B-Group Vitamins: Chemoprevention?

The importance of vitamins in the prevention of cancer has attracted the attention of consumers, nutritionists and scientists for decades. The mechanisms of carcinogenesis, extended in the context of the function of vitamins, i.e. regulation of and participation in metabolic processes in the cell, suggest a substantial impact of these compounds on the initial stages of carcinogenesis. One-carbon metabolism involving folic acid, vitamins B2, B6 and B12, and folate metabolism doesn't only generate methyl groups, thus determining epigenetic processes, modifications of the genome and carcinogenesis. It also provides the compounds involved in the DNA synthesis and repair processes, especially the synthesis of purines and pyrimidines and the conversion of dUMP (2-deoxyuridine monophosphate) to dTMP (2-deoxythymidine monophosphate). In light of these pathways, folate, together with vitamins B2, B6 and B12, became a subject of interest as compounds whose deficit or surplus can potentially have an impact on the processes of carcinogenesis. Literature reports, however, do not fully confirm that the influence on the synthesis of nucleotides is connected with the inhibition of carcinogenesis. The impact of individual vitamins involved in one-carbon metabolism on carcinogenesis and their role in the prevention of these conditions depend on the type of cancer and the dose administered. Nevertheless, the research conducted makes it possible to conclude a considerable and probably long-underestimated role of these compounds in the prevention of serious, difficult to treat or incurable diseases.

Cytotoxicity Evaluation and Crystallochemical Analysis of a Novel and Commercially Available Bone Substitute Material.

BACKGROUND: Alloplastic biomaterials are an alternative for autologous transplants and xenografts in oral surgery and dental implantology. These non-immunogenic and resorbable materials are becoming the basis for complete and predictable guided bone regeneration in many cases. The chemical composition of a great majority of them is based on calcium phosphate salts. In vivo performance is often variable. OBJECTIVES: The objective was to evaluate the biological and chemical properties of an experimental bone substitute material. MATERIAL AND METHODS: The present research focuses on the cytotoxicity comparison and physiochemical characterization of two biomaterials: a novel chitosan/tricalcium phosphate/alginate composite (CH/TCP/Ag) and a commercially available synthetic bone graft made of HA (60%) and ßTCP (40%) (HA/TCP). The materials were evaluated according to PN-EN ISO 10993 Biological evaluation of medical devices i.e. cytotoxicity on mouse fibroblasts (L929) and, in addition, tests on human osteoblasts (hFOB1.19) and human osteosarcoma (MG-63) were conducted. The crystallochemical analysis was performed using the X-ray powder diffraction method. The Bruker-AXS D8 Advance diffractometer (Karlsruhe, Germany) was used to collect diffractograms. RESULTS: The tested materials showed a close resemblance in chemical composition and a considerable differentiation in cytotoxic response. CONCLUSIONS: The novel composite demonstrated a high degree of cytocompatibility, which is promising in future clinical trials.

[The role of thiamine in neurodegenerative diseases]. / Rola tiaminy w chorobach neurodegeneracyjnych.

Vitamin B1 (thiamine) plays an important role in metabolism. It is indispensable for normal growth and development of the organism. Thiamine has a favourable impact on a number of systems, including the digestive, cardiovascular and nervous systems. It also stimulates the brain and improves the psycho-emotional state. Hence it is often called the vitamin of "reassurance of the spirit". Thiamine is a water-soluble vitamin. It can be present in the free form as thiamine or as its phosphate esters: mono-, di- or triphosphate. The main source of thiamine as an exogenous vitamin is certain foodstuffs, but trace amounts can be synthesised by microorganisms of the large intestine. The recommended daily intake of thiamine is about 2.0 mg. Since vitamin B1 has no ability to accumulate in the organism, manifestations of its deficiency begin to appear very quickly. The chronic state of thiamine deficiency, to a large extent, because of its function, contributes to the development of neurodegenerative diseases. It was proved that supporting vitamin B1 therapy not only constitutes neuroprotection but can also have a favourable impact on advanced neurodegenerative diseases. This article presents the current state of knowledge as regards the effects of thiamine exerted through this vitamin in a number of diseases such as Parkinson's disease, Alzheimer's disease, Wernicke's encephalopathy or Wernicke-Korsakoff syndrome and Huntington's disease.

