Tumor Biol. DOI 10.1007/s13277-015-3332-3
RESEARCH ARTICLE
Vitamin D receptor gene polymorphisms and breast cancer risk among postmenopausal Egyptian women Eman Abd-Elkader Abd-Elsalam 1 & Nadia A. Ismaeil 2 & Hoda Sibai Abd-Alsalam 3
Received: 4 January 2015 / Accepted: 12 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Many studies reported that vitamin D can protect against various types of cancers. The mechanism of vitamin D action is mediated by the vitamin D receptor (VDR). VDR may have anti-stress function because it has been identified as p53 direct target gene. This research was designed to investigate the role of VDR polymorphisms BsmI (rs 1544410), ApaI (rs 7975232), TaqI (rs 731236), and FokI (rs 10735810) in pathogenesis of breast cancer using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The study included 130 postmenopausal breast cancer cases aged 49 to 65 years and 100 controls aged 50 to 72 years. A significantly increased risk of breast cancer among carriers of BsmI bb genotype was observed (OR=2.5 (1.1–5.6), P=0.025). Also, a significantly increased risk of breast cancer was detected among women carrying ApaI aa genotype (OR=2.2 (1.02–4.5), P=0.04), while no significant associations were observed between breast cancer risk and genotypes and allele frequencies of FokI and TaqI polymorphisms (P>0.05). Our study showed that VDR gene polymorphisms (BsmI and ApaI) may contribute to breast cancer risk among postmenopausal women.
Keywords VDR . Breast cancer . BsmI . ApaI . Polymorphism * Eman Abd-Elkader Abd-Elsalam [email protected] 1
Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2
General Surgery Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
3
Obstetrics and Gynecology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
Abbreviations VDR SNPs PCR RFLP ER PR
Vitamin D receptor Single-nucleotide polymorphisms polymerase chain reaction Restriction fragment length polymorphism Estrogen receptor Progesterone receptor
Introduction The active vitamin D (1,25-dihydroxycholecalciferol) binds the vitamin D receptor (VDR) to induce a cascade of gene regulation and signaling molecules [26], leading to either suppression of cell proliferation by inducing apoptosis, enhancing cell differentiation, blocking cell cycle, inducing expression of inhibitors of cell cycle progression, inhibiting colony formation, reducing cell inflammation, inhibiting cell invasion, or metastasis and downregulating estrogen receptor signaling pathway in breast cancer [7]. During puberty, pregnancy and involution, VDR expression is regulated in the mammary gland and related to proper glandular development [29]. Ductal proliferation and branching induced by estrogen are inhibited by VDR agonists by binding to VDR [30]. Furthermore, [16] reported that VDR agonists protect against chemically induced pre-neoplastic lesions. p53 is a key molecule in stress response and carcinogenesis where the p53 protein levels are elevated leading to cellular DNA repair or apoptosis [17]. VDR gene has been identified as p53 direct target gene [14] so VDR could have anti-stress function [17]. VDR is also transactivated by stress-activated protein kinases p38 and JNK [19]. Furthermore, some factors related to stress response as prohibition and thioredoxin are
Tumor Biol.
VDR target genes, indicating that VDR has an important role in stress-related response [17]. Mathiasen et al. [15] reported that 1,25-dihydroxyvitamin D3 induced a growth arrest followed by apoptosis in human breast cancer cell lines; MCF-7 cells, expressing a wild-type p53 and T47D cells, that lack a functional p53, at concentrations of 1 to 100 nM, denoting that p53 is not necessary for growth-inhibitory effects of vitamin D. Decreased VDR expression has been observed in breast cancer cells [9] which may be caused by VDR gene polymorphisms [6] and/or DNA methylation [11]. More than 470 single-nucleotide polymorphisms (SNPs) have been discovered in the VDR gene, but a few have been well characterized [26]. This research was designed to evaluate the role of wellcharacterized VDR gene polymorphisms, BsmI (rs 1544410), ApaI (rs 7975232), TaqI (rs 731236), and FokI (rs 10735810) in pathogenesis of breast cancer in Egyptian women.
