Role of the 3-mercaptopyruvate sulfurtransferase in colon/colorectal cancers.

The 3-mercaptopyruvate sulfurtransferase (MPST) is a protein persulfidase, occurring mainly in mitochondria. Although function of this protein in cancer cells has been already studied, no clear outcome can be postulated up to now. Therefore, we focused on the determination of function of MPST in colon (HCT116 cells)/colorectal (DLD1 cells) cancers. In silico analysis revealed that in gastrointestinal cancers, MPST together with its binding partners can be either of a high risk or might have a protective effect. Silencing of MPST gene resulted in decreased ATP, while acetyl-CoA levels were elevated. Increased apoptosis was detected in cells with silenced MPST gene, which was accompanied by decrease in mitochondrial membrane potential, but no changes in IP3 receptor's protein. Mitochondria underwent activation of fission and elevated DRP1 expression after MPST silencing. Proliferation and migration of DLD1 and HCT116 cells were markedly affected, showing the importance of MPST protein in colon/colorectal cancer development.

Genome-wide DNA methylome and transcriptome changes induced by inorganic nanoparticles in human kidney cells after chronic exposure.

The unique physicochemical properties make inorganic nanoparticles (INPs) an exciting tool in diagnosis and disease management. However, as INPs are relatively difficult to fully degrade and excrete, their unintended accumulation in the tissue might result in adverse health effects. Herein, we provide a methylome-transcriptome framework for chronic effects of INPs, commonly used in biomedical applications, in human kidney TH-1 cells. Renal clearance is one of the most important routes of nanoparticle excretion; therefore, a detailed evaluation of nanoparticle-mediated nephrotoxicity is an important task. Integrated analysis of methylome and transcriptome changes induced by INPs (PEG-AuNPs, Fe3O4NPs, SiO2NPs, and TiO2NPs) revealed significantly deregulated genes with functional classification in immune response, DNA damage, and cancer-related pathways. Although most deregulated genes were unique to individual INPs, a relatively high proportion of them encoded the transcription factors. Interestingly, FOS hypermethylation inversely correlating with gene expression was associated with all INPs exposures. Our study emphasizes the need for a more comprehensive investigation of INPs' biological safety, especially after chronic exposure.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

KIT Expression Is Regulated by DNA Methylation in Uveal Melanoma Tumors.

Uveal melanoma (UM) is an ocular tumor with a dismal prognosis. Despite the availability of precise molecular and cytogenetic techniques, clinicopathologic features with limited accuracy are widely used to predict metastatic potential. In 51 UM tissues, we assessed a correlation between the expression of nine proteins evaluated by immunohistochemistry (IHC) (Melan-A, S100, HMB45, Cyclin D1, Ki-67, p53, KIT, BCL2, and AIFM1) and the presence of UM-specific chromosomal rearrangements measured by multiplex ligation-dependent probe amplification (MLPA), to find IHC markers with increased prognostic information. Furthermore, mRNA expression and DNA methylation values were extracted from the whole-genome data, achieved by analyzing 22 fresh frozen UM tissues. KIT positivity was associated with monosomy 3, increasing the risk of poor prognosis more than 17-fold (95% CI 1.53-198.69, p = 0.021). A strong negative correlation was identified between mRNA expression and DNA methylation values for 12 of 20 analyzed positions, five located in regulatory regions of the KIT gene (r = -0.658, p = 0.001; r = -0.662, p = 0.001; r = -0.816; p < 0.001; r = -0.689, p = 0.001; r = -0.809, p < 0.001, respectively). DNA methylation ß values were also inversely associated with KIT protein expression (p = 0.001; p = 0.001; p = 0.015; p = 0.025; p = 0.002). Our findings, showing epigenetic deregulation of KIT expression, may contribute to understanding the past failure to therapeutically target KIT in UM.

Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy.

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

Structural and Functional Impact of Seven Missense Variants of Phenylalanine Hydroxylase.

