Lipid peroxidation and glutathione peroxidase activity relationship in breast cancer depends on functional polymorphism of GPX1.

BACKGROUND: Since targeting oxidative stress markers has been recently recognized as a novel therapeutic target in cancer, it is interesting to investigate whether genetic susceptibility may modify oxidative stress response in cancer. The aim of this study was to elucidate whether genetic polymorphism in the antioxidant enzymes is associated with lipid peroxidation in breast cancer. METHODS: We conducted a study among Polish women, including 136 breast cancer cases and 183 healthy controls. The analysis included genetic polymorphisms in five redox related genes: GPX1 (rs1050450), GPX4 (rs713041), SOD2 (rs4880), SEPP1 (rs3877899) and SEP15 (rs5859), lipid peroxidation, the activities of antioxidant enzymes determined in blood compartments as well as plasma concentration of selenium - an antioxidant trace element involved in cancer. Genotyping was performed using the Real Time PCR. Lipid peroxidation was expressed as plasma concentration of thiobarbituric acid reactive substances (TBARS) and measured with the spectrofluorometric method. Glutathione peroxidase activity was spectrophotometrically determined in erythrocytes (GPx1) and plasma (GPx3) by the use of Paglia and Valentine method. Spectrophotometric methods were employed to measure activity of cytosolic superoxide dismutase (SOD1) in erythrocytes (Beauchamp and Fridovich method) and ceruloplasmin (Cp) in plasma (Sunderman and Nomoto method). Plasma selenium concentration was determined using graphite furnace atomic absorption spectrophotometry. RESULTS: Breast cancer risk was significantly associated with GPX1 rs1050450 (Pro198Leu) polymorphism, showing a protective effect of variant (Leu) allele. As compared to the control subjects, lipid peroxidation and GPx1 activity were significantly higher in the breast cancer cases, whereas ceruloplasmin activity was decreased. After genotype stratification, both GPx1 activity and TBARS concentration were the highest in GPX1 Pro/Pro homozygotes affected by breast cancer. At the same time, there was a significant correlation between the level of lipid peroxidation and GPx1 activity among the cancer subjects possessing GPX1 Pro/Pro genotype (r = 0.3043; p = 0.0089), whereas such a correlation was completely absent in the cases carrying at least one GPX1 Leu allele as well as in the controls (regardless of GPX1 genotype). CONCLUSIONS: GPX1 polymorphism may be an important factor modifying oxidative stress response in breast cancer subjects. Further studies are needed to elucidate its potential clinical significance.

Association of microRNAs and pathologic response to preoperative chemotherapy in triple negative breast cancer: preliminary report.

Triple negative breast cancer (TNBC) has caught the attention of oncologists worldwide because of poor prognosis and paucity of targeted therapies. Gene pathways have been widely studied, but less is known about epigenetic factors such as microRNAs (miRNAs) and their role in tailoring an individual systemic and surgical approach for breast cancer patients. The aim of the study was to examine selected miRNAs in TNBC core biopsies sampled before preoperative chemotherapy and the subsequent pathologic response in mastectomy or breast conservation specimens. Prior to treatment, core needle biopsies were collected from 11 female patients with inoperable locally advanced TNBC or large resectable tumors suitable for down-staging. In all 11 TNBC core biopsies we analyzed 19 miRNAs per sample: 512, 190, 200, 346, 148, 449, 203, 577, 93, 126, 423, 129, 193, 182, 136, 135, 191, 122 and 222 (miRCURY LNA™ Universal RT microRNA polymerase chain reaction Custom Pick & Mixpanels). The Wilcoxon signed-rank test was used to compare related samples. Ingenuity pathway analysis was used to evaluate potential functional significance of differentially expressed miRNAs. Statistical analysis showed that 3 of 19 miRNAs differed in relation to pathologic response i.e. good versus poor. These differences failed to reach statistical significance, although a trend was observed (p=0.06). Among these miRNAs, we identified-miR-200b-3p, miR-190a and miR-512-5p. In summary, our results indicate that higher miR-200b-3p, higher miR-190a and lower miR-512-5p expression levels in core biopsies sampled from TNBC patients may be associated with better pathologic response to chemotherapy and the increased feasibility of breast conserving surgery in these patients. Although these results were from a small cohort, they provide an important basis for larger, prospective, multicenter studies to investigate the potential role of miRNAs in neoadjuvant setting.

