Beata Pajak

Molecular basis of parthenolide-dependent proapoptotic activity in cancer cells

This review outlines the molecular events that accompany the anti-tumor action of parthenolide (PN). Parthenolide (PN) is naturally derived compound, isolated from plant Tanacetum parthenium. PN has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate that this sesquiterpene lactone might evoke the above-mentioned effects by indirect action on genes. PN was shown to inhibit NF-kappaB- and STATs-mediated antiapoptotic gene transcription. On one hand, the proapoptotic activity of PN includes stimulation of intrinsic apoptotic pathway with the higher level of intracellular ROS and modifications of Bcl-2 family proteins (conformational changes of Bak and Bax, Bid cleavage). On the other hand, PN amplifies the apoptotic signal through the sensitization of cancer cells to extrinsic apoptosis, induced by TNF-alpha. These effects are specific to tumor cells. Unique properties of PN make this agent a promising metabolic inhibitor to retard tumorigenesis and to suppress tumor growth.

Sodium butyrate sensitizes human colon adenocarcinoma COLO 205 cells to both intrinsic and TNF-α-dependent extrinsic apoptosis

Overexpression of cFLIP protein seems to be critical in the antiapoptotic mechanism of immune escape of human COLO 205 colon adenocarcinoma cells. Actually, cFLIP appears to inhibit the death receptor ligand-mediated cell death. Application of the metabolic inhibitor sodium butyrate (NaBt), short-chain volatile fatty acid, sensitized COLO 205 cells to TNF-α-mediated apoptosis. Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP and simultaneous up-regulation of TNF-R1 protein levels. Additionally, the combined TNF-α and NaBt treatment caused cleavage of Bid and caspase-9 activation, as well as cytochrome c release from mitochondria. Thus, the evidence of this study indicates that NaBt facilitates the death receptor signal evoked by TNF-α. Moreover, NaBt alone initiated intrinsic apoptosis, that in turn was abolished by intracellular BCL-2 delivery. It confirms the involvement of mitochondria in the proapoptotic activity of NaBt. The activation of mitochondrial pathway was substantiated by up-regulated expression of BAK with concomitant reduction of antiapoptotic BCL-xL, XIAP and survivin proteins. These findings suggest that NaBt could represent a good candidate for the new therapeutic strategy aimed to improve chemo- and immunotherapy of colon cancer.

Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways

Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T(202/)Y(204)P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y(705)P-STAT3) and MEK (T(202/)Y(204)P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S(473)) and GSK-3β (S(9)) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S(9)P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y(216)P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost.

Bisindolylmaleimides in anti-cancer therapy - more than PKC inhibitors.

Bisindolylmaleimide derivatives were originally described as protein kinase C inhibitors. However, several studies have shown that bisindolylmaleimides target several other signaling molecules. The review presents bisindolylmaleimide-mediated PKC-dependent and PKC-independent biological effects, such as reversal of MDR and modulation of Wnt signaling through GSK-3b and b-catenin. Importantly, the potent proapoptotic properties of bisindolylmaleimides are also described. Bis-IX appears as the most efficient activator of intrinsic apoptotic pathway and additionally, facilitates extrinsic apoptosis. Presented molecular mechanisms indicate that bisindolylmaleimides could be useful agents in anticancer therapy. They repress uncontrolled proliferation and restore the sensitivity to chemotherapy which allows eradication of cancer cells.

Alzheimer's disease genetic mutation evokes ultrastructural alterations: Correlation to an intracellular Aβ deposition and the level of GSK-3β-P(Y216) phosphorylated form