Materials and methods This research was carried out at the period from March 2013 to August 2014 at the Medical Biochemistry and General Surgery departments, Faculty of Medicine, Zagazig University. A written informed consent was obtained from each participant. Two hundred thirty females were enrolled in this study. They were classified into two groups; group I (control group) that involved 100 apparently healthy volunteers with ages ranging from 50 to 72 years (with mean value±SD of 58.5± 6.9) with no history (either familial or personal) of cancer of any type and group II including 130 females with ages ranging from 49 to 65 years (with mean value±SD of 55.9±4.3) with a clinical and histological diagnoses of breast cancer. All subjects of this study were postmenopausal. The diagnosis of breast cancer was based on history taking, clinical examination to breast and axilla, radiological examination (ultrasonography, mammogram of both breast and axilla, chest X-ray, and abdominal ultrasonography), pathological diagnosis (fine needle aspiration cytology (FNAC) and true cut needle biopsy of breast mass), and estrogen and progesterone receptors evaluation. The breast cancer patients were subclassified into 76 patients without metastasis (M0) and 54 patients suffering from metastatic breast cancer (M1). According to histological grading, patients were graded into grade I (well differentiated), 35 patients; grade II (moderately differentiated), 52 patients; and grade III (poorly differentiated), 43 patients. Estrogen receptor was positive in 73 patients and negative in 57 patients while progesterone receptor was positive in 66 patients and negative in 64 patients.
Research investigations VDR polymorphisms, BsmI (rs 1544410), ApaI (rs 7975232), TaqI (rs 731236), and FokI (rs 10735810), were examined by PCR-RFLP analysis. Blood sampling One milliliter of peripheral venous blood samples was collected in sterile tubes containing EDTA for DNA extraction. DNA extraction DNA was extracted from whole blood samples using TIANamp Genomic DNA Kit that depends on spin column method, according to the protocol supplied by the manufacturer (Tiangen Biotech, Beijing). For the determination of DNA concentration, 1 O.D. unit measured at 260 nm corresponds to 50 ug/ml of DNA. DNA purity was determined by measuring the A260/A280 ratio. The ratio of 1.8–1.9 corresponds to pure double stranded DNA. DNA sample aliquots were stored at −20 °C till the time of use. Analysis of polymorphisms The analysis was performed using: &
& & &
Tiangen 2× taq PCR mastermix (Tiangen Biotech, Beijing) which is optimized mixture of taq DNA polymerase, dNTPs, MgCL2, and reaction buffer. For 25 μl reaction, 12.5 μl mastermix was used then DNA and PCR primers were added. The volume of the reaction was completed with ddH2O to result in a final volume of 25 μl. PCR primers (Table 1) Thermal cycler (Mastercycler nexus gradient). EC 360 Submarine Gel electrophoresis system (Maxicell, EC 360 M-E-C apparatus Cooperation St. Petersburg. Florida, USA). The PCR products were visualized using
Table 1
Primer Sequences
Polymorphism
Sequences
BsmI
Forward 5′-CAACCAAGACTACAAGTACCGCG TCAGTGA-3′ Reverse 5′-AACCAGCGGGAAGAGGTCAAGGG-3′ Forward 5′-CAGAGCATGGACAGGGAGCAAG-3′ Reverse 5′-GCAACTCCTCATGGGCTGAGGTC TCA-3′
ApaI and TaqI FokI
Forward 5′-GATGCCAGCTGGCCCTGGCACTG-3′ Reverse 5′-ATGGAAACACCTTGCTTCTTCTCC CTC-3′
Tumor Biol.
2 % agarose gel containing ethidium bromide under ultraviolet transillumination. The lowercase allele (b, a, t, f) denotes the presence of restriction site while the uppercase letter (B, A, T, F) denotes the lacking of restriction site.