Gene expression and pathologic response to neoadjuvant chemotherapy in breast cancer.

Pathologic complete response after neoadjuvant systemic treatment appears to be a valid surrogate for better overall survival in breast cancer patients. Currently, together with standard clinicopathologic assessment, novel molecular biomarkers are being exhaustively tested in order to look into the heterogeneity of breast cancer. The aim of our study was to examine an association between 23-gene real-time-PCR expression assay including ABCB1, ABCC1, BAX, BBC3, BCL2, CASP3, CYP2D6, ERCC1, FOXC1, GAPDH, IGF1R, IRF1, MAP2, MAPK 8, MAPK9, MKI67, MMP9, NCOA3, PARP1, PIK3CA, TGFB3, TOP2A, and YWHAZ receptor status of breast cancer core biopsies sampled before neoadjuvant chemotherapy (anthracycline and taxanes) and pathologic response. Core-needle biopsies were collected from 42 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan low density arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to multiple hypothesis testing. Statistical analysis showed that seven genes out of a 23-gene real-time-PCR expression assay differed significantly in relation to pathologic response regardless of breast cancer subtypes. Among these genes, we identified: BAX (p = 0.0146), CYP2D6 (p = 0.0063), ERCC1 (p = 0.0231), FOXC1 (p = 0.0048), IRF1 (p = 0.0022), MAP2 (p = 0.0011), and MKI67 (p = 0.0332). The assessment of core biopsy gene profiles and receptor-based subtypes, before neoadjuvant therapy seems to predict response or resistance and to define new signaling pathways to provide more powerful classifiers in breast cancer, hence the need for further research.

The incidence and clinical analysis of non-melanoma skin cancer.

Non-melanoma skin cancers (NMSCs) are the most common malignancies diagnosed in Caucasian populations. Basal cell carcinoma (BCC) is the most frequent skin cancer, followed by squamous cell carcinoma (SCC). Unfortunately, most European cancer registries do not record individual types of NMSC. To evaluate the incidence of primary BCCs and SCCs regarding age, sex, tumour site and tumour subtype to determine trends in epidemiology of both cancers. Retrospective analysis of BCCs and SCCs diagnosed and treated across seven sites in Poland from 1999 to 2019. We recorded 13,913 NMSCs occurring in 10,083 patients. BCC represented 85.2% of all cases. SCC patients were older than BCC patients (77.1 ± 11.3 years vs. 70.1 ± 12.3 years, p < 0.01). The nodular subtype was the most common subtype of BCC, followed by the superficial and infiltrative subtypes. The superficial BCC subtype was more common on photoprotected areas (p < 0.01), whereas the nodular BCC subtype occurred on the face (p < 0.01). The high-risk SCC subtypes were more common on face compared to low-risk SCC subtypes (p < 0.01). BCC and SCC are common malignancies developing at various ages and anatomical sites. These data underline the need for better registration policies regarding NMSC in order to improve prevention and treatment strategies for these tumours.

Efficacy of DNA double-strand breaks repair in breast cancer is decreased in carriers of the variant allele of the UBC9 gene c.73G>A polymorphism.

UBC9 (E2) SUMO conjugating enzyme plays an important role in the maintenance of genome stability and integrity. In the present work we examined the association between the c.73G>A (Val25Met) polymorphism of the UBC9 gene (rs11553473) and efficacy of DNA double-strand breaks (DSBs) repair (DRE) in breast cancer patients. We determined the level of endogenous (basal) and exogenous (induced by γ-irradiation) DSBs and efficacy of their repair in peripheral blood lymphocytes of 57 breast cancer patients and 70 healthy individuals. DNA damage and repair were studied by neutral comet assay. Genotypes were determined in DNA from peripheral blood lymphocytes by allele-specific PCR (ASO-PCR). We also correlated genotypes with the clinical characteristics of breast cancer patients. We observed a strong association between breast cancer occurrence and the variant allele carried genotypes in patients with elevated level of basal as well as induced DNA damage (OR 6.74, 95% CI 2.27-20.0 and OR 5.33, 95% CI 1.81-15.7, respectively). We also found statistically significant (p<0.05) difference in DRE related to the c.73G>A polymorphism of the UBC9 gene in breast cancer patients. Carriers of variant allele have decreased DNA DRE as compared to wild type genotype carriers. We did not find any association with the UBC9 gene polymorphism and estrogen and progesterone receptor status. The variant allele of the UBC9 gene polymorphism was strongly inversely related to HER negative breast cancer patients (OR 0.03, 95% CI 0.00-0.23). Our results suggest that the c.73G>A polymorphism of the UBC9 gene may affect DNA DSBs repair efficacy in breast cancer patients.

