Henrik Horváth

The Candidate Oncogene CYP24A1: A Potential Biomarker for Colorectal Tumorigenesis

The main autocrine/paracrine role of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25-D3), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D3, we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same samples suggests that overexpression of CYP24A1 reduced local 1,25-D3 availability, decreasing its antiproliferative effect.

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.

CYP24A1 splice variants-Implications for the antitumorigenic actions of 1,25-(OH)2D3 in colorectal cancer

25-hydroxyvitamin D3 24-hydroxylase (CYP24A1), the catabolizing enzyme of the active vitamin D3, is often overexpressed in solid tumors. The unbalanced high levels of CYP24A1 seem to be a determinant of vitamin D resistance in tumors. Splice variants of CYP450 enzymes are common. Existence of CYP24A1 isoforms has been reported recently. We have investigated the presence of CYP24A1 splicing variants (SV) in human colon cancer cell lines and tissue samples. Using a set of primer combination we have screened the entire coding sequence of CYP24A1 and identified three splice variants in colon cancer cell lines. The presence of these SVs in human colon tissue samples showed a correlation with histological type of the tissue and gender of patients. The sequencing of the alternatively spliced fragments showed that two have lost the mitochondrial target domain, while the third lacks the heme-binding domain. All SVs retained their sterol binding domain. Translation of these variants would lead to a dysfunctional enzyme without catalytic activity that still binds its substrates therefore they might compete for substrate with the synthesizing and catabolizing enzymes of vitamin D.

The vitamin D system is deregulated in pancreatic diseases

Highlights • During PDAC development CYP24A1 levels are reduced in the endocrine islets.• During malignant transformation pancreatic ducts accumulate CYP24A1 protein.• CYP24A1 expression correlates with VDR in CP patients, but not in PDAC patients.• CYP24A1 overexpressing tumors are highly proliferative.• CaSR and VDR are co-expressed in the endocrine cells of the islets.

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

The 3′UTR NFKBIA Variant Is Associated with Extensive Colitis in Hungarian IBD Patients

Purpose In previous studies the NFKBIA 3′UTR (untranslated region) AA genotype was associated with Crohn’s disease (CD), while the NFKB1-94ins/delATTG mutation increased the risk for ulcerative colitis (UC). The aim of our study was to investigate these two polymorphisms and patients’ response to medical therapy and/or disease phenotype in Hungarian inflammatory bowel disease (IBD) patients. Methods NFKBIA 3′UTR- and NFKB1-94ins/delATTG polymorphisms were investigated in 415 unrelated IBD patients (CD: 266 patients, mean age 35.2 ± 12.1 years, duration 8.7 ± 7.5 years; UC patients: 149, mean age 44.4 ± 15.4 years, duration 10.7 ± 8.9 years) and 149 controls by PCR-restriction fragment length polymorphism (RFLP) analysis. Detailed clinical phenotypes were determined by reviewing the medical charts. Results The NFKBIA 3′UTR and NFKB1-94ins/delATTG genotypes and allele frequencies were not significantly different among IBD and controls. In patients with UC, the 3′UTR GG genotype was associated with extensive colitis (55.3 vs. 29.4%, odds ratio 2.97, 95% confidence interval 1.45–6.08). The presence of variant alleles did not predict response to steroids, infliximab, or need for surgery. Conclusions The NFKBIA 3′UTR GG genotype was associated with an increased risk for extensive colitis in Hungarian patients. In contrast, variant alleles did not predict response to medical therapy or need for surgery.

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...

Hungarian consensus regarding the role of vitamin D in the prevention and treatment of diseases

Takács István dr.1 ■ Benkő Ilona dr.2 ■ Toldy Erzsébet dr.3 Wikonkál Norbert dr.4 ■ Szekeres László dr.5 ■ Bodolay Edit dr.6 Kiss Emese dr.7 ■ Jambrik Zoltán dr.8 ■ Szabó Boglárka dr.8 Merkely Béla dr.8 ■ Valkusz Zsuzsa dr.9 ■ Kovács Tibor dr.10 Szabó András dr.11 ■ Grigoreff Orsolya dr.1 ■ Nagy Zsolt dr.1 Demeter Judit dr.1 ■ Horváth Henrik Csaba dr.1 Bittner Nóra dr.12 ■ Várbíró Szabolcs dr.13 ■ Lakatos Péter dr.1

Impact of CYP3A7*1C polymorphism on bone mineral content in postmenopausal women

INTRODUCTION CYP3A7*1C polymorphism has been shown to be associated with lower levels of serum dehydroepiandrosterone sulphate in men. The age-related decline of dehydroepiandrosterone sulphate levels is believed to contribute to the development of osteoporosis. We hypothesized that CYP3A7*1C may lead to bone loss through decreased levels of dehydroepiandrosterone sulphate in postmenopausal women. PATIENTS AND METHODS 319 postmenopausal women were studied and divided into two subgroups: 217 women with osteoporosis and 102 aged-matched women without osteoporosis. The CYP3A7*1C polymorphism was genotyped. Serum dehydroepiandrosterone sulphate levels and bone mineral density were measured. RESULTS Homozygous CYP3A7*1C carriers had significantly lower bone mineral density at lumbar spine than that of wild type (T-score with CYP3A7*1C mutant type: -3.27 +/- 1.02, T-score with wild type: -1.35 +/- 1.53, p = 0.041) after adjusting for age and DHEAS levels. No association was found between genotypes and dehydroepiandrosterone sulphate levels. CONCLUSION Our data suggest that CYP3A7 polymorphism might have an influence on bone mass at the lumbar spine independently of serum dehydroepiandrosterone sulphate concentrations.