Analysis of BsmI polymorphism (rs 1544410) According to [25], the amplification was carried out using 1 μg DNA and 0.2 mM of each primer. The amplification was performed under the following conditions: 94 °C for 3 min as an initial denaturation then 35 cycles (the conditions of each cycle were 94 °C for 20 s, 62 °C for 40 s, and 72 °C for 1 min) followed by final extension incubation at 72 °C for 6 min. The PCR products were then digested with the restriction enzyme; BsmI. The size of DNA fragments was B (800 bp) and b (650 and 150 bp). Analysis of ApaI (rs 7975232) and TaqI (rs 731236) polymorphisms According to Curran et al. [4], the amplification was carried out using 50 ng DNA and 0.2 μM of each primer. The amplification was performed under the following conditions: 94 °C initial for 4 min as an initial denaturation, then (94 °C for 45 s, 64 °C for 60 s, and 72 °C for 2 min) for 5 cycles, then (94 °C for 30 s, 64 °C for 30 s, and 72 °C for 45 s) for 25 cycles then 5 min at 72 °C as a final extension. Detection of ApaI polymorphism The PCR products were digested with the restriction enzyme; ApaI. The size of DNA fragments was A (740 bp) and a (515 and 225 bp). Detection of TaqI polymorphism The PCR products were digested with the restriction enzyme; TaqI. The size of DNA fragments was T (495 and 245 bp) and t (290, 245, and 205 bp). Analysis of FokI polymorphism (rs 10735810) According to Rezende et al. [20], the amplification was carried out using 0.5 μg DNA and 0.2 μM of each primer. The amplification was performed under the following conditions: initial denaturation at 95 °C for 3 min, followed by 35 cycles (94 °C for 1 min, 69 °C for 30 s, and 72 °C for 30 s) then final extension at 72 °C for 3 min. The amplified products were digested with the restriction enzyme; FokI. DNA fragments were F (272 bp) and f (198 and 74 bp).
Statistical analysis The results were statistically analyzed using SPSS program (version 11). Genotype frequencies were assessed for deviation from Hardy-Weinberg Equilibrium using the χ2 test. Odds ratios and 95 % confidence intervals were calculated to detect the association between VDR polymorphisms and the risk of breast cancer. P
RESEARCH ARTICLE
Vitamin D receptor gene polymorphisms and breast cancer risk among postmenopausal Egyptian women Eman Abd-Elkader Abd-Elsalam 1 & Nadia A. Ismaeil 2 & Hoda Sibai Abd-Alsalam 3
Received: 4 January 2015 / Accepted: 12 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Many studies reported that vitamin D can protect against various types of cancers. The mechanism of vitamin D action is mediated by the vitamin D receptor (VDR). VDR may have anti-stress function because it has been identified as p53 direct target gene. This research was designed to investigate the role of VDR polymorphisms BsmI (rs 1544410), ApaI (rs 7975232), TaqI (rs 731236), and FokI (rs 10735810) in pathogenesis of breast cancer using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The study included 130 postmenopausal breast cancer cases aged 49 to 65 years and 100 controls aged 50 to 72 years. A significantly increased risk of breast cancer among carriers of BsmI bb genotype was observed (OR=2.5 (1.1–5.6), P=0.025). Also, a significantly increased risk of breast cancer was detected among women carrying ApaI aa genotype (OR=2.2 (1.02–4.5), P=0.04), while no significant associations were observed between breast cancer risk and genotypes and allele frequencies of FokI and TaqI polymorphisms (P>0.05). Our study showed that VDR gene polymorphisms (BsmI and ApaI) may contribute to breast cancer risk among postmenopausal women.