Clinical and epidemiological analysis of basosquamous carcinoma: results of the multicenter study.

Basosquamous carcinoma (BSC) is a rare non-melanoma skin cancer that shares the characteristic features of both basal and squamous cell carcinomas (BCC, SCC). Our research enables better characterization of BSC in comparison to high-risk subtypes of BCC and SCC. Paper includes a retrospective analysis of BSC cases regarding sex, age, number of tumors and anatomical distribution in comparison to BCC and SCC evaluating the differences and defining the implications. Histologically confirmed carcinomas recorded between 1999 and 2019 were studied. 181 diagnosed BSC cases were identified, making this study the largest cohorts of BSC patients reported worldwide. Most cases were reported on head and neck. Analysis of facial anatomic distribution shows that most commonly affected sites were the nose (43%) and the cheek (25%). The age at excision of metatypical BCC was higher than those of low-risk BCC (P < 0.05), however similar to high-risk BCC (P = 0.20). We revisited that the concept of BSC is the most similar to high-risk subtypes of BCC. Patients with diagnosed BSC have higher risk of second nonmelanoma skin cancer. Therefore, the frequency of follow-up examination should be adjusted to the individual risk of another skin cancer.

Polymorphism of the homologous recombination repair genes RAD51 and XRCC3 in breast cancer.

The RAD51 protein and its paralog, XRCC3, play an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Since DSBs may contribute to the pathogenesis of breast cancer and variability in DNA repair genes may be linked with some cancers, we performed a case-control study (135 cases and 175 controls) to check the association between the genotypes of the Thr241Met polymorphism of the XRCC3 gene and the 135G>C polymorphism of the RAD51 gene and breast cancer occurrence and progression. Genotypes were determined in peripheral blood lymphocytes by RFLP-PCR. We did not find any association between either polymorphism singly and breast cancer occurrence. Both polymorphisms were not related to tumor size, estrogen and progesterone receptors status, cancer type and grade. However, the Thr241Met genotype of the XRCC3 polymorphism slightly increased the risk of local metastasis in breast cancer patients (OR 2.56, 95% CI 1.27-5.17). The combined Thr241Met/135G>C genotype decreased the risk of breast cancer occurrence (OR 0.22, 95% CI 0.08-0.59). Our results suggest that the variability of the DNA homologous recombination repair genes RAD51 and XRCC3 may play a role in breast cancer occurrence and progression, but this role may be underlined by a mutual interaction between these genes.

Association between DNA damage, DNA repair genes variability and clinical characteristics in breast cancer patients.

The cell's susceptibility to DNA damage and its ability to repair this damage are important for cancer induction, promotion and progression. In the present work we determined the level of basal (total endogenous) and endogenous oxidative DNA damage as well as polymorphism of the DNA repair genes: RAD51 (135 G/C), XRCC3 (Thr241Met), OGG1 (Ser326Cys) and XPD (Lys751Gln) in peripheral blood lymphocytes of 41 breast cancer patients and 48 healthy individuals. DNA damage was evaluated by alkaline comet assay with DNA repair enzymes: Endo III and Fpg, preferentially recognizing oxidized DNA bases. The genotypes of the polymorphisms were determined by restriction fragment length polymorphism PCR. We observed a strong association between breast cancer occurrence and the genotypes C/C of the RAD51-135G/C polymorphism, Ser/Ser of the OGG1-Ser326Cys and Lys/Gln of the XPD-Lys751Gln, whereas the genotypes G/C of the RAD51-135G/C and Lys/Lys of the XPD-Lys751Gln exerted a protective effect against breast cancer. We also found that individuals with the G/C genotype of the RAD51-135G/C polymorphism and with the Lys/Lys genotype of the XPD-Lys751Gln polymorphism displayed a lower extent of basal and oxidative DNA damage. A strong association between higher level of oxidative DNA damage and the Lys/Gln genotype of the latter polymorphism was found. We also correlated genotypes with clinical characteristics of breast cancer patients. We observed a strong association between the G/C genotype of the RAD51-135 G/C polymorphism and the expression of the progesterone receptor and between both alleles of the OGG1-Ser326Cys polymorphism and lymph node metastasis. Our results suggest that the polymorphism of the RAD51, OGG1 and XPD genes may be linked with breast cancer by the modulation of the cellular response to oxidative stress and these polymorphisms may be considered as markers in breast cancer along with the genetic or/and environmental indicators of oxidative stress.

