Jozef Hatok

In vitro assays for the evaluation of drug resistance in tumor cells

Oncologic diseases are among leading cause of mortality in developed countries. Despite significant progress, the use of standard cytotoxic chemotherapy has reached a therapeutical plateau. Currently, the process of selecting chemotherapy represents a trial and error method neglecting biological individuality of tumor and its bearer. The improvement of treatment results is expected from ex vivo drug sensitivity testing which may allow to choose the most effective drug for individual patient and to exclude agents to which the tumor cells exert resistance. New techniques and rapidly increasing knowledge about the molecular basis of malignant diseases provide important opportunities for the future of chemotherapy. This paper reviews current methods used to test the resistance of tumor cells to a panel of anticancer agents in vitro. In addition, we focused on the in vitro MTT assay which represents one of major technique for testing of tumor cell resistance to anticancer agents.

Polymorphisms of glutathione-S-transferase M1, T1, P1 and the risk of prostate cancer: a case-control study

BackgroundIt has been suggested that polymorphisms in glutathione-S-transferases (GST) could predispose to prostate cancer through a heritable deficiency in detoxification pathways for environmental carcinogens. Yet, studies linking GST polymorphism and prostate cancer have so far failed to unambiguously establish this relation in patients. A retrospective study on healthy, unrelated subjects was conducted in order to estimate the population GST genotype frequencies in the Slovak population of men and compare our results with already published data (GSEC project-Genetic Susceptibility to Environmental Carcinogens). A further aim of the study was to evaluate polymorphisms in GST also in patients with prostate cancer in order to compare the evaluated proportions with those found in the control subjects.MethodsWe determined the GST genotypes in 228 healthy, unrelated subjects who attended regular prostate cancer screening between May 2005 and June 2007 and in 129 histologically verified prostate cancer patients. Analysis for the GST gene polymorphisms was performed by PCR and PCR-RFLP.ResultsWe found that the GST frequencies are not significantly different from those estimated in a European multicentre study or from the results published by another group in Slovakia. Our results suggest that Val/Val genotype of GSTP1 gene could modulate the risk of prostate cancer, even if this association did not reach statistical significance. We did not observe significantly different crude rates of the GSTM1 and GSTT1 null genotypes in the men diagnosed with prostate cancer and those in the control group.ConclusionUnderstanding the contribution of GST gene polymorphisms and their interactions with other relevant factors may improve screening diagnostic assays for prostate cancer. We therefore discuss issues of study feasibility, study design, and statistical power, which should be taken into account in planning further trials.

Anti-apoptotic and pro-apoptotic gene expression evaluated from eutopic endometrium in the proliferative phase of the menstrual cycle among women with endometriosis and healthy controls

OBJECTIVE Endometriosis affects 5-15% of women in the general population and 40% of women seeking infertility evaluation. Its etiology and pathogenesis is controversial. Abnormalities of genes involved in the regulation of apoptosis have been thought to play a role in origin. Hence, we investigated the expression of pro-apoptotic and anti-apoptotic genes in eutopic endometrium from women with endometriosis and healthy controls in relation to disease occurrence and severity. STUDY DESIGN A prospective study in women undergoing laparoscopic surgery for pelvic pain was conducted. In total, 45 women (30 healthy controls and 15 patients) matched inclusion criteria. The mRNA expression of apoptotic genes (p53, Bcl-x(L,S) and Bax) from eutopic endometrium was detected by RT-PCR. RESULTS A significant increase in expression of mRNA p53 (1.42 versus 1.02; p<0.05), and Bcl-x(S) (0.41 versus 0.19; p=0.0006) was found in women with endometriosis compared to healthy controls. Insignificantly increased expression was found for Bax (1.22 versus 1.15). The expression of anti-apoptotic Bcl-x(L) was unchanged (1.08 versus 1.07). The Bcl-x(L)/Bcl-x(S) ratio was twofold higher (5.63 versus 2.63) in controls. By stratifying patients by disease stage we have revealed an increased mRNA expression of apoptotic genes in patients with grades III-IV endometriosis compared to those with grades I-II. However, the difference was significant only for Bcl-x(S) expression (p<0.05). CONCLUSIONS Results suggest that an increased transcription of pro-apoptotic genes (p53 and Bcl-x(S)) in eutopic endometrium is significantly associated with endometriosis, which indicates dysregulation of apoptotic gene transcription associated with disease.

