Aldehyde dehydrogenase 1A1 and 1A3 isoforms - mechanism of activation and regulation in cancer.

In some types of human cancer, aldehyde dehydrogenases represent stemness markers and their expression is associated with advanced disease stages and poor prognosis. Although several biological functions are mediated by their product Retinoid acid, the molecular mechanism is tissue-dependent and only partially understood. In this review, we summarize the current knowledge about the role of ALDH in solid tumours, especially ALDH1A1 and ALDH1A3 isoforms, regarding the molecular mechanism of their transcription and regulation, and their crosstalk with main molecular pathways resulting in the excessive proliferation, chemoresistance, stem cells properties and invasiveness. The recent knowledge of the regulatory effect of lnRNA on ALDH1A1 and ALDH1A3 is discussed too.

ALDH1A inhibition sensitizes colon cancer cells to chemotherapy.

BACKGROUND: Recent evidence in cancer research, developed the notion that malignant tumors consist of different subpopulations of cells, one of them, known as cancer stem cells, being attributed many important properties such as enhanced tumorigenicity, proliferation potential and profound multidrug resistance to chemotherapy. Several key stem cells markers were identified in colon cancer. In our study we focused on the aldehyde dehydrogenase type 1 (ALDH1) expression in colon cancer-derived cell lines HT-29/eGFP, HCT-116/eGFP and LS-180/eGFP, and its role in the chemoresistance and tumorigenic potential. METHODS: The effect of pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde (DEAB) and also effect of molecular inhibition by specific siRNA was evaluated in vitro in cultures of human colorectal cell lines. The expression level of different isoenzymes of aldehyde dehydrogenase was determined using qPCR. Changes in cell biology were evaluated by expression analysis, western blot and apoptosis assay. The efficiency of cytotoxic treatment in the presence of different chemotherapeutic drugs was analyzed by fluorimetric assay. Tumorigenicity of cells with specific ALDH1A1 siRNA was tested in xenograft model in vivo. RESULTS: Treatment by DEAB partially sensitized the tested cell lines to chemotherapeutics. Subsequently the molecular inhibition of specific isoforms of ALDH by ALDH1A1 or ALDH1A3 siRNA led to sensitizing of cell lines HT-29/eGFP, HCT-116/eGFP to capecitabine and 5-FU. On the model of athymic mice we observed the effect of molecular inhibition of ALDH1A1 in HT-29/eGFP cells by siRNA. We observed inhibition of proliferation of subcutaneous xenografts in comparison to control cells. CONCLUSION: This research, verifies the significance of the ALDH1A isoforms in multidrug resistance of human colorectal cancer cells and its potential as a cancer stem cell marker. This provides the basis for the development of new approaches regarding the treatment of patients with colorectal adenocarcinoma and potentially the treatment of other tumor malignancies.

Genetically engineered mesenchymal stromal cells in cancer gene therapy.

Based on our experimental data, we aimed to emphasise the perspectives of the use of mesenchymal stromal cells (MSC) in the cancer gene therapy. On the other hand, we would like to point out factors which should be taken into consideration at their clinical use. In this review we define MSC as unique targets for targeted therapy. We proved the efficacy of experimental therapeutic approach utilising enzymatic conversion of non-toxic prodrug into chemotherapeutic by engineered MSC, and we observed significant cytotoxic effect in many preclinical models including metastatic disease. Treatment was enabled by affinity of MSC to tumour tissue and subsequent delivery of therapeutic molecule into the tumour. We also observed decreased efficacy of cell-mediated gene therapy on chemoresistant tumour cells. Moreover MSC can exert a supportive effect on tumour cells as well as to decrease the efficacy of conventional treatment. Besides obvious unique benefits connected to the use of MSC we pointed also to possible risks associated with their clinical application (Ref. 24).

Mesenchymal stromal cells producing TNFα lack inhibitory effect against A375 experimental lung metastases.

Cell-based anticancer therapy using mesenchymal stromal cells (MSCs) engineered to express therapeutic genes has a potential to target the cancer cells in vivo. Metastatic dissemination of melanoma remains a serious problem in the treatment. In our previous work we used MSCs overexpressing gene for tumor necrosis factor α (TNFα; MSCs/TNFα), and we achieved inhibition of melanoma xenograft growth when engineered MCSs/TNFα were coinjected with tumor cells subcutaneously. The TNFα as a pleiotropic cytokine induces apoptosis of tumor cells, creates "tumor resistant" microenvironment, enhances immune response and can have tumor destructive capacity in selected tumor types, especially in tumors of mesodermal origin.In this study we investigated the possibility of intravenously administered MCSs/TNFα to inhibit metastatic spread of A375 melanoma cells in the lungs. We confirmed elevated expression of TNFα transgene in the lung tissue 20 days after MCSs/TNFα intravenous infusion. We also documented that constitutive expression of TNFα transgene is able to neutralize the supportive effect of MSCs on melanoma cells growth. Metastatic spread of A375 melanoma cells in the lung was inhibited approximately to 50% after MCSs/TNFα i.v. administration in comparison to control group with parental MSCs supporting tumor growth. In conclusion, engineered MCSs/TNFα administered intravenously did not demonstrate significant antitumor effect against experimental melanoma lung metastases in this model settings.