Herein we demonstrate that PC12 cells, which overexpress human wild-type amyloid-beta precursor protein (AbetaPPwt) or AbetaPP bearing double Swedish mutation (AbetaPPsw), reveal phenotype characteristic for Alzheimers disease (AD). The examination of cell ultrastructure showed the presence of peptide aggregates within the cells, activation of endosomal-lysosomal system and extensive exocytosis. Furthermore, the autophagy induction was also characteristic hallmark of amyloid-beta-induced cytotoxicity. Morphological changes were positively correlated with the extent of phosphorylated glycogen synthase kinase-3beta (phospho-Tyr(216)-GSK-3beta, GSK-3beta-P(Y216)). The activity of GSK-3beta is believed to cause tau protein hyper-phosphorylation, increased amyloid-beta production and local plaque-associated microglial-mediated inflammatory responses. All of them are symptomatic for AD. In our studies, the highly significant Y216 phosphorylation and over-expression of total GSK-3beta were observed in AbetaPPsw-transfected PC12 cells. In addition, the immuocytochemical analysis showed co-localization of GSK-3beta-P(Y216) and amyloid-beta deposits. Thus, our data support a functional role of GSK-3beta in AbetaPP processing, further implicating this kinase in the amyloid-beta-dependent pathogenesis.

Rapid Differentiation of Mixed Influenza A/H1N1 Virus Infections with Seasonal and Pandemic Variants by Multitemperature Single-Stranded Conformational Polymorphism Analysis

ABSTRACT Mixed infections of a single host with different variants of influenza A virus are the main source of reassortants which may have unpredictable properties when they establish themselves in the human population. In this report we describe a method for rapid detection of mixed influenza virus infections with the seasonal A/H1N1 human strain and the pandemic A/H1N1/v strain which emerged in 2009 in Mexico and the United States. The influenza virus A/H1N1 variants were characterized by the multitemperature single-stranded conformational polymorphism (MSSCP) method. The MSSCP gel patterns of hemagglutinin gene fragments of pandemic A/H1N1/v and different seasonal A/H1N1 strains were easily distinguishable 2 h after completion of reverse transcription-PCR (RT-PCR). Using the MSSCP-based genotyping approach, coinfections with seasonal and pandemic variants of the A/H1N1 subtype were identified in 4 out of 23 primary samples obtained from patients that presented with influenza-like symptoms to hospitals across Poland during the 2009-2010 epidemic season. Pandemic influenza virus strain presence was confirmed in all these primary samples by real-time RT-PCR. The sensitivity level of the MSSCP-based minor genetic variant detection was 0.1%, as determined on a mixture of DNA fragments obtained from amplification of the hemagglutinin gene of seasonal and pandemic strains. The high sensitivity of the method suggests its applicability for characterization of new viral variants long before they become dominant.

Impaired growth hormone-releasing hormone neurons ultrastructure and peptide accumulation in the arcuate nucleus of mosaic mice with altered copper metabolism.

A progressive decrease in body weight and retarded linear growth observed in mosaic male mice with the mutation linked to X-chromosome (Atp7a(mo-ms)) raised the question whether hypophysiotropic growth axis activity may be affected in these animals. A pathologically developed median eminence ultrastructure with very low somatostatin accumulation as well as an intensive phagocytosis of growth hormone cells observed in the anterior pituitary gland raised the question whether hypothalamic growth hormone-releasing hormone (GHRH) neuronal network is also affected in mosaic mice. In this study an arcuate nucleus GHRH neurons ultrastructure as well as GHRH peptide accumulation in normal and mutant mice were compared. An electron microscopic immunocytochemical method with colloidal-gold labeling was applied to compare the ultrastructural morphology of GHRH neuron and intracellular GHRH peptide distribution. Mosaic mice exhibited a pathologically developed ultrastructure of arcuate nucleus GHRH neurons, defective intracellular peptide localization as well as reduced peptide storage. Obtained results support the crucial role of unaltered copper metabolism in physiological development of hypophysiotropic growth axis activity. Consequently, a pathologically developed GHRH hypothalamic network may impact progressive decrease in body weight and retarded length growth observed in mosaic male mice.