STI571 reduces NER activity in BCR/ABL-expressing cells.

Nucleotide-excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) photoproducts. We investigated the influence of an anticancer drug, STI571, on the efficacy of NER in removing UV-induced DNA damage. STI571 is used mostly in the treatment of chronic myeloid leukemia and inhibits activity of the BCR/ABL oncogenic tyrosine kinase, which is a hallmark of this disease. NER activity was examined in the BCR/ABL-expressing cell lines K562 and BV173 of myeloid and lymphoid origin, respectively, as well as in CCRF-CEM cells, which do not express BCR/ABL. A murine myeloid parental 32D cell line and its counterpart transfected with the BCR/ABL gene were also tested. NER activity was assessed in the cell extracts by use of an UV-irradiated plasmid as a substrate and by a modified single-cell gel electrophoresis (comet) assay on UV-treated nucleoids. Additionally, quantitative PCR was performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. Results obtained from these experiments indicate that STI571 decreases the efficacy of NER in leukemic cells expressing BCR/ABL. Therefore, STI571 may overcome the drug resistance associated with increased DNA repair in BCR/ABL-positive leukemias.

DNA damage and repair in children with Down's syndrome.

Down's syndrome (DS) is associated with the presence of a third 21 chromosome and is generally considered as a non-cancer-prone genetic disease. However, leukaemias occur more frequently in children with the syndrome than in general population and there is an open question, whether the presence of an additional chromosome may contribute to genomic instability, which, in turn, may play a role in a higher susceptibility to cancer and leukaemias in particular. In order to assess genomic instability associated with the presence of a third 21 chromosome, we determined the level of endogenous DNA damage and susceptibility to a genotoxic stress-inducing factor, hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidyne (MNNG) as well as the ability to remove DNA damage in the peripheral blood lymphocytes of children with DS and healthy kids. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline comet assay. Oxidative DNA damage was assayed with DNA repair enzymes: endonuclease III-like NTH1 and formamidopyrimidine-DNA glycosylase. The cells taken from children with DS did not display an effective DNA repair after treatment with 10 mM hydrogen peroxide. No difference in the sensitivity to DNA-damaging agents and the efficacy of DNA repair due to age and gender in DS children was observed. These results suggest that children with DS may be characterized by the increased sensitivity to the DNA-damaging agents impaired cellular reaction to DNA damage, which, in turn, may increase the probability of cancers in these children. Therefore, a special care to avoid exposure to potential mutagenic factor my be considered in these children.

The ESR1 and GPX1 gene expression level in human malignant and non-malignant breast tissues.

BACKGROUND: The aim of this study was to establish whether the gene expression of estrogen receptor alpha (encoded by ESR1) correlates with the expression of glutathione peroxidase 1 (encoded by GPX1) in the tumor and adjacent tumor-free breast tissue, and whether this correlation is affected by breast cancer. Such relationships may give further insights into breast cancer pathology with respect to the status of estrogen receptor. METHODS: We used the quantitative real-time PCR technique to analyze differences in the expression levels of the ESR1 and GPX1 genes in paired malignant and non-malignant tissues from breast cancer patients. RESULTS: ESR1 and GPX1 expression levels were found to be significantly down-regulated by 14.7% and 7.4% (respectively) in the tumorous breast tissue when compared to the non-malignant one. Down-regulation of these genes was independent of the tumor histopathology classification and clinicopathological factors, while the ESR1 mRNA level was reduced with increasing tumor grade (G1: 103% vs. G2: 85.8% vs. G3: 84.5%; p<0.05). In the non-malignant and malignant breast tissues, the expression levels of ESR1 and GPX1 were significantly correlated with each other (Rs=0.450 and Rs=0.360; respectively). CONCLUSION: Our data suggest that down-regulation of ESR1 and GPX1 was independent of clinicopathological factors. Down-regulation of ESR1 gene expression was enhanced by the development of the disease. Moreover, GPX1 and ESR1 gene expression was interdependent in the malignant breast tissue and further work is needed to determine the mechanism underlying this relationship.

Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair.

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 microM and its direct metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 microM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.

DNA damage and repair in gastric cancer--a correlation with the hOGG1 and RAD51 genes polymorphisms.

The cell's susceptibility to mutagens and its ability to repair DNA lesions are important for cancer induction, promotion and progression. Both the mutagens' sensitivity and the efficacy of DNA repair may be affected by variation in several genes, including DNA repair genes. The hOGG1 gene encodes glycosylase of base excision repair and RAD51 specifies a key protein in homologues recombination repair. Both can be involved in the repair of oxidative DNA lesions, which can contribute to stomach cancer. In the present work we determined the level of basal and oxidative DNA damage and the kinetics of removal of DNA damage induced by hydrogen peroxide in peripheral blood lymphocytes of 30 gastric cancer patients and 30 healthy individuals. The metrics from DNA damage and repair study were correlated with the genotypes of common polymorphisms of the hOGG1 and RAD51 genes: a G-->C transversion at 1245 position of the hOGG1 gene producing a Ser-->Cys substitution at the codon 326 (the Ser326Cys polymorphism) and a G-->C substitution at position 135 (5'-untranslated region) of the RAD51 gene (the G135C polymorphism). DNA damage and repair were evaluated by alkaline single cell gel electrophoresis (comet assay) assisted by DNA repair enzymes: endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), preferentially recognizing oxidized DNA bases. The genotypes of the polymorphism were determined by restriction fragment length polymorphism PCR. We observed a strong association between gastric cancer occurrence, impaired DNA repair in human lymphocytes and the G/C genotype of the G135C polymorphism of the RAD51 gene. Moreover, there was a strong correlation between that genotype and stomach cancer occurrence in subjects with high level of oxidatively damaged DNA. We did not observe any correlation between the Ser1245Cys polymorphism of the hOGG1 gene and gastric cancer, including subjects with impaired DNA repair and/or high levels of endogenous oxidative DNA lesions. Therefore, our result suggest that the G135C polymorphism of the RAD51 gene may be linked with gastric cancer by the modulation of the cellular response to oxidative stress and this polymorphism may be a useful additional marker in this disease along with the genetic or/and environmental indicators of oxidative stress.

Helicobacter pylori infection can modulate the susceptibility of gastric mucosa cells to MNNG.

The pathogenesis of stomach cells can be associated with their susceptibility to exogenous dietary irritants, like nitrosamines such as dimethylnitrosamines (DMNA), and to the effects of non-dietary factors, including Helicobacter pylori infection. We used N-methyl-N'-nitro N-nitrosoguanidyne (MNNG) as a surrogate agent that induces a spectrum of DNA damage similar to DMNA. Using the alkaline comet assay, we showed that antioxidants--vitamins C and E, quercetin, and melatonin--reduced the genotoxic effect of MNNG in H. pylori-infected and non-infected human gastric mucosa cells (GMCs). To compare the sensitivity of the stomach and the blood, the experiment was also carried out in peripheral blood. We observed a higher level of DNA damage induced by MNNG in H. pylori-infected than in noninfected GMCs. We did not note any difference in the efficacy of the repair of the damage in either type of GMC. H. pylori infection may play an important role in the pathogenesis of GMCs, as it can modulate their susceptibility to dietary mutagens/carcinogens, thus contributing to gastric cancer.

Imatinib (STI571) inhibits DNA repair in human leukemia oncogenic tyrosine kinase-expressing cells.