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.

Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells.

Although the involvement of type 1 (IP3R1) and type 2 (IP3R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP3R (IP3R3) is still elusive. Therefore, in this work we focused on the role of IP3R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP3R3 compared to IP3R1 and IP3R2 in tumor cells. Silencing of IP3R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP3R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP3R3_del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP3R3_del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP3R3_del cells than in DLD1 cells. These results clearly show that IP3R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP3R1.

Endogenous H2S producing enzymes are involved in apoptosis induction in clear cell renal cell carcinoma.

BACKGROUND: Knowledge about the expression and thus a role of enzymes that produce endogenous H2S - cystathionine-ß-synthase, cystathionine γ-lyase and mercaptopyruvate sulfurtransferase - in renal tumors is still controversial. In this study we aimed to determine the expression of these enzymes relatively to the expression in unaffected part of kidney from the same patient and to found relation of these changes to apoptosis. To evaluate patient's samples, microarray and immunohistochemistry was used. METHODS: To determine the physiological importance, we used RCC4 stable cell line derived from clear cell renal cell carcinoma, where apoptosis induction by a mixture of five chemotherapeutics with/without silencing of H2S-producing enzymes was detected. Immunofluorescence was used to determine each enzyme in the cells. RESULTS: In clear cell renal cell carcinomas, expression of H2S-producing enzymes was mostly decreased compared to a part of kidney that was distal from the tumor. To evaluate a potential role of H2S-producing enzymes in the apoptosis induction, we used RCC4 stable cell line. We have found that silencing of cystathionine-ß-synthase and cystathionine γ-lyase prevented induction of apoptosis. Immunofluorescence staining clearly showed that these enzymes were upregulated during apoptosis in RCC4 cells. CONCLUSION: Based on these results we concluded that in clear cell renal cell carcinoma, reduced expression of the H2S-producing enzymes, mainly cystathionine γ-lyase, might contribute to a resistance to the induction of apoptosis. Increased production of the endogenous H2S, or donation from the external sources might be of a therapeutic importance in these tumors.

Comprehensive genetic study of cystic fibrosis in Slovak patients in 25 years of genetic diagnostics.

INTRODUCTION: Cystic fibrosis (CF) has one of the longest histories in hereditary disease molecular diagnostics. However, identification of causative mutations in the CFTR gene is complicated by over 2000 currently identified mutations; with more still being discovered. Knowledge of mutation spectrum may improve effective routine diagnostics and is obligatory in mutation-specific treatment. OBJECTIVES: This study presents comprehensive mutation screening of the CFTR gene; with 275 unrelated, clinically confirmed and treated cystic fibrosis (CF) patients diagnosed in 25 years genetic testing in Slovakia. METHODS: Detection of the most common CFTR mutations was performed by ELUCIGENE 29 and ELUCIGENE CF EU2 kits. HRM and dHPLC mutation screening methods with subsequent Sanger sequencing were applied for minor mutation screening, and MLPA analysis for deletion/duplication detection. RESULTS: A total of 70 different mutations were identified, from which the most common mutation F508del accounted for 60.36% of all disease alleles and 8 mutations have currently been observed only in Slovak patients. Two large deletions identified on chromosomes 2 and 22 were further characterized to identify breakpoints. Based on mutation screening results and neonatal screening we estimated incidence in Slovakian newborns at approximately 1:6000-7000. CONCLUSION: In our study, we identified mutations in 98.54% of all disease chromosomes, while 86.54% were identified using ELUCIGENE kits, 0.54% by MLPA analysis and 11.46% by sequencing analysis. Knowledge of the mutation spectrum in genetically diagnosed patients improves possibilities of genetic counseling and cascade screening in the affected families and Slovak population.

Functional and structural characterisation of 5 missense mutations of the phenylalanine hydroxylase.

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown. In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterize five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients. p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression. Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding. Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro. While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH. Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.