Global and gene specific DNA methylation in breast cancer cells was not affected during epithelial-to-mesenchymal transition in vitro.

Epithelial-to-mesenchymal transition (EMT) significantly affects the risk of metastasising in breast cancer. Plasticity and reversibility of EMT suggest that epigenetic mechanisms could be the key drivers of these processes, but little is known about the dynamics of EMT-related epigenetic alterations. We hypothesised that EMT, mediated by autocrine and paracrine signals, will be accompanied by changes in DNA methylation profiles. Therefore, conditioned medium from adipose tissue-derived mesenchymal stromal cells was used for induction of EMT in human breast cancer SK-BR-3 cell line. EMT-related morphological alterations and changes in gene expression of EMT-associated markers were assessed. To reverse EMT, 20 nm size gold nanoparticles (AuNPs) synthesized by the citrate reduction method were applied. Finally, DNA methylation of LINE-1 sequences and promoter methylation of TIMP3, ADAM23 and BRMS1 genes were quantitatively evaluated by pyrosequencing. Despite the presence of EMT-associated morphological and gene expression changes in tumour cells, EMT induced by adipose tissue-derived mesenchymal stromal cells had almost no effect on LINE-1 and gene-specific DNA methylation patterns of TIMP3, ADAM23 and BRMS1 genes. Although treatment for 24, 48 or 72 hours with 20 nm AuNPs at a concentration of 3 µg/ml slightly decreased gene expression of EMT-associated markers in SK-BR-3 cells, it did not alter global or gene-specific DNA methylation. Our results suggest that changes in DNA methylation are not detectable in vitro in early phases of EMT. Previously published positive findings could represent rather the sustained presence of potent EMT-inducing signals or the synergistic effect of various epigenetic mechanisms. Treatment with AuNPs slightly attenuated EMT, and their therapeutic potential needs to be further investigated.

3D multicellular models reflect the efficiency of MSC-directed enzyme/prodrug treatment.

Mesenchymal stromal cells (MSC) exhibit beneficial properties to serve as cellular vehicles for enzyme/prodrug cancer gene therapy approaches. We have previously shown that engineered human adipose tissue-derived MSC in combination with non-toxic prodrug mediated substantial cytotoxic and antitumor effect. The aim of this study was to develop advanced 3D cultivation method to serve for modelling of the therapeutic outcome in vitro. We have used engineered MSC expressing fusion transgene cytosine deaminase::uracilphosphoribosyltransferase (CD-MSC) in combination with prodrug 5-fluorocytosine (5FC). This therapeutic regimen designated CD-MSC/5FC was combined with the human melanoma cells A375 or EGFP-A375 in both standard monolayer culture and 3-dimensional (3D) multicellular nodules. The extent of cytotoxicity was evaluated by standard viability assay MTS, ATP-based luminescence assay, fluorimetric test, measurement of Caspase-3/7 activation and DNA laddering. The data have shown that the extent of cytotoxic bystander effect mediated by CD-MSC/5FC is significantly lower in 3D culture conditions. However, these data better recapitulate the therapeutic efficiency as observed previously in vivo. We suggest here to use the 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models.

Long-term efficiency of mesenchymal stromal cell-mediated CD-MSC/5FC therapy in human melanoma xenograft model.

