Association between human telomerase reverse transcriptase (hTERT) MNS16A polymorphism and risk of breast cancer.

BACKGROUND: It is well known that telomerase activity is suppressed in normal human tissues and reactivated in tumors, suggesting that the human telomerase reverse transcriptase (hTERT, MIM: 187270) gene may be involved in carcinogenesis. A polymorphic tandem repeat minisatellite located downstream of exon 16 of hTERT and upstream in the putative promoter region of an antisense hTERT transcript, termed MNS16A, results in a functional polymorphism. Because the association between the MNS16A genetic polymorphism and breast cancer (BC) risk remains an open question, the present case-control study was conducted in Shiraz (Fars Province, Southern Iran). METHODS: A total of 711 samples were collected, including 362 BC patients and 349 healthy individuals. Genotyping was performed by polymerase chain reaction method. Alleles were determined by classifying DNA amplicons of less than and greater than 300 bp as short (S) and long (L) alleles, respectively. RESULTS: Different inheritance models (codominant, dominant, recessive, overdominant genotype models and the allele model) were used to evaluate the association between the MNS16A polymorphism and the risk of BC. No significant association was observed in any of the analyses. It should be noted that the statistical power of the comparisons was low. CONCLUSION: The present study did not support the association between hTERT MNS16A polymorphism and breast cancer risk. Similar studies in other populations with larger sample sizes are needed to determine the association between the hTERT MNS16A polymorphism and susceptibility to breast cancer.

Morphine treatment is associated with diminished telomere length together with down-regulated TERT and TERF2 mRNA levels.

BACKGROUND: Drug dependence promotes accelerated aging and higher mortality compare with the general population. Telomere length is a biomarker of determination of cellular aging. Telomere attrition has been reported in heroin dependent patients. To investigate whether telomere length is affected by morphine or not, the expressions of hTERT and TERF2 in morphine treated human SH-SY5Y cells were determined and compared with untreated cells. METHODS: The SH-SY5Y cells were treated with 1 and 5 µM concentrations of morphine for different exposure times (1d, 2d, 3d, 7d and 60 days). The mRNA levels of hTERT and TERF2 were determined using quantitative real-time RCR. The relative telomere length was measured as the ratio of telomere/36B4. RESULTS: The hTERT and TERF2 mRNA levels were down regulated in morphine treated cells as a function of exposure duration. These alterations were reversible if morphine was removed from the culture medium. No reduction in the relative expression of hTERT and TERF2 in the cells exposed to N-acetyl cysteine (NAC) plus morphine was observed. In the SH-SY5Y cells treated by 5 µM morphine for 60 consecutive days, the hTERT and TERF2 mRNA levels and relative telomere lengths remarkably decreased. CONCLUSIONS: Reversible alteration of mRNA levels by removing morphine from culture medium, and effect of NAC in co-treatment of morphine plus NAC, emphasize the role of reactive oxygen species in down-regulation of the expression of hTERT and TERF2 by morphine. Telomere attrition in morphine treated cells is a consequence of down-regulation of the expression of hTERT and TERF2.

Association between three common genetic polymorphisms of XPC and susceptibility to heroin dependency.

As heroin and morphine produce reactive oxygen species and down-regulate several genes involved in cellular detoxification and DNA repair pathways, neurons experience DNA damage. Xeroderma pigmentosum complementation group C (XPC, OMIM: 613208) gene, which is expressed in the brain, is one of the central genes in the nucleotide excision repair pathway. Three common XPC polymorphisms (Lys939Gln, Ala499Val and PAT) are associated with reduced DNA repair capacity. In this study, the relationship between these polymorphisms and the risk of heroin dependency (HD), as well as, age of first use (AFU) for illegal drugs was investigated on 795 healthy individuals and 442 heroin dependent patients. Statistical analyses indicated that there was no significant association between the XPC polymorphisms and the risk of HD. The haplotypic frequencies of the polymorphisms showed significant difference between HD patients and healthy controls (χ2 = 16.38, df = 6, P = 0.012). Analysis indicated that the "Ala + Gln" haplotype increased the HD risk more than the "Ala + Lys" haplotype (OR = 4.21, 95% CI = 1.29-13.7, P = 0.017). In Cox proportional model, there was significant association between AFU and the Ala499Val polymorphism (Hazard ratio = 1.53, 95% CI: 1.02-2.92, P = 0.036). To investigate the effect of the linkage between the polymorphic sites, we compared the AFU among two common diplotypes ("Ala - Lys/Ala - Lys" and "Val - Lys/Val - Lys"). Statistical analysis indicated that AFU was significantly lower in "Val - Lys/Val - Lys" diplotype (t = 2.63, df = 49, P = 0.011). The present findings suggest that the XPC is a candidate polymorphic locus for AFU.

