation
at 95°C for 30 seconds, annealing at 55°C for 35 seconds, and elongation at 72°C for 45 seconds. The final elongation was conducted at 72°C for 5 min.
After completed amplification the PCR product was confirmed by electrophoresis and visualized by Gel Doc.
Once successfully authenticated, the PCR products were digested with the restriction enzyme BsmA I (BsoMA I) for 16 hours at 55°C in the thermoblock. Then the restriction enzyme was inactivated by heating at 65°C for 20 min using a thermoblock. The resulting product was subjected to electrophoresis at 80 V for 40 min using 2% agarose gel with 1μL GelRad added. The results of electrophoresis were visualized using Gel Doc.
The data obtained were analyzed using the Statistical Program for Social Science (SPSS) v.23 to analyze the differences in the genotype distributions of the p21 (C98A) gene polymorphism in HNSCC and control samples.
Fisher exact test and Chi-square testing for Hardy-Weinberg Equilibrium were applied, considering p < 0.05 to imply significance.
RESULTS
After DNA amplification using PCR, the visualization of the PCR product confirmed a bright ribbon of 272 bp (Figure 1).
FIGURE 1. Visualized PCR product of p21(C98A) at 272 bp.
FIGURE 2. Results of RFLP for samples of three genotypes of p21 (C98A) polymorphism.
Figure 2 above shows the results after digestion with restriction enzyme BsmA I (BsoMA I) for three genotypes.
The CC genotype (wild type homozygote) shows two bands of 183 bp and 89 bp; CA genotype (heterozygote variant)
three bands of 272 bp, 183 bp, and 89 bp; and the AA genotype (homozygote variant) one wand of 272 bp. The results are summarized in Table 1.
TABLE 1. Distribution of genotypes and alleles of p21 (C98A) polymorphism p21 (C98A) polymorphism HNSCC group Control group
Genotype CA 29 (58%) 39 (78%)
Genotype AA 20 (40%) 10 (20%)
Genotype CC 1 (2%) 1 (2%)
Allele C 31 (31%) 41 (41%)
Allele A 69 (69%) 59 (59%)
Chi-square analysis of the results on genotype distributions shown in Table 1 showed consistency with the Hardy- Weinberg Equilibrium (HWE) for both HNSCC patients and healthy controls (p > 0.05). The Fisher Exact test on the differences between the genotype and allele distributions of HNSCC and control groups showed p-values higher than 0.05. Therefore, no significant differences are indicated in the polymorphic genotype or allele distributions between the HNSCC and control groups.
DISCUSSION
The p21 gene is a part of CKI (CIP / KIP) family of cyclin-dependent kinase inhibitors that can attach to and inhibit cyclin-CDK2. Through cyclin-CDK inhibition, p21 acts in the mechanisms involved in cell cycle control, DNA repair, and apoptosis. Several studies have suggested that p21 polymorphisms may affect the expression and protein activity of p21, and play a role in susceptibility to various types of cancer, including cancers of the head and neck [8- 9,15-16].
This study was conducted to assess the genotype and allele distributions of the p21 gene (C98A) polymorphism in HNSCC patients and compare them with the corresponding distributions in a healthy control group. The study was using a cross-sectional analysis in the laboratory, using stored biological materials as previously extracted DNA samples.
The genotype distributions of p21 (C98A) polymorphism was analyzed using the PCR-RFLP method with the restriction enzyme BsmA I (BsoMA I).
No significant differences in the polymorphic genotype or allele distributions were indicated in the Fisher exact test between the HNSCC and control groups (p<0.05). The genotype distributions were consistent with the Hardy- Weinberg Equilibrium (p > 0.05 in Chi-square test).
Previously, the corresponding distributions of p21 (C98A) polymorphism for HNSCC and control groups have been done by several studies in the world, showing mixed results (Table 2).
TABLE 2. Comparison of results from studies on p21 (C98A) gene polymorphism and its relationship with cancer Research Population No. of samples
(cases/control) Origin Significance
Li et al., 2005 USA 712/1222 Caucasoid Significant
Bau et al., 2007 China 137/105 Mongoloid Significant
Taghavi, 2010 Iran 126/100 Caucasoid Not significant
Soare et al., 2014 Portugal 102/191 Caucasoid Significant
Present work Indonesia 50/50 Mongoloid Not significant
The results of Taghavi [15] on an Iranian Caucasoid population indicated no significant association of the polymorphism and cancer, unlike the study of Li et al. on a US Caucasian population. The present study was conducted in an Indonesian (mainly Mongoloid) population, and unlike the previous work of Bau et al. on the Chinese Mongoloid population, found no significant association of the tested polymorphism and cancer. Another gene polymorphism that is a possible candidate marker is interleukin-6 gene (-174 G/C) polymorphism [2].The association of the IL-6 -174G/C promoter gene polymorphism and cancer could indicate a protective effect against the risk of HNSCC.
CONCLUSION
Genetic polymorphism of p21 (C98A) or rs180127 was found to occur so that in DNA samples of both HNSCC patients and healthy controls the CA genotype and A allele were most common. However, there was no significant association of the polymorphism with HNSCC in the tested Indonesian population.
ACKNOWLEDGMENT
The authors wish to gratefully acknowledge the financial support from the Indonesian Ministry of Education through the Universitas Indonesia (EIA).
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