Mesenchymal stromal cells (MSC) can be exploited as cellular delivery vehicles for the enzymes converting non-toxic prodrugs to toxic substances. Because of their inherent chemoresistance, they exert potent bystander and antitumor effect. Here we show that the human adipose tissue-derived MSC expressing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD-MSC) in combination with 5-fluorocytosine (5FC) mediated a long-term tumor-free survival in the 83.3% of tumor-bearing animals. CD-MSC/5FC treatment induced cytotoxicity against model human melanoma cells EGFP-A375. Only 4% of the therapeutic CD-MSC cells eliminated >98.5% of the tumor cells in vitro. Long-term tumor-free survival was confirmed in 15 out of the 18 animals. However, repeatedly used CD-MSC/5FC therapeutic regimen generated more aggressive and metastatic variant of the melanoma cells EGFP-A375/Rel3. These cells derived from the refractory xenotransplants exhibited increased resistance to the CD-MSC/5FC treatment, altered cell adhesion, migration, tumorigenic and metastatic properties. However, long-term curative effect was achieved by the augmentation of the CD-MSC/5FC regimen along with the inhibition of c-Met/hepatocyte growth factor signaling axis in this aggressive melanoma derivative. In summary, the CD-MSC/5FC regimen can be regarded as a very effective antitumor approach to achieve long-term tumor-free survival as demonstrated on a mouse model of aggressive human melanoma xenografts.

Mesenchymal stromal cells retrovirally transduced with prodrug-converting genes are suitable vehicles for cancer gene therapy.

Mesenchymal stem/stromal cells (MSC) possess a set of several fairly unique properties which make them ideally suitable both for cellular therapies and regenerative medicine. These include: relative ease of isolation, the ability to differentiate along mesenchymal and non-mesenchymal lineages in vitro and the ability to be extensively expanded in culture without a loss of differentiative capacity. MSC are not only hypoimmunogenic, but they mediate immunosuppression upon transplantation, and possess pronounced anti-inflammatory properties. They are able to home to damaged tissues, tumors, and metastases following systemic administration. The ability of homing holds big promise for tumor-targeted delivery of therapeutic agents. Viruses are naturally evolved vehicles efficiently transferring their genes into host cells. This ability made them suitable for engineering vector systems for the delivery of genes of interest. MSC can be retrovirally transduced with genes encoding prodrug-converting genes (suicide genes), which are not toxic per se, but catalyze the formation of highly toxic metabolites following the application of a nontoxic prodrug. The homing ability of MSC holds advantages compared to virus vehicles which display many shortcomings in effective delivery of the therapeutic agents. Gene therapies mediated by viruses are limited by their restricted ability to track cancer cells infiltrating into the surrounding tissue, and by their low migratory capacity towards tumor. Thus combination of cellular therapy and gene delivery is an attractive option - it protects the vector from immune surveillance, and supports targeted delivery of a therapeutic gene/protein to the tumor site.

Interaction of human adipose tissue-derived mesenchymal stromal cells with breast cancer cells.

Human adipose tissue was shown to be a very attractive source of mesenchymal stromal cells that have a wide scale of potential applications in reconstructive plastic surgery and regenerative medicine. However, these cells were described to have profound effects on biological behaviour of tumour cells. The aim of this study was to analyze the influence of adipose tissue-derived human mesenchymal stromal cells (AT-MSC) on the proliferation of breast cancer cells. We have tested proliferation of three different human breast cancer cell lines under the influence of AT-MSC derived soluble factors as well as in the direct cocultures. These data were supplemented with the expression analysis of cytokines and their cognate receptors on the target cells. We have observed stimulation of proliferation in breast cancer cells MDA-MB-361, T47D and EGFP-MCF7. AT-MSC were found to secrete wide scale of cytokines, chemokines and growth factors, thus we concluded that this pro-proliferative effect was a result of their synergistic action. These data bring out a need to evaluate whether primary breast tumour derived human cells would respond to these type of stimuli in a similar manner in order to exclude any potential clinical risk related to the application of human mesenchymal stromal cells under the context of patient with history of breast cancer malignancy.

Genotoxic damage of human adipose-tissue derived mesenchymal stem cells triggers their terminal differentiation.

Human adipose tissue-derived mesenchymal (stromal) stem cells (AT-MSCs) and genetically modified to express cytosine deaminase:uracil phosphoribosyltransferase (CDy-AT-MSCs) were treated with hydrogen peroxide in order to induce DNA damage and subsequently evaluate their genetic stability by single cell gel electrophoresis. Both cells types (parental and transgene modified) did not differ in the sensitivity to DNA breaks induction. Potential tumorigenicity of AT-MSCs and CDy-AT-MSCs was tested by subcutaneous inoculation of cell suspension into flank of immunocompromised mice. Dose of 15x10(6) cells was not found to be tumorigenic in given experimental setup. AT-MSCs, CDy-AT-MSCs and MSCs isolated from human lipoma were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) in attempts to transform them. Surviving cells after genotoxic stress were not transformed but underwent replicative senescence. Irreparable DNA damage caused triggered adipogenic terminal differentiation, rather than apoptosis induction in all kinds of cells tested.

First confirmed sheep scrapie with A136R154Q171 genotype in Slovakia.