Association between Genetic Polymorphisms in Superoxide Dismutase Gene Family and Risk of Gastric Cancer.

To determine the association between the SOD1 (Ins/Del), SOD2 (rs2758339, rs5746136), and SOD3 (rs2536512) polymorphisms and the risk of gastric cancer the present study performed. This is a case-control study, including 159 patients with gastric cancer and 242 healthy controls. All subjects were Persian Muslims living in Shiraz (south west Iran). Frequency matching by age and gender was performed. Genomic DNA was extracted from whole blood. Genotypes of the study polymorphism were determined using polymerase chain reaction based methods. The SOD1 Ins/Del and SOD3 rs2536512 polymorphisms did not appear to have relationship with gastric cancer risk. Both SOD2 polymorphisms (rs2758339, rs5746136) showed significant association with the risk of gastric cancer, under assumption that the variant alleles act as dominant alleles. There was significant association between smoking habit and the risk of gastric cancer (OR = 2.54, 95% CI = 1.61-4.02, P < 0.001). Smoker individuals having two putative high-risk genotypes showed elevated risk of gastric cancer compared with nonsmokers without high-risk genotypes, (OR = 5.75, 95% CI = 1.59-20.6, P = 0.007). Assuming that smoking habit and the genotypes are independent risk factors, there was a significant linear trend for the numbers of risk factors and gastric cancer risk (χ2 = 22.9, P < 0.001). This study indicates that the SOD2 polymorphism (rs2758339, rs5746136) is associated with increased risk of gastric cancer, especially in smoker individuals.

Association Between GSTP1 Ile105Val Genetic Polymorphism and Dependency to Heroin and Opium.

Relationship between glutathione S-transferase P1 (GSTP1, OMIM: 134660) variants and the risk of drug dependency is unknown. Chronic use of illegal drugs leads to oxidative stress, which can be alleviated by cellular detoxification mechanisms. There are several polymorphisms in the GSTP1, including Ile105Val (rs1695). This polymorphism leads to an Ile105Val amino acid change and may alter the GSTP1 enzyme activity. There is no study on the association between this polymorphism and risks of heroin (HD) or opium (OD) dependency. This paper consists of two case-control studies. The first study consisted of 442 HD subjects and 794 healthy controls. The second study consisted of 143 cases with OD and 565 healthy blood donors as controls. Genotyping were carried out using PCR based method. The Ile/Val (OR 0.84, 95% CI 0.65-1.07, P = 0.165) and Val/Val (OR 0.87, 95% CI 0.56-1.36, P = 0.879) genotypes did not show significant association with the risk of HD. Neither the Ile/Val (OR 0.72, 95% CI 0.49-1.06, P = 0.103) nor the Val/Val (OR 0.61, 95% CI 0.29-1.30, P = 0.209) was associated with the risk of OD. The GSTP1 Ile105Val polymorphism was not associated with the risk of dependency to opium and heroin.

Evaluating mRNA Expression Levels of the TLR4/IRF5 Signaling Axis During Hepatic Ischemia-Reperfusion Injuries.