A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications.

Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM.

Deregulation of energetic metabolism in the clear cell renal cell carcinoma: A multiple pathway analysis based on microarray profiling.

Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. In order to better understand the biology of ccRCC, we accomplished the gene profiling of fresh tissue specimens from 11 patients with the renal tumors (9 ccRCCs, 1 oncocytoma and 1 renal B-lymphoma), in which the tumor-related data were compared to the paired healthy kidney tissues from the same patients. All ccRCCs exhibited a considerably elevated transcription of the gene coding for carbonic anhydrase IX (CAIX). Moreover, the ccRCC tumors consistently displayed increased expression of genes encoding the glycolytic pathway enzymes, e.g. hexokinase II (HK2) and lactate dehydrogenase A (LDHA) and a decreased expression of genes for the mitochondrial electron transport chain components, indicating an overall reprogramming of the energetic metabolism in this tumor type. This appears to be accompanied by altered expression of the genes of the pH regulating machinery, including ion and lactate transporters. Immunohistochemical staining of tumor tissue sections confirmed the increased expression of CAIX, HK2 and LDHA in ccRCC, validating the microarray data and supporting their potential as the energetic metabolism-related biomarkers of the ccRCC.

Augmenting clinical interpretability of thiopurine methyltransferase laboratory evaluation.

OBJECTIVE: Individuals with decreased thiopurine methyltransferase (TPMT) activity are at risk of adverse effects of thiopurine administration whereas its increased activity may inactivate drugs faster. We evaluated genotype-phenotype correlations in patients with suspected hematological malignancies and inflammatory bowel disease from our region based on findings of nonlinear TPMT enzyme kinetics previously unreported. PATIENTS AND METHODS: The study group comprised 267 individuals. They were screened for the most common variants of low TPMT activity. TPMT activity was measured in erythrocytes using the HPLC rate-blanked method. RESULTS: Thirty-three patients (12.4%) were heterozygous (26 were TPMT*1/*3A, 5 TPMT*1/*2, 2 TPMT *1/*3C) and 1 was a compound heterozygote (*2/*3A). Normal and low normal TPMT activities substantially overlapped in wild-type and heterozygous individuals, whereas high activities were found in 29 wild-type genotyped patients. Extreme and life-threatening toxicity was observed in the compound heterozygote patient. CONCLUSION: Activity measurement performed at diagnosis provides clinicians with information on immediate pharmacokinetic-related adverse events and/or hypermetabolism, and genotyping may indicate the rate of pharmacodynamic thioguanine nucleotide accumulation due to slower overall thiopurine metabolism.

Triptolide induces apoptosis through the SERCA 3 upregulation in PC12 cells.

Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transporter in three pheochromocytoma cell lines. This transporter is involved in the apoptosis induction through the inhibition of a transcription factor NF-kappa B. Nevertheless, calcium release from the endoplasmic reticulum can also induce inner mitochondrial pathway of apoptosis in variety of cells. Therefore, the aim of this work was to evaluate an involvement of calcium and, more specifically, the intracellular calcium transport systems in the apoptosis induction in pheochrocytoma cell line PC12. We observed significantly increased amount of reticular calcium in TTL-treated cells compared to control, untreated cells. Surprisingly, gene expression of the IP3 receptors was not changed after the TTL treatment, but ryanodine receptor of the type 2 (RyR2) was downregulated and sarco/endoplasmic reticulum calcium ATPase type 3 (SERCA 3) was upregulated in TTL- treated cells, compared to untreated controls. SERCA 3 blocking with the specific blocker thapsigargin prevented increase in apoptosis observed by the TTL treatment. Decrease in the ATP production by a replacement of glucose in the cultivation medium for its nonutilizable analog 2-deoxyglucose also prevented induction of the apoptosis in TTL-treated PC12 cells. Thus, these results suggest that upregulation of the SERCA 3 is ultimately involved in the TTL-induced apoptosis in PC12 cells.

