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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 1  |  Page : 8-11

The relationship of age and serum prostate-specific antigen with FAS 1377 G/A in prostate cancer


1 Department of Genetics, Islamic Azad University, Domghan, Iran
2 Department of Biology, Islamic Azad University, Roudehen, Iran

Date of Web Publication27-Mar-2018

Correspondence Address:
Dr. Zahra Tahmasebi Fard
Islamic Azad University, Roudehen Branch, Roudehen
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/LJMS.LJMS_37_17

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  Abstract 


Introduction: Apoptosis or programmed cell death is triggered by Fas-Fas ligand (FasL) binding. Mutation in the active sites of these genes blocks death signal transmission and ultimately causes carcinogenesis. The present case–control study was conducted to compare the relationship of age and serum prostate-specific antigen (PSA) with FAS-1377 G/A (rs2234767) polymorphism as one of the best-known polymorphisms of FAS in patients with prostate cancer and a group of controls. Materials and Methods: A total of 100 cases diagnosed with prostate cancer by a specialist and 100 healthy controls were selected from those presenting to the Urogenital Research Unit of Imam Khomeini Hospital in Tehran, Iran. The individual's blood samples were taken, and the enzyme-linked immunosorbent assay method was then used to isolate their blood serum and measure its PSA levels. The saturated salt solution method was used to extract the leukocyte DNA, and the individual's genotype was determined using the Restriction fragment length polymorphism-polymerase chain reaction technique. The data collected were analyzed in IBM SPSS-23. Results: The genotype count found genotype AA in 42% of the cancer patients and 36% of the controls (P = 0.384, odds ratio [OR] = 1.28 confidence interval [CI] 95%: 0.728–2.274), genotype GG in 57% of the cancer patients and 64% of the controls (P = 0.311, OR = 0.746, CI 95%: 0.422–1.317), and genotype AG in a single patient (1%) (P = 0.316, OR: 0.990, CI 95%: 0.971–1.010). No significant relationships were observed between the two groups in terms of these genotypes. Age and serum PSA were found to have significant relationships with both the AA and GG genotypes. Discussion: Older age and elevated serum PSA levels were found to be significantly related to the mutant genotype AA in the cancer patients. Given the lack of significant relationships between the risk allele A and prostate cancer, conducting a study with a larger sample size in different Iranian ethnic groups seems necessary, as sample size can affect the results obtained on this polymorphism.

Keywords: Fas gene, polymorphism Rs 2,234,767, prostate cancer, restriction fragment length polymorphism-polymerase chain reaction


How to cite this article:
Sabour R, Fard ZT. The relationship of age and serum prostate-specific antigen with FAS 1377 G/A in prostate cancer. Libyan J Med Sci 2018;2:8-11

How to cite this URL:
Sabour R, Fard ZT. The relationship of age and serum prostate-specific antigen with FAS 1377 G/A in prostate cancer. Libyan J Med Sci [serial online] 2018 [cited 2018 Oct 16];2:8-11. Available from: http://www.ljmsonline.com/text.asp?2018/2/1/8/228676




  Introduction Top


Prostate cancer is the second most common cancer in the world in men and the single most common in American and European men; in Iran, it is the third most common cancer in men and the sixth most common cancer overall.[1] There is little information about the etiology of prostate cancer in Iran.[2]

Apoptosis or the process of programmed cell death occurs in multicellular organisms. Cell death occurs following biochemical events and changes in cell morphology, such as blabbing of the cell membrane, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation.[3] Malignant cells can suppress apoptosis stimulants and change the apoptosis pathway and thus lead to the development of a variety of human diseases, including cancer.[4]

Fas-Fas ligand binding initiates apoptotic cascade signals. Studies suggest that the under-expression of Fas or the overexpression of Fas ligand occur in many human tumors.[5] Fas receptor is widely expressed in most cells, while Fas ligand expression is limited to immune system cells, including activated T cells, natural killer cells, and peripheral immune cells in parts of the eye and the germinal centers. Nevertheless, the underexpression of Fas and the overexpression of Fas ligand have been identified in most cancers and show that the Fas/FasL system plays a key role in the development of cancer. There is strong evidence suggesting that the underexpression of Fas can keep the deformed cells from being eliminated while the overexpression of FasL enables tumor cells to counterattack the immune system to kill Fas-sensitive lymphocytes and Fas thus plays a role in the development of cancer.[6]