OBJECTIVES: Hepatic ischemia and reperfusion during liver transplant surgery result in hepatocellular damage. Toll-like receptors, especially TLR4, have a fundamental basic role in the inflammatory phase of ischemia-reperfusion injuries. The effect of the TRIF-dependent signaling pathway downstream of TLR4 in hepatic ischemia-reperfusion injury has been well established. However, the role of TLR4-MyD88-dependent signal transduction in hepatic ischemia-reperfusion injury has not yet been clarified. The interferon regulatory factor 5 was introduced as the main regulator of the TLR4-MyD88 signaling pathway for activating proinflammatory cytokines. The present study was carried out to investigate the functional impact of the TLR4/IRF5 signaling axis in hepatic ischemia-reperfusion injury. MATERIALS AND METHODS: mRNA expression levels of TLR4, IRF5, tumor necrosis factor α, interleukin 1ß, and interleukin 6 were measured using real-time polymerase chain reaction after short (3 h) and long (168 h) reperfusion periods in a hepatic mouse model of ischemia-reperfusion injury in the presence and absence of N-acetylcysteine. Liver damage was evaluated by plasma levels of alanine aminotrans-ferase and histopathology. RESULTS: Our results show that mRNA levels of TLR4/IRF5 and its downstream cytokines were significantly elevated 3 hours after reperfusion and had drastically fallen to baseline levels 168 hours after reperfusion. Plasma levels of alanine aminotransferase showed the same pattern. Histopathologic study of the samples revealed significant hepatic cell infiltration and necrosis 168 hours after reperfusion. Pretreatment with N-acetylcysteine significantly decreased the mRNA levels of TLR4/IRF5 and its downstream cytokines 3 hours after reperfusion and subsequently improved the previously mentioned hepatic damages 168 hours after reperfusion. CONCLUSIONS: This study suggests a possible role for the TLR4/IRF5 signaling pathway in hepatic ischemia-reperfusion injury. Furthermore, it reveals that N-acetylcysteine may suppress this inflammatory axis and consequently improve hepatic injuries.

Effects of electromagnetic field, cisplatin and morphine on cytotoxicity and expression levels of DNA repair genes.

Morphine (Mor) is widely used as an analgesic drug in cancers and in combination with chemotherapy is known to have DNA damaging effects on non-targeted cell. This study surveyed the effect of Mor in combination with 50-Hz electromagnetic field (EMF) and co-treatment of cisplatin in combination with Mor and EMF on the expression of genes involved in DNA repair pathways. MCF-7 and SH-SY5Y cells were treated with 5.0 µM Mor and then exposed to 50-Hz 0.50 mT EMF in the intermittent pattern of 15 min field-on/15 min field-off. Gene expression, cisplatin and bleomycin cytotoxicity were measured using real-time PCR and MTT assay. Mor treated cells showed significant down-regulation of the examined genes, while in "Mor + EMF" treatments the genes were not significantly changed. IC50 of cisplatin was significantly elevated in both cell lines when co-treated with "Mor + EMF" compared with Mor treated cells. Non-homologous end joining (NHEJ) related genes were significantly decreased in co-treatment of cisplatin and "Mor + EMF" which led to bleomycin higher cytotoxicity in SH-SY5Y not in MCF-7. Our data is promising for providing a cell line-specific sensitization by combination of cisplatin and "Mor + EMF" treatment with local administration of double strand breaking agents.

Expressions of some antioxidant genes in SH-SY5Y cells treated with ß-lapachone, morphine and electromagnetic field.

ß-Lapachone (ß-Lap), morphine (Mor), and electromagnetic field (EMF) generate reactive oxygen species. The goal of the present study was to examine the effects of Mor and EMF, in combination with ß-Lap on the cell growth inhibition and expression of several antioxidant genes. The 0.50 mT intensity of 50 Hz EMF and two exposure conditions ("15 min field-on/15 min field-off" and "30 min field-on continuously") on SH-SY5Y cells were used. The effects of Mor and EMF, in combination with ß-Lap on cell growth inhibition and the expression levels of several antioxidant genes (NQO1, NQO2, SOD1, SOD2, CAT, GSTO1, GSTM2, GSTM3, GSTP1, MGST1, MGST3) in SH-SY5Y cells were measured. The relative mRNA levels were calculated according to the [Formula: see text]. Whereas NQO1 mRNA level decreased in the "15 min field-on/15 min field-off" condition, the expression level of NQO2 was increased. Both NQO1 and NQO2 expressions increased in Mor treated cells. IC50 values of ß-Lap in combination with Mor, EMF, and "Mor + EMF" were higher than cells treated only with ß-Lap. The NQO1 expression level in the cells treated with ß-Lap was higher than the other treatments, indicating that ß-Lap induces the expression of NQO1. Moreover, multiple linear regression analysis indicated that NQO1 mRNA levels were associated positively with ß-Lap and negatively with EMF. At least in part, the mRNA levels of NQO1 were associated with IC50 values of ß-Lap in designed treatments. There is a negative association between mRNA levels of NQO1 and IC50 values of ß-Lap but not NQO2.

