Bernadett Balla

Effects of the lactase 13910 C/T and calcium-sensor receptor A986S G/T gene polymorphisms on the incidence and recurrence of colorectal cancer in Hungarian population.

BackgroundEpidemiological studies suggested the chemopreventive role of higher calcium intake in colorectal carcinogenesis. We examined genetic polymorphisms that might influence calcium metabolism: lactase (LCT) gene 13910 C/T polymorphism causing lactose intolerance and calcium-sensing receptor (CaSR) gene A986S polymorphism as a responsible factor for the altered cellular calcium sensation.Methods538 Hungarian subjects were studied: 278 patients with colorectal cancer and 260 healthy controls. Median follow-up was 17 months. After genotyping, the relationship between LCT 13910 C/T and CaSR A986S polymorphisms as well as tumor incidence/progression was investigated.Resultsin patient with colorectal cancer, a significantly higher LCT CC frequency was associated with increased distant disease recurrence (OR = 4.04; 95% CI = 1.71–9.58; p = 0.006). The disease free survival calculated from distant recurrence was reduced for those with LCT CC genotype (log rank test p = 0.008). In case of CaSR A986S polymorphism, the homozygous SS genotype was more frequent in patients than in controls (OR = 4.01; 95% CI = 1.33–12.07; p = 0.014). The number of LCT C and CaSR S risk alleles were correlated with tumor incidence (p = 0.035). The CCSS genotype combination was found only in patients with CRC (p = 0.033).ConclusionLCT 13910 C/T and CaSR A986S polymorphisms may have an impact on the progression and/or incidence of CRC.

The protective role of bone morphogenetic protein-8 in the glucocorticoid-induced apoptosis on bone cells

One of the side effects associated with glucocorticoid therapy is glucocorticoid-induced bone loss. Glucocorticoids partly detain bone formation via the inhibition of osteoblastic function, however, the exact mechanism of this inhibition remains elusive. In this study, we examined the effect of dexamethasone, an active glucocorticoid analogue, on cell viability and expression of bone remodelling-related genes in primary mouse calvarial and cloned MC3T3-E1 osteoblasts. Using sensitive biochemical assays, we demonstrated the apoptotic effect of dexamethasone on osteoblastic cells. Then, utilizing Taqman probe-based quantitative RT-PCR technology, gene expression profiles of 111 bone metabolism-related genes were determined. As a result of dexamethasone treatment we have detected significant apoptotic cell death, and six genes, including Smad3, type-2 collagen α-1, type-9 collagen α-1, matrix metalloproteinase-2, bone morphogenetic protein-4 and bone morphogenetic protein-8 showed (BMP-8) significant changes in their expression on a time- and concentration-dependent manner. BMP-8, (a novel player in bone-metabolism) exhibited a two orders of magnitude elevation in its mRNA level and highly elevated protein concentration by Western blot in response to dexamethasone treatment. The knockdown of BMP-8 by RNA interference significantly increased dexamethasone-induced cell death, confirming a protective role for BMP-8 in the glucocorticoid-induced apoptosis of osteoblasts. Our results suggest that BMP-8 might be an essential player in bone metabolism, especially in response to glucocorticoids.

CYP3A7*1C Polymorphism, Serum Dehydroepiandrosterone Sulfate Level, and Bone Mineral Density in Postmenopausal Women

The CYP3A7 enzyme metabolizes some steroid hormones, including dehydroepiandrosterone sulfate (DHEAS). The age-related decline of serum DHEAS levels is believed to contribute to osteoporosis. Previously, the CYP3A7*1C polymorphism has been shown to cause a persistent high CYP3A7 enzyme activity, resulting in lower levels of DHEAS in men. We hypothesized that the CYP3A7*1C polymorphism might contribute to bone loss through decreased levels of serum DHEAS in postmenopausal women. Postmenopausal women (n = 319) were divided into two subgroups: 217 with osteoporosis and 102 healthy controls. Genotyping, serum DHEAS measurement, and osteodensitometry of the lumbar spine and femoral neck were carried out in all subjects. Homozygous CYP3A7*1C carriers had significantly lower BMD at the lumbar spine compared to wild types (T score −3.27 ± 1.02 in CYP3A7*1C homozygous mutants vs. −1.35 ± 1.53 in wild types, P = 0.041). This association remained significant after adjustment for menopausal age, serum DHEAS level, alcohol consumption, steroid intake, smoking habits, and previous fractures. No association was found between genotypes and serum DHEAS levels in the total study population or in the subgroups. Serum DHEAS levels correlated positively with bone mineral density at the lumbar spine (r = 0.59, P = 0.042) after correction for age. Our data suggest that the CYP3A7 polymorphism might have an influence on bone mass at the lumbar spine independently of serum DHEAS concentrations.