BCR/ABL oncogene, as a result of chromosome aberration t(9;22), is the pathogenic principle of almost 95% of human chronic myeloid leukemia (CML). Imatinib (STI571) is a highly selective inhibitor of BCR/ABL oncogenic tyrosine kinase used in leukemia treatment. It has been suggested that BCR/ABL may contribute to the resistance of leukemic cells to drug and radiation through stimulation of DNA repair in these cells. To evaluate further the influence of STI571 on DNA repair we studied the efficacy of this process in BCR/ABL-positive and -negative cells using single cell electrophoresis (comet assay). In our experiments, K562 human chronic myeloid leukemia cells expressing BCR/ABL and CCRF-CEM human acute lymphoblastic leukemia cells without BCR/ABL expression were employed. The cells were exposed for 1 h at 37 degrees C to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 5 microM, mitomycin C (MMC) at 50 microM or to gamma-radiation at 15 Gy with or without a 24 h preincubation at 1 microM of STI571. The MTT cells survival after 4 days of culture showed that STI571 enhanced the cytotoxity of the examined compounds in the K562 line. Further it was found, that the inhibitor decreased the efficacy of DNA repair challenged by each agent, but only in the K562 expressing BCR/ABL. Due to the variety of DNA damage induced by the employed agents in this study we can speculate, that BCR/ABL may stimulate multiple pathways of DNA repair. These results extend our previous studies performed on BCR/ABL-transformed mouse cells onto human cells. It is shown that BCR/ABL stimulated DNA repair in human leukemia cells. In conclusion we report that STI571 was found to inhibit DNA repair and abrogate BCR/ABL-positive human leukemia cells therapeutic resistance.

Genetic Polymorphism of SUMO-Specific Cysteine Proteases - SENP1 and SENP2 in Breast Cancer.

SENP proteases take part in post-translational modification of proteins known as sumoylation. They catalyze three distinct processes during sumoylation: processing of SUMO protein, deconjugation of SUMO from the target protein, and chain editing which mentions to the dismantling of SUMO chain. Many proteins that are involved in the basic processes of cells, such as regulation of transcription, DNA repair or cell cycle control, are sumoylated. The aim of these studies was to investigate an association between polymorphic variants (SNPs) of the SENP1 gene (c.1691 + 36C > T, rs12297820) and SENP2 gene (c.902C > A, p.Thr301Lys, rs6762208) and a risk of breast cancer occurrence. We performed a case-control study in 324 breast cancer cases and 335 controls using PCR-RLFP. In the case of the SENP1 gene polymorphism we did not find any association between this polymorphism and breast cancer risk. In the case of SENP2 gene polymorphism we observed higher risk of breast cancer for carriers of the A allele (OR =1.33; 95 % CI 1.04-1.69). Our analysis also showed the genotype C/C (OR =0.67, 95 % CI 0.48-0.93) and the allele C (OR =0.75, 95 % CI 0.59-0.69) of this polymorphism decrease a risk of breast cancer. We also checked the distribution of genotypes and frequency of alleles of the SENP1 and SENP2 genes polymorphisms in groups of patients with different hormone receptor status, patients with positive and negative lymph node status and patients with different tumor grade. Odds ratio analysis showed a higher risk of metastases in women with the genotype C/C (OR =2.07, 95 % CI 1.06-4.05) and allele C (OR =2.10 95 % CI 1.10-4.01) of the c.1691 + 36C > T SENP1 gene polymorphism. Moreover, we observed reduced risk in women with the allele T (OR =0.48, 95 % CI 0.25-0.91) in this polymorphic site. In the case of SENP2 gene polymorphism we observed that the A/A genotype correlated with the lack of estrogen receptor (OR =1.94, 95 % CI 1.04-3.62). Our results suggest that the variability of the SENP1 and SENP2 genes may play a role in breast cancer occurrence. Further studies are needed to clarify their biological functions in breast cancer.

Expression of MMP and TIMP mRNA in peripheral blood leukocytes of patients with invasive ductal carcinoma of the breast.