[The phenomenon of vitamin D]. / Fenomen witaminy D

The receptor of vitamin D (VDR) is present in most non-skeletal human cells, suggesting its role beyond the bone and calcium metabolism. The relationship between vitamin D and the respiratory tract is a consequence of its activity in the immune system. Some gastrointestinal diseases, such as inflammatory bowel disease, coeliac disease, liver, pancreas or cardiac diseases, lead to vitamin D deficiency. Many studies indicate a correlation between vitamin D and diabetes. VDR and 1α-hydroxylase have been detected in the cutaneous capillary vessels, endothelium, vascular smooth muscles, myocytes and cardiac fibroblasts. The influence of vitamin D on the expression of genes related to the vascular walls implies its role in the pathomechanisms of vascular diseases and the cardiovascular system. Due to the VDR detected in most immunocompetent cells, calcitriol can modulate the congenital and acquired immune system. The correlation between vitamin D and cancer development is also not surprising because of many functions which vitamin D has in the organism. The vitamin D-regulated genes encode the proteins which participate in differentiation, proliferation or apoptosis. This paper aims to focus on the less well known roles of vitamin D in the organism, especially considering that most "sun consumers" know only its antirachitic and bone reinforcing action. So, this article may be surprising, and first of all it should convince everyone to vitamin D supplemention.

Formation and preclinical evaluation of a new alloplastic injectable bone substitute material.

Alloplastic bone substitute materials are raising some more interest as an alternative for autologic transplants and xenogenic materials especially in oral surgery over the last few years. These non-immunogenic and completely resorbable biomaterials are the basis for complete and predictable guided bone regeneration. In the majority of cases, such a material is chosen because of its convenient application by surgeons. The main objective of our project was to design and fabricate an osteoconductive, injectable and readily tolerable by human tissues biomaterial for guided bone regeneration. For this purpose, a self-setting composite consisting of chitosan/tricalcium phosphate microparticles and sodium alginate was made. The material obtained was characterized by microsphere and agglomerate morphology and microstructure. Its features relating to setting time and mechanical properties were precisely investigated. Our material was also evaluated according to PN-EN ISO 10993 Biological evaluation of medical devices, i.e., the in vitro tests for genotoxicity and cytotoxicity were conduced. Then, the following examinations were performed: subchronic systemic toxicity, skin sensitization, irritation and delayed-type hypersensitivity and local effects after implantation. The material tested showed a high degree of cytocompatibility, fulfilled the requirements of International Standards and seemed to be a "user friendly" material for oral surgeons.

Modulation of apoptosis protein profiles--role of P-gp in HeLa cells exposed to doxorubicin.

As shown previously doxorubicin (1 µM) plus sulindac (50 µM) reduced the expression of ABCB1 (ATP-binding cassette, sub-family B (MDR/TAP), member 1) mRNA in HeLa cells and this effect was accompanied by increased apoptosis. The aim of this study was to define if the decrease of ABCB1 expression or blocking of P-glycoprotein (P-gp) can affect the expression of the apoptotic genes determined with use of quantitative real time polymerase chain reaction (qRT-PCR). Western blot was used for visualization of chosen pro- and antiapoptotic proteins. Doxorubicin was the main compound which affected the apoptotic genes. The effectiveness of the drugs in reducing of P-gp function has been shown as not being related to the regulation of apoptotic gene transcription. In this experimental scheme, regulation of apoptotic gene transcription depended on the kind of P-gp modulator.

[Epigenetics and etiology of neurodegenerative diseases]. / Epigenetyka a etiologia chorób neurodegeneracyjnych.

Determination of specific gene profile expression is essential for morphological and functional differentiation of cells in the human organism. The human genome consists of 25-30 thousands genes but only some of them are expressed in each cell. Epigenetic modifications such as DNA methylation, histone and chromatin modifications or non-coding RNA functions are also responsible for the unique gene expression patterns. It is suggested that transcriptional gene activation is related to hypomethylation and the transcriptionally non-active sequences are hypermethylated. Covalent histone modifications and DNA methylation are correlated and interacting. Chromatin modeling is regulated not only by specific enzymes but also by protein kinases or phosphatases and coactivators, such as CBP. Such interaction makes the "histone code" which with the chromatin proteins determines gene expression patterns as the response to external agents. Evidence of a major role for epigenetic modifications in neurological disease has come from three converging lines of enquiry: high conservation throughout evolution of the histone residues that are the target for epigenetic modifications; association between mutations in epigenetic components and multisystem disease syndrome in the nervous system; and broad efficacy of small-molecule epigenetic modulators, e.g. histone deacetylase inhibitors, in models of neurological diseases incurable up to now, such as Huntington's disease, (HD), Parkinson's disease (PD) and Alzheimer's disease (AD). This article is a survey of the literature concerning the characterization of gene expression patterns correlated with some neurodegenerative diseases. The processes of DNA hypomethylation and histone acetylation are emphasized. The histone deacetylases are indicated as the basis for design of potential drugs.