Association of the SOD2 (rs2758339 and rs5746136) polymorphisms with the risk of heroin dependency and the SOD2 expression levels.

BACKGROUND: Superoxide dismutase-2 (SOD2, OMIM: 147460) is involved in the detoxification of superoxide anions. The SOD2 mRNA level is down-regulated in cells exposed to morphine. Oxidative stress plays an important role in drug dependency. The rs2758339 (A/C) is located in the vicinity of SP1 and NF-κB transcription element sequences and the rs5746136 (A/G) creates a new glucocorticoid receptor binding site. Taken together, it is hypothesized that these polymorphisms might be associated with the risk of heroin dependency (HD) and the SOD2 expression level. METHODS: To investigate the association between the SOD2 polymorphisms and the risk of HD, 442 heroin dependent persons and 799 healthy controls were included. This study also, consisted of 77 healthy students of Shiraz University (Iran) to investigate the association between the SOD2 polymorphisms and the gene expression level. RESULTS: The AC (OR = 0.72, 95% CI = 0.56-0.93, P = 0.013) and CC (OR = 0.64, 95% CI = 0.45-0.92, P = 0.015) genotypes of the rs2758339 were negatively associated with the risk of HD. The AA genotype of the rs5746136 increased the risk of HD (OR = 1.46, 95% CI = 1.03-2.07, P = 0.031). The AA haplotype was associated with the risk of HD (OR = 1.31, 95% CI = 1.09-1.58, P = 0.004). There was no relationship between the study genotypes (or diplotypes) and the SOD2 expression level. CONCLUSION: The rs2758339 and rs5746136 polymorphisms of the SOD2 are associated with the risk of HD but not associated with the SOD2 expression level.

Electromagnetic Field Could Protect SH-SY5Y Cells Against Cisplatin Cytotoxicity, But Not MCF-7 Cells.

Cisplatin [cis-dichlorodiammine platinum (II), CDDP], morphine (Mor), and electromagnetic field (EMF) induced oxidative stress. In this study, we tried to increase the cytotoxicity of CDDP in combination with Mor and/or EMF in MCF-7 and SH-SY5Y cells. Furthermore, we evaluate the expression levels of 11 antioxidant genes in both cell lines. We designed four treatments: CDDP alone, "CDDP+Mor," "CDDP+EMF," and "CDDP+Mor+EMF." Serial dilutions of CDDP, Mor (5.0 µM), and EMF (50 Hz, 0.50 mT, "15 min field-on/15 min field-off") were used for estimation of relative IC50 values. The mRNA expression levels of antioxidant genes were determined by real-time PCR. The IC50 value of CDDP in "CDDP+Mor+EMF" treatment was significantly higher than CDDP alone and "CDDP+Mor" treatments in both cell lines. Whereas the expression levels of antioxidant genes in the four treatments showed similar patterns in MCF-7 cells, in SH-SY5Y cells, most of the antioxidant genes showed an upregulation with "CDDP+EMF" and "CDDP+Mor+EMF" treatments. Moreover, significant differences in the number of upregulated genes were observed between different treatments in SH-SY5Y cells. The molecular mechanism of CDDP-reduced cytotoxicity in our designed combinations is probably different in MCF-7 and SH-SY5Y cells. CDDP in combination with EMF could protect SH-SY5Y cells from the cytotoxicity, whereas it has no significant change in MCF-7 cells.

Influence of a 50bp Ins/Del polymorphism at promoter of the superoxide dismutase-1 on gene expression and risk of heroin dependency.

