Miroslav Červinka

Hydrogels in endovascular embolization: VI. Toxicity tests of poly(2-hydroxyethyl methacrylate) particles on cell cultures

Cytotoxicity of poly(2-hydroxyethyl methacrylate) [poly(HEMA)] hydrogel spherical particles, prepared by radical suspension polymerization and designed for endovascular occlusion, was studied in vitro on cell cultures. Testing methods included a direct contact test and extraction test. No inhibition of growth of cells surrounding the poly(HEMA) beads and a very low inhibition of cell viability, only in concentrated extracts in long-term contact, were observed. As a result, poly(HEMA) beads can be considered non-toxic.

Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes

Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.

Polyphenolic Coumpounds in Chemoprevention of Colon Cancer - Targets and Signaling Pathways

Colorectal cancer is one of the leading causes of premature death in people worldwide. Due to the fact that malignant conversion of normal colonic cells requires several steps and often proceeds over considerable time periods, primary prevention of this process should include several approaches, with optimization of nutrition and diet being among most important. During past decades, several groups of chemicals (both naturally occurring as well as synthetic) have been studied in terms of their potential chemopreventive role in colorectal cancer development. Naturally occurring plant polyphenols have recently come into scientific focus because of their presence in various popular natural products (wine grapes, teas, berries, peanuts) and, more importantly, due to their reported antiproliferative and cytostatic abilities in various in vitro and in vivo models. This review seeks to summarize the currently known targets and mechanisms whereby polyphenolic compounds interfere with colonic cancer cells while evaluating their chemopreventive potential in vivo.

Selenite-induced apoptosis and autophagy in colon cancer cells.

Sodium selenite (Se) is known to induce diverse stress responses in malignant cells which may lead to various types of cell death including apoptosis and/or autophagy. In colon cancer cells, Se activates several signaling pathways whose interactions and ultimate endpoints may vary in individual study models. In our previous work we showed differences in Se-dependent growth inhibition, cell cycle alterations and apoptosis in colon cancer cells with functional (HCT-116) and deleted (HCT-116-p53KO) p53. Moreover, detailed morphological and biochemical analyses revealed the presence of autophagy in Se-treated cells. Thus the aim of this study was to investigate in detail mechanisms, relationship and crosstalk between apoptosis and autophagy in Se-treated HCT-116 cancer cells differing in p53 status since p53 has been shown to play a well-known role in apoptosis but dichotomous role in autophagy. We report that the absence of p53 in malignant colonocytes changes patterns of response to Se-induced stress which include differential activation of MAP kinases (p38 - HCT-116 and JNK - HCT-116 p53KO) including their respective roles in the process of apoptosis and autophagy as well as the involvement of mTOR or PI3K signaling. Our results seem to suggest that deletion of p53 inevitably leads to a higher level of instability and delays in an individual cell decision in the face of stress whether to activate apoptosis or autophagy which may consequently occur simultaneously with mutual dichotomous relationship.

Sulforaphane induces cytotoxicity and lysosome- and mitochondria-dependent cell death in colon cancer cells with deleted p53

Mechanisms and pathways responsible for cytotoxicity of sulforaphane (SF) in colon cancer cells with deleted p53 were investigated during 48 h of exposure. SF showed dose-dependent cytotoxicity and proapoptotic activity in the present model. In addition, in HCT-116 p53KO cells SF induced DNA damage with the subsequent cellular response and signaling not including p53 and caspase-2 pathways. Conversely, in SF-treated cells JNK was activated which led to an early lysosomal membrane permeabilization, release of cathepsin B and D and activation of Bid by specific cleavage. Concomitantly, the expression of Bax increased in the presence of JNK-mediated Bcl-2 inhibition which was followed by mitochondrial release of cytochrome c and activation of apoptosis. These results suggest that SF may be useful as a chemopreventive agent in colon cancer with inactivated or lost p53.

Camptothecin induces p53-dependent and -independent apoptogenic signaling in melanoma cells

Various DNA-targeting agents may initiate p53-dependent as well as p53-independent response and subsequent apoptosis via alternative cellular systems which include for instance p73, caspase-2 or Bcl-2 family proteins. The scope of involvement of individual molecules in this process and the mechanisms governing their potential interplay are still not entirely understood, in particular in highly aggressive cancers such as in malignant melanoma. In this work we investigated the role and involvement of both p53-dependent and -independent mechanisms in selected melanoma cell lines with differing status of p53 using a model DNA topoisomerase I inhibitor camptothecin (CPT). Here we report that CPT induced in Bowes melanoma cells apoptosis which is essentially p53 and mitochondria-dependent but with some involvement of caspase-2 and p73. Conversely, in mutant p53 melanoma cells overall levels of CPT-induced apoptosis are significantly lower, with p73 and caspase-2 signaling playing important roles. In addition, in these cells the expression of micro RNAs family 34 (miR-34) were low compared to wild-type p53 cells. The ectopic expression of wild type p53 than restored apoptotic response of cells to CPT despite the fact that the expression of miR-34 and miR-155 were not influenced. These results suggest that CPT induces multivariate cellular stress responses including activation of DNA-damage response-p53 pathway as well as p53-independent signaling and their mutual crosstalk play the decisive role in the efficient triggering of apoptosis in melanoma cells.