[The role of the proteasome for therapy of incurable diseases]. / Rola proteasomu w terapii chorób nieuleczalnych.

Proteins constitute the basic building elements of living organisms. Proteins have a limited lifetime in a cell. The so called "half-life period" of proteins is diverse and lasts from several minutes to several days. Regulatory proteins appear in a cell for a definite time and are short-lived. The proteins responsible for basic cell functions are stable and long-lived. Once their functions are fulfilled or because of their surfeit or damage, proteins are eliminated by degradation. Transformation processes of proteins are precisely controlled. There is also a strict association between protein metabolism and the energetic state of a cell. The main proteolytic cell systems are lysosomal and proteasome ones. The first (lysosomes) function in a simple and transparent way. The second system (proteasomes) is highly organized; by using ubiquitin it delivers "molecular label" and sends a marked protein for degradation. Efficient degradation of cellular proteins by the UPS route (ubiquitin - proteasome system) is essential for signal transduction, transcription adjustment, response to stress and control of receptors' activity. The dysfunction of the UPS route is crucial in the development of tumors, neurodegenerative diseases and diseases of immunological and infectious origin. Therefore, it is a challenge to elaborate methods of pharmacological intervention within this system involving, for example, the use of specific, low molecular-weight proteasome inhibitors and enzymes catalyzing the ubiquitination process. The article presents a review of advances in the field, including description of the lysosomal protein degradation route, proteasome model, and the phenomenon of protein aggregation. The summary of the experience on applied therapies, which use the processes of protein degradation as a basis, were also presented.

Possible role of HSP60 in synergistic action of anthracyclines and sulindac in HeLa cells.

As was observed in the earlier studies, doxorubicin (DOX) induced apoptosis in HeLa cells and that effect was potentiated significantly by sulindac (SUL). The aim of the current work was to study the effects of DOX and SUL on HSP60, HSF1 and HSP60 expression and the influence of DOX and SUL on HSP60 translocation. Expression of HSP60 and HSF1 was determined with QRT-PCR; the expression and localization of HSP60 were evaluated with Western blot. The 24-h cell cultures were co-incubated with DOX - 1 microM and/or SUL - 50 microM. The significant induction of HSF1 and HSP60 mRNA level was observed after exposure of the cells to DOX 1 microM. SUL 50 microM alone caused moderate increase in mRNA level. The significant decrease in expression of HSF1 and HSP60 was noted after DOX 1 microM and SUL 50 microM simultaneous treatment. HSP60 appeared in the higher levels in cytosol than in mitochondria No intracellular translocation was noted under treatment of the cells to DOX and/or SUL. In conclusion, the effects of HSP60 and HSF1 evoked in the cells depend on the inducer, proapoptotic action of DOX + SUL may correlate to the increased expression of HSF1 and HSP60; HSP60 mRNA level and the regulation of that protein expression depend on the apoptotic inducer; the role of HSP60 in apoptosis expressed in potential shift between mitochondria and cytosol is determined by the apoptotic inducer and the cell type.

NFkappaB activation and drug sensitivity in human neoplastic cells treated with anthracyclines.

NFkappaB (nuclear factor kappaB) is a transcription factor controlling, among others, cell proliferation and apoptosis. The potent activators of NFkappaB are anthracyclines which can activate apoptotic processes. As shown by some authors, NFkappaB activated by these drugs well correlated with their cytotoxic activity. The aim of this study was to assess the effects of doxorubicin (DOX) and its analogs (annamycin, WP903) on the NFkappaB activity in human melanoma cells: a sensitive (ME18) and a resistant to DOX (ME18/R) and its possible correlation with cell sensitivity to these drugs. In the studies, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, ELISA test and confocal microscopy were used. As was shown, DOX, 1.7; 8.6 microM, strongly induced NFkappaB in ME18 cells. Annamycin (ANN), 0.3; 3.0 microM and WP903, 3.0 microM induced NFkappaB in ME18/R cells. PDTC (pyrrolidine dithiocarbamate)--NFkappaB inhibitor made ME18/R cells more sensitive to ANN and WP903 but did not affect cytotoxicity of DOX in ME18 cells. These results suggest that the influence of NFkappaB activation on cytotoxicity of anthracyclines is highly drug- and cell-specific.