Keywords VDR . Breast cancer . BsmI . ApaI . Polymorphism * Eman Abd-Elkader Abd-Elsalam [email protected] 1
Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2
General Surgery Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
3
Obstetrics and Gynecology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
Abbreviations VDR SNPs PCR RFLP ER PR
Vitamin D receptor Single-nucleotide polymorphisms polymerase chain reaction Restriction fragment length polymorphism Estrogen receptor Progesterone receptor
Introduction The active vitamin D (1,25-dihydroxycholecalciferol) binds the vitamin D receptor (VDR) to induce a cascade of gene regulation and signaling molecules [26], leading to either suppression of cell proliferation by inducing apoptosis, enhancing cell differentiation, blocking cell cycle, inducing expression of inhibitors of cell cycle progression, inhibiting colony formation, reducing cell inflammation, inhibiting cell invasion, or metastasis and downregulating estrogen receptor signaling pathway in breast cancer [7]. During puberty, pregnancy and involution, VDR expression is regulated in the mammary gland and related to proper glandular development [29]. Ductal proliferation and branching induced by estrogen are inhibited by VDR agonists by binding to VDR [30]. Furthermore, [16] reported that VDR agonists protect against chemically induced pre-neoplastic lesions. p53 is a key molecule in stress response and carcinogenesis where the p53 protein levels are elevated leading to cellular DNA repair or apoptosis [17]. VDR gene has been identified as p53 direct target gene [14] so VDR could have anti-stress function [17]. VDR is also transactivated by stress-activated protein kinases p38 and JNK [19]. Furthermore, some factors related to stress response as prohibition and thioredoxin are
Tumor Biol.
VDR target genes, indicating that VDR has an important role in stress-related response [17]. Mathiasen et al. [15] reported that 1,25-dihydroxyvitamin D3 induced a growth arrest followed by apoptosis in human breast cancer cell lines; MCF-7 cells, expressing a wild-type p53 and T47D cells, that lack a functional p53, at concentrations of 1 to 100 nM, denoting that p53 is not necessary for growth-inhibitory effects of vitamin D. Decreased VDR expression has been observed in breast cancer cells [9] which may be caused by VDR gene polymorphisms [6] and/or DNA methylation [11]. More than 470 single-nucleotide polymorphisms (SNPs) have been discovered in the VDR gene, but a few have been well characterized [26]. This research was designed to evaluate the role of wellcharacterized VDR gene polymorphisms, BsmI (rs 1544410), ApaI (rs 7975232), TaqI (rs 731236), and FokI (rs 10735810) in pathogenesis of breast cancer in Egyptian women.
Materials and methods This research was carried out at the period from March 2013 to August 2014 at the Medical Biochemistry and General Surgery departments, Faculty of Medicine, Zagazig University. A written informed consent was obtained from each participant. Two hundred thirty females were enrolled in this study. They were classified into two groups; group I (control group) that involved 100 apparently healthy volunteers with ages ranging from 50 to 72 years (with mean value±SD of 58.5± 6.9) with no history (either familial or personal) of cancer of any type and group II including 130 females with ages ranging from 49 to 65 years (with mean value±SD of 55.9±4.3) with a clinical and histological diagnoses of breast cancer. All subjects of this study were postmenopausal. The diagnosis of breast cancer was based on history taking, clinical examination to breast and axilla, radiological examination (ultrasonography, mammogram of both breast and axilla, chest X-ray, and abdominal ultrasonography), pathological diagnosis (fine needle aspiration cytology (FNAC) and true cut needle biopsy of breast mass), and estrogen and progesterone receptors evaluation. The breast cancer patients were subclassified into 76 patients without metastasis (M0) and 54 patients suffering from metastatic breast cancer (M1). According to histological grading, patients were graded into grade I (well differentiated), 35 patients; grade II (moderately differentiated), 52 patients; and grade III (poorly differentiated), 43 patients. Estrogen receptor was positive in 73 patients and negative in 57 patients while progesterone receptor was positive in 66 patients and negative in 64 patients.