The Fas cell surface receptor, also known as APO-1 or CD95, is a tumor necrosis factor receptor family member and a neuron growth factor that was first identified by antibodies. This receptor induces apoptosis in human cell lines. Fas expression has been observed in many immune and nonimmune cells.[7]

The Fas gene is mapped on chromosome 10q24.1, has nine exons and spans more than 26 kb. Moreover, its promoter region is rich in GC and has binding sequences for transcription factors.[8] The guanine-to-adenine (G → A) transition in the Fas promoter region (position 1377) may reduce stimulatory protein 1 transcription factor binding and thus reduce Fas expression.[9]

Identifying the genes and chromosomes involved in prostate cancer is crucial for obtaining an accurate understanding of the pathophysiology of prostate cancer to properly identify and treat those at risk. Given the genetic structure of the Iranian population and the specificity of polymorphisms in each race, the present study was conducted to compare age and serum prostate-specific antigen (PSA) levels as variables related to prostate cancer and the Fas 1377 G/A polymorphism in patients with prostate cancer and the control group.


  Materials and Methods Top


Sample study and DNA extraction

A hundred patients diagnosed with prostate cancer both by a specialist and based on clinical examinations were assigned to the case group, and 100 healthy controls were assigned to the control group after their health was ensured. To make the most accurate assessments and interpret the overall results of the study, pathological and other data related to the disease were extracted from the patients' records. After giving their consent for participation in the study, 5–7 ml of blood was taken from the participants, and part of this blood sample was used for isolating their serum and the other part for isolating DNAs using the salting out method. The samples were examined using spectrophotometry and agarose gel to ensure the quality of the extracted DNA. The target region was then amplified for all the samples using polymerase chain reaction with specific primers.[8]

Enzyme-linked immunosorbent assay technique

After isolating the blood samples' serum using the kit (GenWay, San Diego), the serum level of PAS was measured in all the samples in according to the manufacturer's instructions.

Restriction fragment length polymorphism-polymerase chain reaction method

A reaction volume of 23 μl was selected for amplification. For each reaction, 10 μl of Amplicon master mix, 1 μl of both the forward and reverse primers (10 pmol), 10 μl of distilled water, and 100 ng of the extracted DNA were transferred to each of the microtubes, which were then placed in a thermocycler with a thermal cycle as follows: One cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, at 60°C for 40 s, at 72°C for 40 s, and ultimately at 72°C for 5 min. Part of the products was run on 1.5% gel and the amplification was verified when a 122 bp band was observed next to the marker [Table 1]. Next, 5 μl of the amplified products was digested with 5 U of the restriction enzyme BstuI (made by Thermo Fisher Scientific), and the participants' genotype was determined on observing the size of the fragments formed on the 2% gel. If nucleotide G is observed in the target region, the amplified fragment will be digested, yielding two fragments with lengths of 108 and 14 bp, which indicate the genotype GG, but if the target nucleotide has been transformed into A, the enzyme will not be able to detect the digestion site, and the same amplified 122-bp fragment will appear on the gel, indicating the homozygous genotype AA, and three fragments with lengths of 122, 108, and 14 bp are observed on the gel if the individual is heterozygous.
Table 1: Sequences of primers, temperature of primers, restriction enzyme, and amplified fragment

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Statistical analysis

Following genotype counting, the percentage and frequency of genotypes in the case and control groups were determined. The allele frequency in both groups was determined according to the Hardy-Weinberg equilibrium using IBM SPSS version 23 (IBM Corporation /United States). Chi-square test was used to determine the relationship between genotypes and risk of prostate cancer in the groups.