The first confirmed evidence of scrapie in Slovakia was demonstrated in one sheep of the autochthonous Merino breed from the southeastern part of the country. The reported scrapie was diagnosed during compulsory transmissible spongiform encephalitis (TSE) screening of sheep over 9 months of age assigned for consumption. The positive ewe was 5-year-old, which did not show any clinical signs of scrapie. The presence of the proteinase-resistant prion protein (PrP) in brain was proved independently by two laboratories using two different immunochemical screening systems, namely the Prionics Check (Western blot analysis) and Enfer TSE enzyme-linked immunosorbent assay (ELISA). In addition, the genotyping analysis of PrP gene demonstrated the presence of PrP genotype from the high risk group R4. The affected sheep was homozygous for the allele PrP(ARQ) (ARQ/ARQ) coding for alanine (A), arginine (R) and glutamine (Q) at three most relevant codons (136, 154 and 171, respectively). The healthy sister of the positive ewe was heterozygous in the PrP locus and carried alleles ARQ/ARR.

Antigenic relationship between five isolates of murine gammaherpesvirus analysed with monoclonal antibodies.

A panel of six monoclonal antibodies (mAbs) specific for murine gammaherpesvirus (MHV) was used for analysis of the antigenic relationship between five MHV-isolates (MHV 68, MHV 72, MHV 76, MHV 78, MHV S). Two mAbs raised against MHV 72 and four raised against MHV S were used in the study. Antibody-virus interactions were tested using immunochemical (ELISA, Western blot, immunofluorescence) and biological (virus-neutralization) assays. Immunoanalysis by ELISA showed a close antigenic relationship between the five viruses, nevertheless, some antigenic individuality of the isolate MHV S was observed. This isolate originated from a geographically distinct area in Czechia relative to the other four isolates from Slovakia. In Western blot analysis, antibodies to MHV 72 recognized viral antigens with the relative molecular mass about 116,000. Of four mAbs against MHV S, only two reacted with denatured viral antigen in Western blot and showed specificity for the 50-55,000 protein. These findings suggested that both isolates, besides of minor antigenic variability, could differ also in immunodominant proteins. Mabs to MHV S exhibited much stronger virus-neutralizing potency than mAbs to MHV 72, indicating thus that the 50-55,000 antigen might be more relevant for the infectivity of MHV-virus. Immunofluorescence with mAbs allowed specific localization of antigens in virus-infected VERO cells.

Risk factors of cardiovascular diseases.

BACKGROUND: Cardiovascular diseases are the leading cause of premature mortality of women and men in Slovak republic. THE AIM: The occurrence and mean values of major CVD risk factors were assessed in a population of 2480 Bratislava citizens (2/3 of them were women) interested in health promotion and primary CVD prevention activities in Community Health Promotion Center. METHODS: Major CVD risk factors were assessed, using standard methods, and criteria, in accordance with the guidelines of European and national medical societies. MAIN RESULTS: The greatest proportion of visitors, both women and men, were in their forties. 73% of all women and 70% of all men were aged 30-59 yrs. The most frequent risk factors, were overweight and obesity, present in 64% of men and 59% of women. Of them, central type obesity was found in 30% of men, 15% of women. In 52% of men and 32% of women elevated casual blood pressure was assessed at the first visit. Of the total, in 27% of men and 21% of women, the BP elevation was within the range of borderline hypertension. In 25% of men and 11% women the BP values were within the mild to severe hypertension range. Elevated blood cholesterol was assessed in 53% of men and 54% of women, lowered HDL cholesterol in 55% of men and 43% of women. Elevated TC/HDL-C ratio was found in 60% of men and in 35% of women. Triglyceride level elevation was assessed in 24% of men and in 17% of women, with TGL/HDL-C ratio raised in 66% of these men and in 40% of these women. CONCLUSIONS: In assessment of CVD risk factors clustering, our results are different from the results of CINDI SR screening from 1992. In our study, only 13% persons were free of any CVD risk factors. One risk factor was found in 21.1%, two of them in 29.9%, three in 29.8% and four in 6.2% of the population screened. Evaluation of the effect of complex individual intervention in our center will be the subject of our next study. (Fig. 5, Tab. 5, Ref. 11.)

Specific endonucleases in Streptomyces aureofaciens.

Two strains of Streptomyces aureofaciens were found to contain restriction endodeoxynucleases; S. aureofaciens IKA 18/4 contains Sau I which splits lambda DNA into three fragments, S. aureofaciens IKA 22201 Sau Ii which splits lambda DNA into more than 15 fragments.