CYP24A1 inhibition facilitates the anti-tumor effect of vitamin D3 on colorectal cancer cells

AIM The effects of vitamin D3 have been investigated on various tumors, including colorectal cancer (CRC). 25-hydroxyvitamin-D3-24-hydroxylase (CYP24A1), the enzyme that inactivates the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25-D3), is considered to be the main enzyme determining the biological half-life of 1,25-D3. During colorectal carcinogenesis, the expression and concentration of CYP24A1 increases significantly, suggesting that this phenomenon could be responsible for the proposed efficacy of 1,25-D3 in the treatment of CRC. The aim of this study was to investigate the anti-tumor effects of vitamin D3 on the human CRC cell line Caco-2 after inhibition of the cytochrome P450 component of CYP24A1 activity. METHODS We examined the expression of CYP24A1 mRNA and the effects of 1,25-D3 on the cell line Caco-2 after inhibition of CYP24A1. Cell viability and proliferation were determined by means of sulforhodamine-B staining and bromodeoxyuridine incorporation, respectively, while cytotoxicity was estimated via the lactate dehydrogenase content of the cell culture supernatant. CYP24A1 expression was measured by real-time reverse transcription polymerase chain reaction. A number of tetralone compounds were synthesized to investigate their CP24A1 inhibitory activity. RESULTS In response to 1,25-D3, CYP24A1 mRNA expression was enhanced significantly, in a time- and dose-dependent manner. Caco-2 cell viability and proliferation were not influenced by the administration of 1,25-D3 alone, but were markedly reduced by co-administration of 1,25-D3 and KD-35, a CYP24A1-inhibiting tetralone. Our data suggest that the mechanism of action of co-administered KD-35 and 1,25-D3 does not involve a direct cytotoxic effect, but rather the inhibition of cell proliferation. CONCLUSION These findings demonstrate that the selective inhibition of CYP24A1 by compounds such as KD-35 may be a new approach for enhancement of the anti-tumor effect of 1,25-D3 on CRC.