Effect of structural modification at the 4, 3', and 2' positions of doxorubicin on topoisomerase II poisoning, apoptosis, and cytotoxicity in human melanoma cells.

INTRODUCTION: The mechanism of the cytotoxicity of anthracyclines is pleiotropic and its significance in cell growth inhibition seems to be highly specific and dependent on cell type and anthracycline drug. Resistance and the high cardiotoxicity of anthracyclines have stimulated many studies aimed at identifying critical substituents required for optimal activity. Many authors point to the fact that the double-strand breaks, the consequence of the activity of topoisomerase II poisons, and the inability of cells to repair the DNA lesions are the signal for apoptosis. The aim of this study was to define the influence of 4-demetoxy 2'-halogenated analogs with altered basicity at the 3'-position on topoisomerase II and the relationship of that interaction with apoptosis and the cytotoxicity of these novel anthracyclines. Parental human ME18 melanoma cells and the ME18/R subline, obtained experimentally, resistant to doxorubicin (DOX), exposed to 1.7 and 8.6 microM DOX or its analogs, annamycin and WP903 (both 0.3 and 3.0 microM) were studied. MATERIALS AND METHODS: The MTT test was used to assay cytotoxicity. Interaction of the drugs with topoisomerase II and apoptosis were done by Western blot and fluorescence microscopy using Hoechst 33342. RESULTS: The structural changes at positions 4, 2', and 3' can influence topoisomerase II interaction and apoptotic activity, although correlation between these events and cytotoxic consequences has not been proved. CONCLUSIONS: The biological response of the cells to the structurally similar anthracyclines may be variable and probably depends on the cell type which seems to be an additional problem in the multifactorial resistance of tumor cells to anthracyclines.

Topoisomerase II alpha expression and cytotoxicity of anthracyclines in human neoplastic cells.

Topoisomerase II is ATP dependent enzyme that catalyzes DNA strand passage, the pivotal process in replication, transcription, recombination etc. As part of this breakage and religation process the intermediate generated is a cleavable complex between DNA and topoisomerase II. This complex is the target for topoisomerase II inhibitors like epipodophyllotoxins, actinomycin D or anthracyclines. Stabilization of cleavable complexes by the topoisomerase II inhibitors leading to DNA lesions and next to apoptosis is the most common mechanism of drug resistance reflected in reduced formation of the complexes due to decreased amounts and/or activity of topoisomerase II. The aim of this study was to characterize human melanoma and human cervix carcinoma cells differing in sensitivity to doxorubicin and anthracycline analogs, annamycin and WP903 for topoisomerase IIalpha protein and gene expression with use of Western blot and RT- PCR. As shown, no significant differences in topoisomerase IIalpha protein level were noted between the cell lines tested. These results were confirmed at the gene expression level. The current study points to the fact that topoisomerase IIalpha protein or gene expression are not the reliable marker of cell sensitivity to anthracyclines but these observations do not exclude the potential mutations in topoisomerase IIalpha gene or some postranslational changes in that protein which requires further studies.

Relationship between topoisomerase II-DNA cleavable complexes, apoptosis and cytotoxic activity of anthracyclines in human cervix carcinoma cells.