Sodium/calcium exchanger is upregulated by sulfide signaling, forms complex with the ß1 and ß3 but not ß2 adrenergic receptors, and induces apoptosis.

Hydrogen sulfide (H2S) as a novel gasotransmitter regulates variety of processes, including calcium transport systems. Sodium calcium exchanger (NCX) is one of the key players in a regulation calcium homeostasis. Thus, the aims of our work were to determine effect of sulfide signaling on the NCX type 1 (NCX1) expression and function in HeLa cells, to investigate the relationship of ß-adrenergic receptors with the NCX1 in the presence and/or absence of H2S, and to determine physiological importance of this potential communication. As a H2S donor, we used morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate-GYY4137. We observed increased levels of the NCX1 mRNA, protein, and activity after 24 h of GYY4137 treatment. This increase was accompanied by elevated cAMP due to the GYY4137 treatment, which was completely abolished, when NCX1 was silenced. Increased cAMP levels would point to upregulation of ß-adrenergic receptors. Indeed, GYY4137 increased expression of ß1 and ß3 (but not ß2) adrenergic receptors. These receptors co-precipitated, co-localized with the NCX1, and induced apoptosis in the presence of H2S. Our results suggest that sulfide signaling plays a role in regulation of the NCX1, ß1 and ß3 adrenergic receptors, their co-localization, and stimulation of apoptosis, which might be of a potential importance in cancer treatment.

APEX microarray panel for genotyping polymorphisms in cancer chemotherapy and estimation frequencies in a Slovak population.

AIM: Many studies focus on monitoring response to chemotherapy, adverse effects and prediction of therapeutic effects, which depend on individual gene variability. The amount of various polymorphisms in genes involved in the folate cycle, and other metabolic pathways involved in the metabolism of chemotherapeutic drugs, are an essential topic of such studies. This work focuses on the design and establishment of a pharmacogenetically relevant panel, which could be applied to the rapid genotyping of patients treated with thiopurines, 5-fluorouracil, methotrexate, irinotecan and glucocorticoids. MATERIALS & METHODS: A total of 97 variations in 36 genes associated with side effects of chemotherapeutic treatment were selected. Of these, 94 SNPs were genotyped by the arrayed primer extension (APEX; Asper Biotech Ltd) microarray method or direct sequencing. Variations of tandem repeats or gene deletions were genotyped by capillary electrophoresis and PCR detection. A total of 300 DNA samples from healthy volunteers were tested to estimate genotype frequencies for a Slovak population. All data were checked for Hardy-Weinberg equilibrium and genetic linkage between variations. RESULTS: We designed an APEX microarray for genotyping pharmacologically relevant polymorphisms in patients undergoing chemotherapy. We estimated genotype frequencies for all 97 polymorphisms testing 300 individuals from the Slovak population, which may also serve as an estimate of central European frequencies. These data also allowed for the testing of genetic linkage between loci. Many of the determined genotype frequencies in this study were in similar ranges found in other European populations but four SNPs, rs11760837 (p = 0.018), rs1801265 (p = 0.0375), rs1801394 (p = 0.0066) and rs182455 (p = 0.0083), demonstrated stronger deviation. CONCLUSION: Genetic variability in genes involved in metabolic pathways of chemotherapeutic drugs, such as methotrexate, 5-fluorouracil, thiopurines or irinotecan, is responsible for individual therapy response and development of side effects. A comprehensive approach in genotyping of numerous variants is aimed to improve individual access to patients and the selection of appropriate drugs for treatment. The APEX microarray method is a valuable tool for fast, reliable and cost-effective genotyping of variants which can be used for the typing of known variants in patients prior to treatment as well as in studies searching for new genotype-phenotype associations. The opportunity of adding additional variants during the study makes the APEX microarray technology flexible and suitable for such trials. Original submitted 4 October 2010; Revision submitted 23 November 2010.