Marked Increase in CYP24A1 Gene Expression in Human Papillary Thyroid Cancer

The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-D3) inhibits cell growth and induces cell differentiation and apoptosis in numerous tumors, such as colon, breast, and prostate cancers (1–3). However, the anticancer effect of 1,25-D3 cannot always be seen in a clinical setting. In case of colon cancers, a marked increase in the expression of the 1,25D3 inactivating enzyme, the 24-hydroxylase (CYP24A1), has been observed (4). The role of 1,25-D3 in the development of thyroid carcinomas has not yet been established. However, Sharma et al. (5) observed the correlation of high baseline CYP24A1 and relatively low stimulation after calcitriol treatment (compared with sensitive cells) with lack of growth inhibition in numerous thyroid cancer cell lines. We have also examined the gene expression of the inactivating CYP24A1 and the activating 1-alpha-hydroxylase (CYP27B1) enzyme in human thyroid cancers. To date, gene expression analysis of thyroid samples (both normal and papillary tumor tissue of the same patient) was carried out in six unrelated, consecutive, Hungarian, Caucasian patients at our Thyroid Clinic. All surgically removed thyroid tissue samples underwent histological examination, and papillary tumor was identified in all cases. The study was approved by the Regional Committee of Science and Research Ethics, Semmelweis University (SOTE-TUKEB 1160-0/20101018EKU), and all patients gave written informed consent. Total RNA was isolated from each sample with Roche High Pure Total RNA Isolation kit. Five hundred nanograms of total RNA was reverse-transcribed to cDNA. The expression differences of selected genes were analyzed by Taqman probe-based quantitative real-time reverse transcriptase– polymerase chain reaction. Relative quantification was carried out from collected data (threshold cycle numbers) by Applied Biosystems 7500 System SDS software 1.3. CYP24A1 mRNA expression was markedly increased in all but one papillary cancer compared with that of normal thyroid tissue, sometimes reaching 300-fold elevation (Supplementary Fig. S1; Supplementary Data are available online at www.liebertonline.com/thy). No significant alteration was seen in CYP27B1 gene activity between neoplastic and normal tissues. There was no significant difference in serum 25-OHvitamin D3 concentrations among patients (mean: 28.03 ng/ mL, range: 22.7–31.9 ng/mL). In this letter, we report marked increases in the 1,25-D3neutralizing CYP24A1 gene expression in human papillary thyroid cancer. The tumor cell growth-inhibiting role of vitamin D3 has been extensively studied in different malignancies (1–3). The interest in the association of vitamin-D3 serum levels with various cancer incidences has dramatically increased. However, only very limited data are available on the relationship between serum vitamin D3 concentrations and malignant thyroid conditions (6,7). The pilot study of Laney et al. showed levels of vitamin D and rates of vitamin D deficiency to be similar between patients with thyroid nodules, thyroid cancer in remission, and active thyroid cancer (8). However, the results of Stepien et al. revealed significantly lower calcitriol concentrations in patients with papillary, follicular, and anaplastic thyroid cancers (7). This finding could be attributable to higher CYP24A1 levels cleaving calcitriol, as there were no differences in serum 25-OH-vitamin D3. Earlier, calcitriol had also been shown to attenuate thyrotropinstimulated growth and iodide uptake by rat thyroid cells (FRTL-5) (9,10). Papillary cancer is the most common thyroid malignancy. The role of vitamin D3 in the prevention or development of this disorder has to be yet determined. The increase in CYP24A1 expression in this cancer type could be a ‘‘protective’’ mechanism against the well-known anticancer effect of calcitriol. The observation of Sharma et al. (5) regarding increased expression of CYP24A1 in thyroid cancer cell lines corroborates this notion. One of the cell lines they used was human papillary thyroid carcinoma cell line, whereas the other ones showed the features of human anaplastic thyroid carcinoma. Also, the most resistant cell lines to calcitriol had the highest baseline levels of CYP24A1. In our study at this stage, the number of examined tumor tissues does not allow drawing of statistically significant conclusion about the correlation of tumor histopathological stage or aggressiveness with CYP24A1 levels. Khadzkou et al. demonstrated enhanced 1-alphahydroxylase expression but concluded that this elevation did not show large differences in CYP27B1 expression in papillary thyroid cancer and its metastasis compared with normal

Fast and robust next-generation sequencing technique using ion torrent personal genome machine for the screening of neurofibromatosis type 1 (NF1) gene

Neurofibromatosis type 1 (NF1) gene exhibits one of the highest spontaneous mutation rates in the human genome. Identification of the NF1 mutation is challenging because the NF1 gene is very large and complex, lacking mutational “hot spots.” There is no clustering of mutations, there are several pseudogenes, and a wide spectrum of different types of mutation has been recognized. To date, NF1 mutations or deleted regions have been detected with a number of techniques. With the appearance of next-generation sequencing (NGS) machines, molecular biology is in a new revolutionary phase. Our aim was to work out a method to use the high-throughput NGS machine, Ion Torrent PGM, in diagnostic settings for neurofibromatosis type 1. In our examination, we could reveal 21 distinct variations in NF1 gene in seven patients. This is an absolutely new method for exploring the genetic background of neurofibromatosis type 1 exhibiting the extremely high throughput of NGS in a diagnostic setting.

Effect of Vertebroplasty Filler Materials on Viability and Gene Expression of Human Nucleus Pulposus Cells

Consequences of intradiscal cement leakage—often occurring after vertebral cement augmentation for the treatment of vertebral compression fractures—are still unknown. In this study, we have investigated the influences of vertebroplasty filler materials (polymethylmethacrylate‐, calcium phosphate‐ and calcium sulfate‐based bone cement) on isolated nucleus pulposus cells. Cell viability of cultured human nucleus pulposus cells were measured after treatment with vertebroplasty filler materials. Gene expression profile of selected genes was determined with quantitative real‐time PCR. The widely used polymethylmethacrylate and calcium phosphate cement significantly decreased cell number in a dose‐ and time‐dependent manner while calcium sulfate cement affected cell viability less. Expression of genes involved in matrix metabolism of nucleus pulposus—aggrecan, collagens, small proteoglycans—as well as important transcription factors have also significantly changed due to treatment (e.g., 2.5‐fold decrease in aggrecan expression was determined in cultures due to polymethylmethacrylate treatment). Our results suggest that vertebroplasty filler materials—depending on the type of applied material—can accelerate the degeneration of nucleus pulposus cells resulting in a less flexible disc in case of intradiscal cement leakage. This process may increase the risk of a subsequent new vertebral fracture, the main complication of vertebral augmentation.