Antiproliferative effects of selenium compounds in colon cancer cells: Comparison of different cytotoxicity assays

A number of cytotoxicity assays are currently available, each of them using specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. In this study we compared the potential of five commonly employed cytotoxicity assays (WST-1, XTT, MTT, Brilliant blue and Neutral red assay) to detect antiproliferative effects of three selenium compounds, sodium selenite, seleno-L-methionine (SeMet) and Se-(Methyl)selenocysteine (SeMCys) on three colorectal cancer cell lines in vitro. Cells were exposed to the selected selenium compounds in the concentration range of 0-256 microM during 48 h. WST-1 and XTT failed to detect cytotoxic effect, with the exception of the highest concentration of selenium compounds tested. Conversely, the metabolic activity of selenium treated cells measured by WST-1 and XTT significantly increased in comparison to untreated controls. MTT, Neutral red and Brilliant blue assays were more sensitive and yielded mutually comparable results, with significant decrease of measured parameters in a concentration-dependent manner. To a smaller extent, the results were affected by the different chemical nature of the selenium compounds tested as well as by the biological properties of individual cell lines.

Sulforaphane-induced apoptosis involves p53 and p38 in melanoma cells

In malignant melanoma complex reprogramming of cell death and survival pathways leads to increased chemoresistance and poor longer-term survival. Sulforaphane (SF) is a promising isothiocyanate compound occurring in cruciferous plants with reported antiproliferative and proapoptotic activity in several tumor cell lines including melanoma. In this work we investigated the effects of SF in several melanoma cell lines and fresh melanoma cultivates. We found that SF is cytotoxic and induces mitochondrial, caspase-dependent apoptosis in our study model, however with lower efficiency in fresh melanoma cultivates. Moreover, our results indicate that in melanoma cell lines and fresh melanoma cultivates SF induces multiple signaling including oxidative stress-mediated activation of DNA-damage response pathway, changes in p38 kinase activity and enhanced expression of Bax and Puma proapoptotic proteins. In addition, in SF-exposed p53-mutant melanoma cells Puma expression seem to be under p38 control and acts as a compensatory proapoptotic mechanism. Conversely, decreased apoptosis in SF-exposed melanoma cultivates might be attributed to Akt-mediated suppression of p38 as well as p53 activity. Together, our results suggest that SF inhibits growth and proliferation and induces mitochondrial apoptosis both in melanoma cell lines as well as in fresh melanoma cultivates. This proapoptotic effect might be enhanced in combination with Akt inhibitors, in particular in melanoma samples. SF is thus commendable for further preclinical testing, both as a single agent as well as in combination regimens.

Stress responses of human dermal fibroblasts exposed to zinc pyrithione.

Zinc pyrithione is used as a topical agent in a range of medicinal and cosmetic applications. Despite its extensive use and reported beneficial effects in treatment of various dermal problems, its potential toxicity towards skin cells remains relatively underexplored. In this work we investigated effects of nM zinc pyrithione on cell stress response pathways of primary human skin fibroblasts during 24h of exposure. We demonstrate that zinc pyrithione-induced cytotoxity in dermal fibroblasts is dose-dependent and it associates with increased intracellular zinc concentrations and activated stress response pathways including p53 and stress kinase p38. Higher zinc pyrithione concentrations (500nM and above) stimulate oxidative stress and moderate DNA damage which occur in the presence of activated p38 kinase. Cells further upregulate the expression of p53 which increases its transcriptional activity while mitogenic signaling exemplified by mTOR (mammalian target of rapamycin) expression is suppressed and these steps lead to mitochondrial, caspase-dependent apoptosis. Conversely, lower zinc concentrations (125nM) fail to induce oxidative stress and significant DNA damage; however, treated cells still activate p38 and upregulate the expression and transcriptional activity of p53 and its target gene p21 as well as the expression of p16 in the presence of active mTOR pathway and a changed DNA methylation pattern. The end result is premature senescence phenotype. Specific pharmacological inhibitors as well as gene knockdown technology prove that an interaction between p38, p53 and mTOR might be responsible for these observed endpoints. Taken together, exposure of dermal fibroblasts to varying concentrations of zinc pyrithione may result in either cell death-apoptosis or cellular premature senescence which attests to the ability of this compound to affect this type of cells in an in vitro model system.

Nickel modifies the cytotoxicity of hexavalent chromium in human dermal fibroblasts.

We investigated combined effects of hexavalent chromium and nickel on viability, intracellular signaling and cell death of primary human skin fibroblasts during 24 h of exposure. We show that nickel at non-toxic concentrations prevents hexavalent chromium-induced cell damage and apoptosis, mainly by overexpression of heat shock proteins (HSPs), in particular HSP27 and activation of nuclear factor kappa B (NFkappaB) as demonstrated by specific knockdown of HSPs or NFkappaB. Conversely, cytotoxic nickel concentrations which induce apoptosis in dermal fibroblasts by themselves act to enhance hexavalent chromium effects in the same cells by stimulating oxidative stress and depleting ATP leading to rapid necrosis as demonstrated by markedly increased LDH release in exposed cells. Using specific pharmacological inhibitors it was further demonstrated that oxidative stress and PARP-1 activity are responsible for rapid necrosis. In conclusion, exposure of dermal fibroblasts to high nickel concentrations in combination with hexavalent chromium may result in rapid cell damage leading to necrosis while low nickel concentrations may prevent hexavalent chromium-induced cell death with potential accumulation of damaged but otherwise viable cells.