Bcl-2 family proteins: master regulators of cell survival.

Abstract The most prominent function of proteins of the Bcl-2 family is regulation of the initiation of intrinsic (mitochondrial) pathways of apoptosis. However, recent research has revealed that in addition to regulation of mitochondrial apoptosis, proteins of the Bcl-2 family play important roles in regulating other cellular pathways with a strong impact on cell survival like autophagy, endoplasmic reticulum (ER) stress response, intracellular calcium dynamics, cell cycle progression, mitochondrial dynamics and energy metabolism. This review summarizes the recent knowledge about functions of Bcl-2 family proteins that are related to cell survival.

Age-related Oxidative Modifications of Proteins and Lipids in Rat Brain

Oxidants have been shown to play a major role in ageing and ageing-related neurodegenerative diseases. In the present study, we investigated the effect of ageing on oxidative damage to lipids and proteins in brain homogenate, mitochondria and synaptosomes of adult (6-month-old), old (15-month-old), and senescent (26-month-old) Wistar rats. There was a significant increase in thiobarbituric acid-reactive substances and conjugated dienes in homogenates, which indicate increased lipid peroxidation (LPO). Oxidative modifications of homogenate proteins were demonstrated by a loss of sulfhydryl content, accumulation of dityrosines and formation of protein conjugates with LPO-end products. Increase in protein conjugates with LPO-end products and a decrease in SH groups were observed also in mitochondria and synaptosomes, but dityrosine content was elevated only in synaptosomes. Protein surface hydrophobicity, measured by fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS), was increased only in homogenate. These results suggest that besides mitochondria and synaptosomes other cellular compartments are oxidatively modified during brain ageing.

Endometrial aromatase mRNA as a possible screening tool for advanced endometriosis and adenomyosis.

Endometriosis (ENDs) and adenomyosis (ADNs) are estrogen-dependent diseases. The aim of this study was to determine the clinical usefulness of examining endometrial biopsy specimens for aromatase cytochrome P450 (CYP19) as a diagnostic importance for endometriosis and adenomyosis. In general, the RT-PCR analyses of 101 samples revealed increased aromatase mRNA expression in eutopic endometrium in women with endometriosis and adenomyosis compared to healthy controls (p = 0.0002). The highest number of positive cases (93.3%) of CYP19 mRNA expression was detected in women with advanced disease stages. Concrete expression of CYP19 mRNA level in controls was 0.68 compared to patients with ADNs (1.21), ENDsL stage I-II (1.15) and ENDsA stage III-IV (1.65) (p < 0.0001), respectively. The possible influence of increased body mass index on aromatase expression in each group showed in controls an insignificant slight increase of aromatase expression, contrary to cases where this trend was the opposite. The results point to the higher (2.45-fold) difference in aromatase expression in patients with endometriosis stage III-IV compared to controls and provide direct evidence that screening for eutopic endometrial aromatase expression in combination with clinical data could be of discriminative value in the prediction of estrogen-dependent diseases, independent from body mass index.