Research investigations VDR polymorphisms, BsmI (rs 1544410), ApaI (rs 7975232), TaqI (rs 731236), and FokI (rs 10735810), were examined by PCR-RFLP analysis. Blood sampling One milliliter of peripheral venous blood samples was collected in sterile tubes containing EDTA for DNA extraction. DNA extraction DNA was extracted from whole blood samples using TIANamp Genomic DNA Kit that depends on spin column method, according to the protocol supplied by the manufacturer (Tiangen Biotech, Beijing). For the determination of DNA concentration, 1 O.D. unit measured at 260 nm corresponds to 50 ug/ml of DNA. DNA purity was determined by measuring the A260/A280 ratio. The ratio of 1.8–1.9 corresponds to pure double stranded DNA. DNA sample aliquots were stored at −20 °C till the time of use. Analysis of polymorphisms The analysis was performed using: &
& & &
Tiangen 2× taq PCR mastermix (Tiangen Biotech, Beijing) which is optimized mixture of taq DNA polymerase, dNTPs, MgCL2, and reaction buffer. For 25 μl reaction, 12.5 μl mastermix was used then DNA and PCR primers were added. The volume of the reaction was completed with ddH2O to result in a final volume of 25 μl. PCR primers (Table 1) Thermal cycler (Mastercycler nexus gradient). EC 360 Submarine Gel electrophoresis system (Maxicell, EC 360 M-E-C apparatus Cooperation St. Petersburg. Florida, USA). The PCR products were visualized using
Table 1
Primer Sequences
Polymorphism
Sequences
BsmI
Forward 5′-CAACCAAGACTACAAGTACCGCG TCAGTGA-3′ Reverse 5′-AACCAGCGGGAAGAGGTCAAGGG-3′ Forward 5′-CAGAGCATGGACAGGGAGCAAG-3′ Reverse 5′-GCAACTCCTCATGGGCTGAGGTC TCA-3′
ApaI and TaqI FokI
Forward 5′-GATGCCAGCTGGCCCTGGCACTG-3′ Reverse 5′-ATGGAAACACCTTGCTTCTTCTCC CTC-3′
Tumor Biol.
2 % agarose gel containing ethidium bromide under ultraviolet transillumination. The lowercase allele (b, a, t, f) denotes the presence of restriction site while the uppercase letter (B, A, T, F) denotes the lacking of restriction site.
Analysis of BsmI polymorphism (rs 1544410) According to [25], the amplification was carried out using 1 μg DNA and 0.2 mM of each primer. The amplification was performed under the following conditions: 94 °C for 3 min as an initial denaturation then 35 cycles (the conditions of each cycle were 94 °C for 20 s, 62 °C for 40 s, and 72 °C for 1 min) followed by final extension incubation at 72 °C for 6 min. The PCR products were then digested with the restriction enzyme; BsmI. The size of DNA fragments was B (800 bp) and b (650 and 150 bp). Analysis of ApaI (rs 7975232) and TaqI (rs 731236) polymorphisms According to Curran et al. [4], the amplification was carried out using 50 ng DNA and 0.2 μM of each primer. The amplification was performed under the following conditions: 94 °C initial for 4 min as an initial denaturation, then (94 °C for 45 s, 64 °C for 60 s, and 72 °C for 2 min) for 5 cycles, then (94 °C for 30 s, 64 °C for 30 s, and 72 °C for 45 s) for 25 cycles then 5 min at 72 °C as a final extension. Detection of ApaI polymorphism The PCR products were digested with the restriction enzyme; ApaI. The size of DNA fragments was A (740 bp) and a (515 and 225 bp). Detection of TaqI polymorphism The PCR products were digested with the restriction enzyme; TaqI. The size of DNA fragments was T (495 and 245 bp) and t (290, 245, and 205 bp). Analysis of FokI polymorphism (rs 10735810) According to Rezende et al. [20], the amplification was carried out using 0.5 μg DNA and 0.2 μM of each primer. The amplification was performed under the following conditions: initial denaturation at 95 °C for 3 min, followed by 35 cycles (94 °C for 1 min, 69 °C for 30 s, and 72 °C for 30 s) then final extension at 72 °C for 3 min. The amplified products were digested with the restriction enzyme; FokI. DNA fragments were F (272 bp) and f (198 and 74 bp).
Statistical analysis The results were statistically analyzed using SPSS program (version 11). Genotype frequencies were assessed for deviation from Hardy-Weinberg Equilibrium using the χ2 test. Odds ratios and 95 % confidence intervals were calculated to detect the association between VDR polymorphisms and the risk of breast cancer. P