The use of anthracyclines as antitumor drugs dates back to the 1970s, but the mechanism of the cytotoxicity of these compounds has long been a matter of debate. There is increasing evidence indicating that drug-induced cytotoxicity commonly converges on the induction of apoptosis. Many authors point to the fact that double-strand breaks, resulting from stabilization of cleavable complexes, are the signal for the initiation of the apoptotic cascade. In this work, the possible correlation between stabilization of topoisomerase II (topoII)-DNA complexes, apoptosis induction and cytotoxicity was studied. Parental human cervix carcinoma cells, HeLa, and its subline resistant to vinblastine, KB-V1, were exposed to doxorubicin (DOX) and the novel anthracyclines annamycin and WP903, given at the concentrations 0.2 and 2.0 microg/ml (DOX and annamycin) or 0.2 and 1.0 microg/ml (WP903). It was found that annamycin was the strongest topoII poison in HeLa cells at both concentrations used, whereas poly (ADP-ribose) polymerase (PARP) cleavage was observed dose-dependently in KB-V1 cells treated with annamycin or WP903. Simultaneously, apoptosis, observed as cell morphology or phosphatidylserine translocation, was evident in both cell types exposed to the novel anthracyclines, independent of concentration. DOX appeared to be the weakest apoptotic inducer. On the basis of these studies, it can be suggested that topoII poisoning is not the key process leading to apoptosis and seems to be cell specific. PARP cleavage is probably not an evident marker of anthracycline-induced apoptosis which, in turn, does not seem to be the determinant in the cytotoxic action of these compounds. The efficiency of anthracycline antibiotics, interpreted as cytotoxic action, was dependent on cell type.

Constitutive activation of transcription factor NFkappaB as a possible marker of sensitivity of neoplastic cells to anthracyclines.

NFkappaB, a member of the Rel family of transcription factors has been found to be critically important for control of cell proliferation and apoptosis. Although rare, there are systems in which NFkappaB has occurred as pro-apoptotic factor. The potent activators of NFkappaB are anthracycline anticancer drugs which induce the events of apoptosis. These results could point to the pro-apoptotic role of NFkappaB. Recent studies have shown that activation of that transcription factor well correlated with cytotoxic activity of anthracyclines. Potential mechanism through which NFkappaB could play a role in tumorigenesis involves its constitutive activation which was shown in a wide variety of tumour types. The aim of this study was to define the level of activity of NFkappaB in the native human tumour cells differing in sensitivity to anthracycline analogs to study the mechanism responsible for cytotoxic action of these drugs. Constitutive activation of NFkappaB was determined with use of ELISA test and confocal microscopy. As was shown, both methods have occurred as quite comparable. Constitutive activation of NFkappaB was observed in all neoplastic cells tested independently on sensitivity to anthracyclines. However, these results do not exclude the supportive role of NFkappaB in apoptotic activity of these drugs. It is necessary to study the influence of the compounds tested here on NFkappaB activation and on the induction and intensity of apoptotic processes.

The effect of new anthracycline derivatives on the induction of apoptotic processes in human neoplastic cells.

This study was an attempt to determine the effect of a selected anthracycline derivative, WP903, on apoptotic processes in human melanoma cells depending on intracellular concentrations of the compound, and to evaluate the significance of apoptosis induction for the cytotoxic effect of anthracycline antibiotics. It was found that the WP903, contrary to ADR (adriamycin) is a strong inducer of apoptotic processes in ME18 human melanoma cells regardless of their susceptibility to adriamycin and WP903. The cells were treated for 24 h with ADR (1 and 5 microg/ml) or WP903 (0.2 and 2 microg/ml). Apoptosis was detected with the use of annexin V-FITC and PI (propidium iodide) and with TUNEL assay. WP903 used at 0.2 microg/ml induced early apoptosis in 23% of ME18 cells and in 60% of ME18/R cells; at 2 microg/ml in 70% of each of cell line tested. Significant late apoptotic effect was observed in ME18 cells. In contrast, ADR was found to be a weak inducer of apoptotic events. The results suggest that apoptosis is not a mechanism directly related to the cytotoxic effect of anthracycline antibiotics.

Studies on the influence of vitamin D3 metabolites on apoptosis induction in human neoplastic cells.

The vitamin D is involved in essential cell regulatory processes such as proliferation and differentiation in a number of different cell types including cancer cells. Adriamycin is one of the most effective agents in the treatment of many types of solid tumours and leukemias. The common features in the biological activity, expressed by vitamin D family members and adriamycin such as: apoptosis induction, influence on antioxidant enzymes activity etc., created the suggestion of synergistic effects of these compounds combination. In the earlier studies the antiproliferative activity of vitamin D3 metabolites was shown in ME18 cells, but without any correlation with sensitivity of the cells to adriamycin. In the current work the possible role of 25-hydroxycholecalciferol (calcidiol) and 1,25-dihydroxycholecalciferol (calcitriol) as the apoptotic inducers was studied in human melanoma cells. As was shown in these experiments, vitamin D3 metabolites did not stimulate apoptotic events in the cells studied and did not influence apoptosis induction in the cells treated with adriamycin.