  Results Top


The demographic data of all the patients were obtained, and their medical records and pathology reports helped determined the stage of cancer in the patients with adenocarcinoma. Of the 100 healthy controls, 72 cases had benign prostatic hyperplasia with no symptoms of cancer in their biopsy specimens; the other 28 cases were completely healthy. [Table 2] presents the mean values of the study variables. Of all the participants examined in the two groups, 65 never smoked, 76 smoked occasionally, and 59 were regular smokers, and the two groups were not significantly different in terms of this variable (P = 0.530). In the case group, 47 cases (19.7%) were at Stage I, 38 (15.9%) at Stage II, seven (2.9%) at Stage III, and four (3.3%) at Stage IV of the disease, although no statistically significant relationships were observed between the progression of the disease and any of the genotypes.
Table 2: Means of variables in case and control groups

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The genotype count revealed the mutant genotype AA in 42% of the cancer patients and 36% of the controls, genotype GG in 57% of the cancer patients and 64% of the controls and genotype AG in only one patient. [Table 3] presents the allele frequencies. Genotype AA was found to be significantly related to age (P = 0.035) and serum PSA (P = 0.045), but it had no significant relationships with body mass index (BMI) (P = 0.629), as was the case for genotype GG, which showed a significant relationship with age (P = 0.042) and serum PSA (P = 0.040) but not with BMI (P = 0.689). Genotype AG had no statistically significant relationships with any of the variables. [Figure 1] presents the genotype of some of the samples digested with the enzyme.
Table 3: The frequency of alleles and statistically the odds ratio and confidence interval in case and control group

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Figure 1: The picture was shown parts of the analysis of polymerase chain reaction-restriction fragment length polymorphism in samples. Well numbers 1 and 3: Genotype GG. Well numbers 2, 4, 6, 7, 8, and 9: Genotype AA Well number 5: Genotype AG/GA Wells number 10: Marker 100 bp

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  Discussion Top


Preliminary cancer research in the past decade has vastly improved our understanding of cancer biology and genetics, mainly in relation to apoptosis and genes that can profoundly affect the phenotype of malignancy if not controlled. For example, it is correctly understood that mutation in certain cell protection genes disrupts apoptosis and develops or causes the progress and metastasis of tumors.[10] One study showed that the in vitro treatment of prostate cancer is possible with a combination of standard chemotherapeutic agents and Fas; the study found that early prostate cancer cells are sensitive to Fas-induced apoptosis.[11] Fas is one of the candidate genes associated with a number of cancers and its functional polymorphisms in the promoter region of the gene are reported to be associated with an increased risk of breast, gastric and esophageal cancer, especially in Asian populations.[12] Numerous relationships have been identified between the genetic polymorphisms of cell death pathway genes, namely, Fas and Fas ligand, and the risk of the development of cancer.[13] Mutations in the active sites of these genes damage the transmission of the apoptotic signal [14] and play a key role in the initiation, development and progression of cancer. Single-nucleotide polymorphisms also play a key role in developing genetic susceptibility to cancer.[9]

In recent years, Fas 1377 polymorphism has been reported to be associated with many types of cancer, including gastric cancer,[15],[16] kidney cancer,[17] oral squamous cell carcinoma,[18] nasopharyngeal cancer,[19] ovarian cancer,[20] and breast cancer,[21] in different Asian populations. Fas 1377 G/A polymorphism was found to be associated with prostate cancer in a study conducted in 2011 by Shao on 602 patients and 703 healthy controls of Chinese origin.[22] No such significant relationship was observed in the present study or the one conducted by Mandal and Mittal in 2012 on 192 patients and 224 healthy controls of Indian origin.[23] The disparity of findings may be attributed to the small sample size of the present study and the one by Mandal compared to the one by Shao.

The present study found that age and serum PSA are significantly related to the mutant genotype AA, which may suggest the contribution of this genotype as well as some other variables to the development of prostate cancer. Further studies are recommended to be conducted with larger sample sizes to ensure of the role of this polymorphism in the development of prostate cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Pakzad R, Rafiemanesh H, Ghoncheh M, Sarmad A, Salehiniya H, Hosseini S,et al. Prostate cancer in Iran: Trends in incidence and morphological and epidemiological characteristics. Asian Pac J Cancer Prev 2016;17:839-43.  Back to cited text no. 1
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O'Connell J, O'Sullivan GC, Collins JK, Shanahan F. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med 1996;184:1075-82.  Back to cited text no. 5
    
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Zhang X, Miao X, Sun T, Tan W, Qu S, Xiong P,et al. Functional polymorphisms in cell death pathway genes FAS and FASL contribute to risk of lung cancer. J Med Genet 2005;42:479-84.  Back to cited text no. 6
    