PURPOSE: An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) appears critical for tumor progression and metastasis. This study aimed to determine whether gene expression of MMP1, MMP2, MMP9, TIMP1 and TIMP3 and the MMP/TIMP expression ratio in peripheral blood leukocytes (PBLs) and the MMP1 and TIMP1 contents or MMP1/TIMP1 ratio in plasma were associated with clinicopathological characteristics in invasive ductal carcinoma (IDC) of the breast. MATERIALS AND METHODS: Blood samples were collected from women newly diagnosed with IDC who had not received prior treatment (n = 102). Gene expression in PBLs was analyzed by quantitative real-time polymerase chain reaction. Concentrations of MMP1 and TIMP1 in plasma were measured using ELISA. RESULTS: In univariate analysis the expression levels of MMP2 and TIMP1 mRNA were significantly higher in premenopausal compared to postmenopausal patients (p<0.001 and p = 0.014, respectively). MMP2 mRNA expression negatively correlated with age (p<0.001, r = -0.43). We found that the MMP2/TIMP3 expression ratio was significantly higher in women after menopause (p = 0.007). The MMP2/TIMP1 expression ratio was higher in human epidermal growth factor receptor 2 (HER2)-positive patients (p = 0.022). Low-grade tumors had significantly lower MMP1/TIMP1 and MMP2/TIMP1 expression ratios (p = 0.047 and p = 0.048, respectively). TIMP1 plasma concentration was significantly higher in small tumors compared with T2-T3 tumors (p = 0.013). CONCLUSIONS: These findings reveal an important association between tumor characteristics and expression ratios of MMP1/TIMP1 and MMP2/TIMP1 in PBLs and TIMP1 concentration in plasma. Menopausal status may influence the mRNA expression levels of MMP2 and TIMP1 as well as the MMP2/TIMP3 expression ratio in IDC of the breast.

Immune checkpoints: Cytotoxic T-lymphocyte antigen 4 and programmed cell death protein 1 in breast cancer surgery.

Immune checkpoints refer to a plethora of inhibitory pathways built into the immune system, and recent studies have emphasized the role of these checkpoints in carcinogenesis. The aim of the present study was to evaluate two major immune checkpoints, the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), in the serum of 35 patients with stage I and II breast cancer. Serum concentrations of CTLA-4 and PD-1 were measured at three time points: i) Preoperatively; ii) during anesthesia following the harvesting of sentinel nodes (SNs); and iii) 24 h postoperatively. Control samples were obtained from 25 healthy, age-matched females. Assessment of CTLA-4 and PD-1 expression levels was conducted using flow cytometry. A statistically significant difference in PD-1 expression was identified between breast cancer patients preoperatively and healthy controls (26.31±11.87 vs. 12.72±8.15; P<0.0001). In addition, a statistically significant association was found between CTLA-4 and PD-1 levels prior to surgery (P=0.0084). In addition, CTLA-4 expression was associated with age (P=0.0453), with elevated levels of CTLA-4 detected in older breast cancer patients. Higher PD-1 expression levels were observed in T2 tumors compared with T1 tumors prior to surgery and intraoperatively; however, the differences were not statistically significant. Furthermore, a decrease in PD-1 levels was observed subsequent to harvesting SNs with metastasis, but not in SN-negative patients (P=0.05). A negative correlation was also observed between PD-1 expression and progesterone receptor (PR) status following surgery (P=0.024). These results provided a basis for further investigation of immune checkpoints in breast cancer. Breast cancer patients exhibit an altered profile of immune checkpoint markers, with higher concentrations of PD-1 observed in larger, PR-negative tumors.

Analysis of the G/C polymorphism in the 5'-untranslated region of the RAD51 gene in breast cancer.

The breast cancer suppressor proteins BRCA1 and BRCA2 interact with RAD51, a protein essential for maintaining genomic stability by playing a central role in homology-dependent recombinational repair of the DNA double-strand breaks. Therefore, genetic variability in the RAD51 gene may contribute to the appearance and/or progression of breast cancer. A single nucleotide polymorphism in the 5'- untranslated region of RAD51 (a G to C substitution at position 135, the G/C polymorphism) is reported to modulate breast cancer risk. We investigated the distribution of genotypes and frequency of alleles of the G/C polymorphism in breast cancer. Tumor tissues were obtained from postmenopausal women with node-negative and node-positive breast carcinoma with uniform tumor size. Blood samples from age matched healthy women served as control. The G/C polymorphism was determined by PCR-based MvaI restriction fragment length polymorphism. The distribution of the genotypes of the G/C polymorphism did not differ significantly (P > 0.05) from those predicted by the Hardy-Weinberg distribution. There were no differences in the genotype distribution and allele frequencies between node-positive and node-negative patients. There were no significant differences between distributions of the genotypes in subgroups assigned to histological grades according to Scarf-Bloom-Richardson criteria and the distribution predicted by Hardy-Weinberg equilibrium (P > 0.05). Our study implies that the G/C polymorphism of the RAD51 gene may not be directly involved in the development and/or progression of breast cancer and so it may not be useful as an independent marker in this disease.