The p53 Codon 72 Exon 4 BstUI Polymorphism and Endometrial Cancer in Caucasian Women

Objectives: Studies on the association between the p53 Arg72Pro polymorphism and endometrial cancer have reported contrasting conclusions. With the exception of Asian subjects, data demonstrating the influence of this polymorphism on endometrial carcinogenesis in other races/ethnic groups, including Caucasians, are scarce. Thus, we aimed to investigate its role in the development of endometrial cancer and its association with prognostic markers. Methods: A case-control study examining the p53 codon 72 polymorphism in a total of 451 samples (121 cancer patients, 330 healthy controls) using polymerase chain reaction and sequencing techniques was conducted. Genotypes were correlated with clinico-pathological factors and age. Logistic regression analyses were used to adjust for possible confounding variables, and data were evaluated using the Pearson χ2 test. Results: We found the Pro allele and genotype frequency to be insignificantly higher in cases than controls (Pro allele: 24.8 and 22.3%, respectively; genotypes: Arg/Pro 36.36 and 34.24%, Pro/Pro 6.61 and 5.15%, respectively). Logistic regression analysis revealed an increased risk for disease in carriers of the Pro allele, with an odds ratio (OR) of 1.13 [95% confidence interval (CI) 0.73–1.76] for heterozygotes and an OR of 1.36 (95% CI 0.56–3.30) for homozygotes. Furthermore, we noted a trend for Arg/Pro + Pro/Pro towards poor tumour differentiation, angioinvasion, pelvic lymph node spread and type II carcinomas, with ORs of 1.27 (95% CI 0.60–2.66), 1.24 (95% CI 0.67–2.30), 1.21 (95% CI 0.53–2.75) and 1.69 (95% CI 0.70–4.10), respectively. Additionally, the Pro genotype was associated with a lower risk for cancer in women with early menarche (OR 1.17, 95% CI 0.60–2.28) and late menopause (OR 0.70, 95% CI 0.30–1.63). Despite the increased or decreased risk observed for some variables, none of these trends were significant. Conclusions: Our data did not demonstrate any significant difference in the prevalence of the p53 Arg72Pro genotype between patients and controls, providing evidence that this polymorphism is only weakly associated with the risk of endometrial cancer and prognostic factors in Caucasian women.

The expression profile of acid-sensing ion channel (ASIC) subunits ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3 in the esophageal vagal afferent nerve subtypes

Acid-sensing ion channels (ASICs) have been implicated in esophageal acid sensing and mechanotransduction. However, insufficient knowledge of ASIC subunit expression profile in esophageal afferent nerves hampers the understanding of their role. This knowledge is essential because ASIC subunits form heteromultimeric channels with distinct functional properties. We hypothesized that the esophageal putative nociceptive C-fiber nerves (transient receptor potential vanilloid 1, TRPV1-positive) express multiple ASIC subunits and that the ASIC expression profile differs between the nodose TRPV1-positive subtype developmentally derived from placodes and the jugular TRPV1-positive subtype derived from neural crest. We performed single cell RT-PCR on the vagal afferent neurons retrogradely labeled from the esophagus. In the guinea pig, nearly all (90%-95%) nodose and jugular esophageal TRPV1-positive neurons expressed ASICs, most often in a combination (65-75%). ASIC1, ASIC2, and ASIC3 were expressed in 65-75%, 55-70%, and 70%, respectively, of both nodose and jugular TRPV1-positive neurons. The ASIC1 splice variants ASIC1a and ASIC1b and the ASIC2 splice variant ASIC2b were similarly expressed in both nodose and jugular TRPV1-positive neurons. However, ASIC2a was found exclusively in the nodose neurons. In contrast to guinea pig, ASIC3 was almost absent from the mouse vagal esophageal TRPV1-positive neurons. However, ASIC3 was similarly expressed in the nonnociceptive TRPV1-negative (tension mechanoreceptors) neurons in both species. We conclude that the majority of esophageal vagal nociceptive neurons express multiple ASIC subunits. The placode-derived nodose neurons selectively express ASIC2a, known to substantially reduce acid sensitivity of ASIC heteromultimers. ASIC3 is expressed in the guinea pig but not in the mouse vagal esophageal TRPV1-positive neurons, indicating species differences in ASIC expression.

Gene expression profiling of histologically normal breast tissue in females with human epidermal growth factor receptor 2‑positive breast cancer.