OBJECTIVE: Superoxide dismutase-1 (SOD1, OMIM: 147450) is one of the major antioxidant enzymes, which plays a vital role in clearance of reactive oxygen species. A genetic polymorphism of 50 bp insertion/deletion (Ins/Del) in the promoter region of the SOD1 was reported. The aims of the present study are to evaluate the influence of this polymorphism on the SOD1 mRNA levels in human peripheral blood cells and its association with risk of heroin dependency. METHODS: The present study consisted of 47 healthy students of Shiraz University (south-west Iran) for investigating the association between the Ins/Del polymorphism on expression level of SOD1, also a total of 442 heroin dependent and 799 healthy controls were included in a case-control study investigating the association between the study polymorphism and risk of dependency to heroin. The quantitative SOD1 mRNA expression levels were investigated using quantitative real-time PCR. RESULTS: Statistical analysis revealed a significant difference between the study genotypes (t = 5.17; df = 45; P < 0.001). The Del allele of the study polymorphism decreased approximately 33% of the SOD1 mRNA levels of the gene in the heterozygote individuals. Statistical analysis indicating that in both genders, neither the Ins/Del nor the Del/Del genotypes was associated with the risk of heroin addiction. CONCLUSIONS: The present study indicating that although the Ins/Del polymorphism of SOD1 is associated with the SOD1 expression levels, this polymorphism is not associated with the risk of dependency to heroin.

Different profiles of the mRNA levels of DNA repair genes in MCF-7 and SH-SY5Y cells after treatment with combination of cisplatin, 50-Hz electromagnetic field and bleomycin.

Neurotoxicity is known to be a major dose-limiting adverse effect of cisplatin (CDDP), alone or in combination with other chemicals. DNA repair capacity serve as a neuroprotective factor against CDDP. The purpose of this study was to evaluate the effect of 50-Hz electromagnetic field (EMF) in combination with CDDP and bleomycin (Bleo) on expression of some of DNA repair genes (GADD45A, XRCC1, XRCC4, Ku70, Ku80, DNA-PKcs and LIG4) in MCF-7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. MCF-7 and SH-SY5Y cells were pre-treated with CDDP in the presence or absence of EMF and then exposed to different concentration of Bleo. EMF (0.50mT intensity) was used in the intermittenet pattern of "15min field on/15min field off" with 30min total exposure. Cell viability assay was done and then the transcript levels of the examined genes were measured using quantitative real-time PCR in "CDDP+Bleo" and "CDDP+EMF+Bleo" treatments. Our results indicated that MCF-7 cells treated with "CDDP+EMF+Bleo" showed more susceptibility compared with "CDDP+Bleo" treated ones, while SH-SY5Y susceptibility was not changed between the two treatments. The represented data indicated that MCF-7 and SH-SY5Y cells showed non-random disagreement in DNA repair gene expression in 11 conditions (out of 14 conditions) with each other (χ2=4.52, df=1, P=0.033). This finding can be promising for sensitizing breast cancer cells while protecting against CDDP induced neuropathy in cancer patients.

Effect of Sodium Arsenite on the Expression of Antioxidant Genes (SOD2 and CAT) in MCF-7 and Jurkat Cell Lines.

BACKGROUND: Sodium arsenite (NaAsO2) has potent cytotoxic activity in human cancer cells. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. The purpose of the present study was to evaluate the alteration of mRNA levels of catalase (CAT) and superoxide dismutase 2 (SOD2) in MCF-7 and Jurkat cells after exposure to NaAsO2. METHODS: Methylthiazol tetrazolium (MTT) viability assay was performed to evaluate cytotoxicity of NaAsO2 in MCF-7 and Jurkat cells. For evaluating the expression levels of the CAT and SOD2, we used two concentrations of NaAsO2 (5 and 15 µM), lower than the concentrations at which 50% of cell viability were lost. The cells were treated with co-treatment of NaAsO2 (15 µM) and N-acetyl-cysteine (NAC; 5 µM) in the media for 24 h. The control cells were maintained in sodium arsenite free growth medium. The experiments were done triplicate. Using quantitative real-time PCR, the expression levels of CAT and SOD2 were quantified. One sample student's t test was performed for comparisons of mRNA levels between treatment groups and their corresponding untreated control cells. RESULTS: CAT mRNA level decreased significantly in both cell lines following exposure to NaAsO2 (P<0.05). Expression levels of SOD2 decreased in Jurkat cells and increased in MCF-7 cells after treatment with NaAsO2 (P<0.05). CONCLUSION: After cells exposure to NaAsO2, CAT mRNA level decreased in both examined cell lines but the alterations of SOD2 mRNA level is cell specific. The NAC modulated the NaAsO2 associated alterations of CAT and SOD2 mRNA levels, therefore, the NaAsO2 might act through inducing reactive oxygen species.