Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely.

Intracellular glutathione level and efflux in human melanoma and cervical cancer cells differing in doxorubicin resistance.

INTRODUCTION: Drug resistance continues to be a major problem in cancer treatment. Occurrence of this phenomenon is often associated with altered levels of glutathione (GSH) and GSH-related enzymes. The aim of the study was to evaluate the possible involvement of GSH and GSH-related enzymes in doxorubicin (DOX) resistance in two types of cancer cells of different etiology, from both parental and DOX-resistant sublines. MATERIALS AND METHODS: The human melanoma (ME18 and ME18/R) and cervical cancer cells (HeLa and KB-V1) were tested in terms of their DOX sensitivity (EZ4U test), GSH level (HPLC) and its efflux (spectrofluorometrically). The effects of inhibition of the GSH-related enzymes γ-glutamylcysteine synthetase (γ-GCS) and glutathione S-transferase (GST) were also evaluated. RESULTS: Exposure to DOX caused an increase of GSH levels in all tested cells except for HeLa cells. However, depletion of GSH did not have a significant influence on the sensitivity of the cells to DOX. Inhibition of the activity of GST also did not have a major effect on DOX sensitivity, although it caused changes of the GSH content. Our attempts to use the spectrofluorometric method for measurements of GSH efflux were not successful. It could be suggested that in ME18 and HeLa cells treated with DOX, GSH efflux does occur. DISCUSSION: The obtained results seem to refute the hypothesis of a central role of GSH in DOX resistance of the tested cells. Despite observations of different effects related to GSH, they do not seem to be essential in terms of DOX resistance. The mechanisms underlying DOX resistance are highly cell-specific.

Assessment of the genotoxic activity of alpha-asarone and its derivatives in the comet assay.

In our previous paper we examined mutagenic and genotoxic activity of 4 alpha-asarone isomers 2-5 exhibiting relatively high hypolipidemic activity. In the present paper, we examined genotoxic activity of alpha-asarone and its isomers as the ability to damage cellular DNA, evaluated in the comet assay. Additionally, mutagenic activity of alpha-asarone in Ames test has been examined. The Ames test for alpha-asarone was carried out in accordance with the guidelines of the PN-EN ISO 10993-3 standard. Compounds 4 and 5 were found to be devoid of any genotoxic activity while maintaining their hypolipemic potential. Because mutagenic activity of compound 4 was also minor it could be considered as a candidate for further pharmacological evaluation. Genotoxic but not mutagenic activity of alpha-asarone has been confirmed.

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.

Assessment of cytotoxic and genotoxic activity of alcohol extract of Polyscias filicifolia shoot, leaf, cell biomass of suspension culture and saponin fraction.

Some medicinal plants are the object of biotechnologists' special interest owing to their content of secondary metabolites, which have a strong pharmacological effect. Polyscias filicifolia is a plant known for long in traditional medicine of the Southeast Asia. Literature data suggest that it acts on the endocrine system, has adaptogenic and antiulcerative activity, shows bactericidal and insecticidal properties, restores the activity of the protein synthesis system in the conditions of long- and short-term anoxia, as well as reduces the effect of many mutagens in vitro. The purpose of the studies was to assess the cytotoxic and genotoxic effect of ethanol extracts from Polyscias filicifolia dry shoots and leaves obtained in vitro, as well as cell biomass from suspension culture. Saponin fraction from dried shoots was also tested. Initially, the cytotoxic effect was evaluated using the murine connective tissue cell line C3H/AN - L929. The genotoxic properties of the extracts were assessed using standard screening tests: the Ames test and the micronucleus test. Based on the obtained results it can be concluded that none of the extracts increases the number of revertants, both in tests with and without metabolic activation. The lack of in vitro genotoxic and mutagenic activity of tested shoot, dried leaf, cell biomass extracts, as well as the saponin fraction from dried shoots allows us to hope that Polyscias filicifolia could be used as a possible pharmaceutical raw material showing therapeutic properties.

Genotoxicity of alpha-asarone analogues.

The role of cholesterol in the formation of atherosclerotic lesions during hypercholesterolemia has been confirmed. alpha-Asarone is a substance of a potent hypolipidemic activity which is isolated from plants. We previously described the synthesis of several alpha-asarone analogues exhibiting hypolipidemic and antiplatelet activity. Genotoxic activity of four selected alpha-asarone analogues was theoretically evaluated based on quantum-mechanical method for calculation of enthalpy of carbocations formation (DeltaH(R)) according to the Testa's method. In the present paper, we evaluated the mutagenic and genotoxic activity of alpha-asarone isomers 2-5 based on the reference Ames test and micronucleus test. Results obtained in the study show that tested isomers were non-mutagenic, however, they exhibited growing cytotoxic activity. Relationship between the heat of formation of their putative metabolic intermediates and mutagenic/genotoxic activity was not confirmed.

Estimation of DNA damage and cytotoxicity of anthracycline analogs in human melanoma cells on early and late passages.

Human malignant melanoma is a major problem characterized by both rapid rising incidence and strong chemoresistance. The aim of present experiments was to estimate the effects on DNA of new anthracycline analogs in melanoma cells obtained from various patients, cultured on early (E) and late (L) passages. For determination of cytotoxic effect, MTT assay was used and comet assay was used for the detection of DNA damage. The discrepancy between the intensity of DNA damage processes and IC50 values may indicate that there are some critical loci in the genome, responsible for cell death.

Evaluation of mutagenic and genotoxic activities of new derivatives of anthracyclines.

The phenomenon of multidrug resistance is connected with an increased efflux of cytotoxic agents from the cells and is a serious problem in the treatment with anthracycline antibiotics. Search for new antitumor drugs from the anthracycline group are focused on the syntheris of derivatives with changes in saccharide and anthracycline parts. The genotoxic activity of selected nine new derivatives of anthracycline was evaluate basing on reference tests: micronucleus test in vitro and the Ames test. A correlation was observed between the results obtained in the Ames test and those received in the micronucleus test. In both tests, derivative WP903 demonstrated a strong genotoxic action.

The application of the sister chromatid exchange test for evaluation of the novel anthracycline derivatives. Part I.

In the first part of our study, which involved the evaluation of the potential cytogenetic activity of the novel anthracycline derivatives with respect to adriamycin in the sister chromatid exchange (SCE) test, we explored the possibilities of using the test in our laboratory and attempted to determine the range of investigated concentrations. We found that adriamycin at concentrations of 0.05 microg/mL and 0.1 microg/mL leads to a statistically significant increase in SCE versus control (p<0.01). We also found that adriamycin levels exceeding 0.2 microg/mL were toxic and suppressed lymphocyte proliferation.

Study on the interaction of ions of transient metals with ascorbic acid in the presence of different scavengers of active oxygen species in SOS chromotest.

SOS chromotest was employed to study the interaction of ascorbic acid with free ions of transient metals in the presence of added catalase, superoxide dismutase or D-mannitol. Catalase diminished the genotoxic activity of the mixture of ascorbic acid with copper ions in E. coli strains PQ37 and PQ 300, but genotoxicity of this mixture was not suppressed by superoxide dismutase and D-mannitol. The results suggest that copper ions diminished the content of peroxide generated by ascorbic acid.