Gene expression profile‑based taxonomy of breast cancer (BC) has been described as a significant breakthrough in comprehending the differences in the origin and behavior of cancer to allow individually tailored therapeutic approaches. In line with this, we hypothesized that the gene expression profile of histologically normal epithelium (HNEpi) could harbor certain genetic abnormalities predisposing breast tissue cells to develop human epidermal growth factor receptor 2 (HER2)‑positive BC. Thus, the aim of the present study was to assess gene expression in normal and BC tissue (BCTis) from patients with BC in order to establish its value as a potential diagnostic marker for cancer development. An array study evaluating a panel of 84 pathway‑ and disease‑specific genes in HER2‑positive BC and tumor‑adjacent HNEpi was performed using quantitative polymerase chain reaction in 12 patients using microdissected samples from frozen tissue. Common prognostic and predictive parameters of BC were assessed by immunohistochemistry and in situ hybridization. In the BCTis and HNEpi samples of 12 HER2‑positive subjects with BC, the expression of 2,016 genes was assessed. A total of 39.3% of genes were deregulated at a minimal two‑fold deregulation rate and 10.7% at a five‑fold deregulation rate in samples of HNEpi or BCTis. Significant differences in gene expression between BCTis and HNEpi samples were revealed for BCL2L2, CD44, CTSD, EGFR, ERBB2, ITGA6, NGFB, RPL27, SCBG2A1 and SCGB1D2 genes (P<0.05), as well as GSN, KIT, KLK5, SERPINB5 and STC2 genes (P<0.01). Insignificant differences (P<0.07) were observed for CCNA1, CLU, DLC1, GABRP and IL6 genes. The ontological gene analyses revealed that the majority of the deregulated genes in the HNEpi samples were part of the functional gene group directly associated with BC origin and prognosis. Functional analysis showed that the most frequent gene deregulations occurred in genes associated with apoptosis and cell cycle regulation in BCTis samples, and with angiogenesis, regulation of the cell cycle and transcriptional activity in HNEpi samples. The molecular profiling of HNEpi breast tissue revealed gene expression abnormalities that may represent potential markers of increased risk for HER2‑positive malignant transformation of breast tissue, and may be able to be employed as predictors of prognosis.

The role of p21 3'UTR gene polymorphism in the risk of prostate cancer: A pilot study

The cell cycle regulator p21 plays an important role in regulating critical cell activities including cell cycle control, DNA repair and apoptosis. Consequently, it may affect the efficacy of the response to DNA damage and tumor development. The aim of our study was to evaluate the frequencies of the p21 C70T polymorphism, the association between this genetic variant and smoking status, and the serum prostate-specific antigen (PSA) levels and Gleason score in 118 prostate cancer patients and 130 males routinely screened for prostate cancer in the Slovak population. Blood samples were collected from all individuals for DNA isolation, used for subsequent genotyping assays via PCR-RFLP methods. Overall, we did not observe any significant association between this polymorphism and prostate cancer risk. An analysis of the association between the p21 genotypes and smoking was then conducted. Among smokers, CC and CT genotypes were associated with a non‑significant increased risk (OR=1.48; 95% CI, 0.80-2.76 and OR=1.15; 95% CI, 0.27‑4.77, respectively; p>0.05) in comparison to non-smokers with the CC genotype. Patients with a CT genotype and serum PSA levels≥10 ng/ml had an 84% decrease in prostate cancer risk (OR=0.16; 95% CI, 0.03-0.75; p<0.05) compared to cases with serum PSA levels <10 ng/ml and the CC genotype. No significant association was detected between Gleason score and prostate cancer risk. Based on these results, we concluded that the p21 C70T polymorphism is associated with decreased risk of prostate cancer in Slovak men. To confirm these findings, a systematic approach is required to identify sequence variants in this and other related genes, and subsequently, to test for an association between such variants, smoking status and tumor-specific clinicopathological characteristics in large samples.