Bisindolylmaleimide IX facilitates extrinsic and initiates intrinsic apoptosis in TNF-α-resistant human colon adenocarcinoma COLO 205 cells

Human COLO 205 colon adenocarcinoma cells are immune to extrinsic apoptosis induced by immunomodulatory cytokines. Among the antiapoptotic mechanisms responsible for the immune escape, the overexpression of the cFLIP protein seems to be critical. cFLIP appears to inhibit the TNF-α-induced death receptor signal. The application of the metabolic inhibitor bisindolylmaleimide IX (Bis-IX), known as a potent PKC repressor, sensitized COLO 205 cells to TNF-α-mediated apoptosis. The Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIPL and TRADD protein levels. At the same time, the level of FADD protein was up-regulated. Additionally, the combined TNF-α and Bis-IX treatment caused cleavages of Bid and procaspase-9, as well as cytochrome c release. Thus, the evidence of this study indicates that Bis-IX facilitates the death receptor signal mediated by TNF-R1. Moreover, Bis-IX alone initiated intrinsic apoptosis, which could be abolished by Bcl-2 delivery. It heralds the involvement of mitochondria in caspase-8-independent intrinsic apoptosis. In turn, the treatment with bisindolylmaleimide III (Bis-III) did not assist TNF-α-dependent apoptosis.

Chapter 4: Regulation of Clusterin activity by calcium.

In this chapter, the attention is put on Ca(2+) effect on Clusterin (CLU) activity. We showed that two CLU forms (secreted and nuclear) are differently regulated by Ca(2+) and that Ca(2+) fluxes affect CLU gene expression. A secretory form (sCLU) protects cell viability whereas nuclear form (nCLU) is proapoptotic. Based on available data we suggest, that different CLU forms play opposite roles, depending on intracellular Ca(2+) concentration, time-course of Ca(2+) current, intracellular Ca(2+) compartmentalization, and final Ca(2+) targets. Discussion will be motivated on how CLU acts on cell in response to Ca(2+) waves. The impact of Ca(2+) on CLU gene activity and transcription, posttranscriptional modifications, translation of CLU mRNA, and posttranslational changes as well as biological effects of CLU will be discussed. We will also examine how Ca(2+) signal and Ca(2+)-dependent proteins are attributable to changes in CLU characteristics. Some elucidation of CLU gene activity, CLU protein formation, maturation, secretion, and intracellular translocations in response to Ca(2+) is presented. In response to cell stress (i.e., DNA damage) CLU gene is activated. We assume that commonly upregulated mRNA for nCLU versus sCLU and vice versa are dependent on Ca(2+) accessibility and its intracellular distribution. It looks as if at low intracellular Ca(2+) the delay in cell cycle allows more time for DNA repair; otherwise, cells undergo nCLU-dependent apoptosis. If cells are about to survive, intrinsic apoptosis is abrogated by sCLU interacting with activated Bax. In conclusion, a narrow range of intracellular Ca(2+) concentrations is responsible for the decision whether nCLU is mobilized (apoptosis) or sCLU is appointed to improve survival. Since the discovery of CLU, a huge research progress has been done. Nonetheless we feel that much work is left ahead before remaining uncertainties related to Ca(2+) signal and the respective roles of CLU proteins are unraveled.

Abundance of some skeletal muscle mitochondrial proteins is associated with increased blood serum insulin in bovine fetuses

The aim of this study was to investigate the evolution of the abundance of cytochrome oxidase c subunit IV (NCOIV) and beta subunit of ATP synthase (β-ATP) during the last third of gestation in bovine skeletal muscles. Semitendinosus, longissimus thoracis and rectus abdominis muscles were chosen for the immunoblotting of the respective protein levels. Muscle and blood samples from bovine fetuses of randomly selected breeds were collected at 180, 210, and 260 days post-conception (dpc). The muscle tissue expressions of NCOIV, β-ATP were compared to blood glucose and insulin. At 260 dpc, protein levels of NCOIV raised in skeletal muscles. Additionally, β-ATP in semitendinosus and longissimus thoracis were elevated and paralleled by higher concentrations of blood serum insulin. It corroborates our previous observations indicating that accelerated metabolic differentiation of bovine skeletal muscles is associated with elevated blood insulin and occurs during the last trimester of gestation. Our observations point to the connection between insulin-sensitivity and the molecular mechanisms of mitochondrial contribution to ontogenesis of skeletal muscles.