Identifying novel genes involved in both deer physiological and human pathological osteoporosis

Osteoporosis attacks 10% of the population worldwide. Humans or even the model animals of the disease cannot recover from porous bone. Regeneration in skeletal elements is the unique feature of our newly investigated osteoporosis model, the red deer (Cervus elaphus) stag. Cyclic physiological osteoporosis is a consequence of the annual antler cycle. This phenomenon raises the possibility to identify genes involved in the regulation of bone mineral density on the basis of comparative genomics between deer and human. We compare gene expression activity of osteoporotic and regenerating rib bone samples versus autumn dwell control in red deer by microarray hybridization. Identified genes were tested on human femoral bone tissue from non-osteoporotic controls and patients affected with age-related osteoporosis. Expression data were evaluated by Principal Components Analysis and Canonical Variates Analysis. Separation of patients into a normal and an affected group based on ten formerly known osteoporosis reference genes was significantly improved by expanding the data with newly identified genes. These genes include IGSF4, FABP3, FABP4, FKBP2, TIMP2, TMSB4X, TRIB, and members of the Wnt signaling. This study supports that extensive comparative genomic analyses, here deer and human, provide a novel approach to identify new targets for human diagnostics and therapy.

New approach to analyze genetic and clinical data in bisphosphonate‐induced osteonecrosis of the jaw

OBJECTIVES Osteonecrosis of the jaw (ONJ) is a major complication associated with long-term use of bisphosphonates (BP). We aimed to investigate the effect of CYP2C8 rs1934951 SNP and its relationship to a number of clinical and biochemical factors in 46 Hungarian subjects with bisphosphonate-induced ONJ. METHODS Blood samples were collected from each subject and genomic DNA was extracted. SNP analysis of CYP2C8 gene was carried out by predesigned TaqMan primer/probe sets. The genetic data together with clinical and biochemical variables were evaluated by chi-square test, logistic regression, and principal component analysis (PCA). RESULTS The risk of mandibular localization of ONJ was 19.2-fold higher in subjects with AG genotype than in normal GG genotype. PCA revealed strong positive correlations between maxillar localization of ONJ and a group of variables including intravenous BP application and serum lipid markers. Mandibular localization of ONJ was correlated positively with serum calcium, 25-hydroxy-vitamin D and PTH levels, oral BP application, and the length of BP therapy. The degree of the disease and the number of recurrences were correlated with the application of hormone-deprivation therapy for breast cancer patients. CONCLUSION The statistical approach applying PCA to our data may contribute to the better understanding of factors playing role in the development of bisphosphonate-induced ONJ.

Importance of dehydroepiandrosterone and dehydroepiandrosterone sulfate in different diseases

A dehidroepiandroszteron es szulfatalt szarmazeka androgen es osztrogen hormonok előanyaga, a gonadok szexualszteroid termelesehez jarul hozza. A dehidroepiandroszteron-szulfat csak deszulfatalodasa utan kepes reszt venni a folyamatban. A harmadik dekadtol kezdve a szerumszintjuk fokozatosan csokken, idősebb korban a maximalis ertek csupan 10–20%-a. Az alacsonyabb koncentracio kulonboző korallapotokhoz vezethet, mint a csontritkulas, a lipidmetabolizmus romlasa, sziv-errendszeri betegsegek, 2-es tipusu cukorbetegseg. Autoimmun betegsegben, szexualis diszfunkciok eseten szinten alacsonyabb szintet talaltak. Az intrakrinologia az endokrinologia specialis aga, jelentese, hogy a hormonszintezis es hatas ugyanazon szovetben, szekrecio nelkul jon letre. A magasabb helyi androgen- es/vagy osztrogenkoncentracio a hirsutismus, acne, seborrhea, az emlő es prosztatadaganatok patomechanizmusaban jelentős tenyező lehet. A dehidroepiandroszteron-potlas kedvező hatasat lattak posztmenopauzas csontritkulasban, lupus eryt...