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Kokkonen TS, Karttunen TJ. Fas/Fas ligand-mediated apoptosis in different cell lineages and functional compartments of human lymph nodes. J Histochem Cytochem 2010;58:131-40.  Back to cited text no. 7
    
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Tahmasbifard Z, Hasanzad M, Nafisi N. Study of Fas 1377 G>A polymorphism in breast cancer of Iranian patients. ISMJ 2016;18:1132-9.  Back to cited text no. 8
    
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Huang QR, Morris D, Manolios N. Identification and characterization of polymorphisms in the promoter region of the human apo-1/Fas (CD95) gene. Mol Immunol 1997;34:577-82.  Back to cited text no. 9
    
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Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485-95.  Back to cited text no. 10
    
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Symes JC, Kurin M, Fleshner NE, Medin JA. Fas-mediated killing of primary prostate cancer cells is increased by mitoxantrone and docetaxel. Mol Cancer Ther 2008;7:3018-28.  Back to cited text no. 11
    
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Xu Y, He B, Li R, Pan Y, Gao T, Deng Q,et al. Association of the polymorphisms in the Fas/FasL promoter regions with cancer susceptibility: A systematic review and meta-analysis of 52 studies. PLoS One 2014;9:e90090.  Back to cited text no. 12
    
13.
Sun T, Miao X, Zhang X, Tan W, Xiong P, Lin D,et al. Polymorphisms of death pathway genes FAS and FASL in esophageal squamous-cell carcinoma. J Natl Cancer Inst 2004;96:1030-6.  Back to cited text no. 13
    
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Wajant H. The Fas signaling pathway: More than a paradigm. Science 2002;296:1635-6.  Back to cited text no. 14
    
15.
Zhou RM, Wang N, Chen ZF, Duan YN, Sun DL, Li Y,et al. Polymorphisms in promoter region of FAS and FASL gene and risk of cardia gastric adenocarcinoma. J Gastroenterol Hepatol 2010;25:555-61.  Back to cited text no. 15
    
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Wang M, Wu D, Tan M, Gong W, Xue H, Shen H,et al. FAS and FAS ligand polymorphisms in the promoter regions and risk of gastric cancer in Southern China. Biochem Genet 2009;47:559-68.  Back to cited text no. 16
    
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Zhu J, Qin C, Wang M, Yan F, Ju X, Meng X,et al. Functional polymorphisms in cell death pathway genes and risk of renal cell carcinoma. Mol Carcinog 2010;49:810-7.  Back to cited text no. 17
    
18.
Wang LH, Ting SC, Chen CH, Tsai CC, Lung O, Liu TC,et al. Polymorphisms in the apoptosis-associated genes FAS and FASL and risk of oral cancer and malignant potential of oral premalignant lesions in a Taiwanese population. J Oral Pathol Med 2010;39:155-61.  Back to cited text no. 18
    
19.
Cao Y, Miao XP, Huang MY, Deng L, Lin DX, Zeng YX,et al. Polymorphisms of death pathway genes FAS and FASL and risk of nasopharyngeal carcinoma. Mol Carcinog 2010;49:944-50.  Back to cited text no. 19
    
20.
Li Y, Hao YL, Kang S, Zhou RM, Wang N, Qi BL,et al. Genetic polymorphisms in the Fas and FasL genes are associated with epithelial ovarian cancer risk and clinical outcomes. Gynecol Oncol 2013;128:584-9.  Back to cited text no. 20
    
21.
Wang W, Zheng Z, Yu W, Lin H, Cui B, Cao F,et al. Polymorphisms of the FAS and FASL genes and risk of breast cancer. Oncol Lett 2012;3:625-8.  Back to cited text no. 21
    
22.
Shao P, Ding Q, Qin C, Wang M, Tang J, Zhu J,et al. Functional polymorphisms in cell death pathway genes FAS and FAS ligand and risk of prostate cancer in a Chinese population. Prostate 2011;71:1122-30.  Back to cited text no. 22
    
23.
Mandal RK, Mittal RD. Are cell cycle and apoptosis genes associated with prostate cancer risk in north Indian population? Urol Oncol 2012;30:555-61.  Back to cited text no. 23
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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