Cytotoxic steroidal saponins from the rhizomes of Tacca integrifolia

Three new steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (1), (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (3), and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (5), as well as the new pregnane glycoside (3beta,16beta)-3-{[6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranosyl]oxy}-20-oxopregn-5-en-16-yl (4R)-5-(beta-D-glucopyranosyloxy)-4-methylpentanoate (6), were isolated from the rhizomes of Tacca integrifolia together with two known (25R) configurated steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (2) and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (4). The cytotoxic activity of the isolated compounds was evaluated in HeLa cells and showed the highest cytotoxicity value for compound 2 with an IC(50) of 1.2+/-0.4 muM. Intriguingly, while compounds 1-5 exhibited similar cytotoxic properties between 1.2+/-0.4 (2) and 4.0+/-0.6 muM (5), only compound 2 showed a significant microtubule-stabilizing activity in vitro.

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

NKp46+ cells express granulysin in multiple cutaneous adverse drug reactions

The spectrum of cutaneous adverse drug reactions (cADRs) ranges from benign presentations to severe life-threatening forms such as toxic epidermal necrolysis (TEN). In TEN, granulysin has been shown to be the key cytotoxic molecule. Still, little is known about the expression of granulysin in other cADRs. As an important source of granulysin, natural killer (NK) cells are of major interest in cADRs. Recently, NKp46 has been identified as the most selective NK-cell marker. However, the role of NKp46(+) cells in cADRs and their contribution to granulysin expression remain to be elucidated.

Cytotoxic effects of polycarbonate-based orthodontic brackets by activation of mitochondrial apoptotic mechanisms

OBJECTIVES: The aim of the study was to evaluate the biological effects of water eluents from polycarbonate based esthetic orthodontic brackets. METHODS: The composite polycarbonate brackets tested were Silkon Plus (SL, fiber-glass-reinforced), Elan ME (EL, ceramic particle-reinforced) and Elegance (EG, fiber-glass-reinforced). An unfilled polyoxymethylene bracket (Brilliant, BR) was used as control. The brackets' composition was analyzed by ATR-FTIR spectrometry. The cytotoxicity and estrogenicity of the eluents obtained after 3months storage of the brackets in water (37°C) were investigated in murine fibroblasts (NIH 3T3), breast (MCF-7) and cervical cancer (CCl-2/Hela) cell lines. RESULTS: SL and EG were based on aromatic-polycarbonate matrix, whereas EL consisted of an aromatic polycarbonate-polyethylene terepthalate copolymer. A significant induction of cell death and a concurrent decrease in cell proliferation was noted in the EG eluent-treated cells. Moreover, EG eluent significantly reduced the levels of the estrogen signaling associated gene pS2, specifically in MCF7 cells, suggesting that cell death induced by this material is associated with downregulation of estrogen signaling pathways. Even though oxidative stress mechanisms were equally activated by all eluents, the EG eluents induced expression of apoptosis inducing factor (AIF) and reduced Bcl-xL protein levels. SIGNIFICANCE: Some polycarbonate-based composite brackets when exposed to water release substances than activate mitochondrial apoptosis.

Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis

Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.

Rapid cytopathic effects of Clostridium perfringens beta-toxin on porcine endothelial cells

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C beta-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

Impaired immune evasion in HIV through intracellular delays and multiple infection of cells

With its high mutation rate, HIV is capable of escape from recognition, suppression and/or killing by CD8(+) cytotoxic T lymphocytes (CTLs). The rate at which escape variants replace each other can give insights into the selective pressure imposed by single CTL clones. We investigate the effects of specific characteristics of the HIV life cycle on the dynamics of immune escape. First, it has been found that cells in HIV-infected patients can carry multiple copies of proviruses. To investigate how this process affects the emergence of immune escape, we develop a mathematical model of HIV dynamics with multiple infections of cells. Increasing the frequency of multiple-infected cells delays the appearance of immune escape variants, slows down the rate at which they replace the wild-type variant and can even prevent escape variants from taking over the quasi-species. Second, we study the effect of the intracellular eclipse phase on the rate of escape and show that escape rates are expected to be slower than previously anticipated. In summary, slow escape rates do not necessarily imply inefficient CTL-mediated killing of HIV-infected cells, but are at least partly a result of the specific characteristics of the viral life cycle.

Synthesis and cytotoxic activity of non-naturally substituted 4-oxycoumarin derivatives

Coumarins are a large family of natural and synthetic compounds exerting different pharmacological effects, including cytotoxic, anti-inflammatory or antimicrobial. In the present communication we report the synthesis of a series of 12 diversely substituted 4-oxycoumarin derivatives including methoxy substituted 4-hydroxycoumarins, methyl, methoxy or unsubstituted 3-aryl-4-hydroxycoumarins and 4-benzyloxycoumarins and their anti-proliferative effects on breast adenocarcinoma cells (MCF-7), human promyelocytic leukemia cells (HL-60), human histiocytic lymphoma cells (U937) and mouse neuroblastoma cells (Neuro2a). The most potent bioactive molecule was the 4-hydroxy-5,7-dimethoxycoumarin (compound 1) which showed similar potency (IC(50) 0.2-2 μM) in all cancer cell lines tested. This non-natural product reveals a simple bioactive scaffold which may be exploited in further studies.

Toxicity of clopidogrel and ticlopidine on human myeloid progenitor cells: importance of metabolites

Ticlopidine and clopidogrel are thienopyridine derivatives used for inhibition of platelet aggregation. Not only hepatotoxicity, but also bone marrow toxicity may limit their use. Aims of the study were to find out whether non-metabolized drug and/or metabolites are responsible for myelotoxicity and whether the inactive clopidogrel metabolite clopidogrel carboxylate contributes to myelotoxicity. We used myeloid progenitor cells isolated from human umbilical cord blood in a colony-forming unit assay to assess cytotoxicity. Degradation of clopidogrel, clopidogrel carboxylate or ticlopidine (studied at 10 and 100 μM) was monitored using LC/MS. Clopidogrel and ticlopidine were both dose-dependently cytotoxic starting at 10 μM. This was not the case for the major clopidogrel metabolite clopidogrel carboxylate. Pre-incubation with recombinant human CYP3A4 not only caused degradation of clopidogrel and ticlopidine, but also increased cytotoxicity. In contrast, clopidogrel carboxylate was not metabolized by recombinant human CYP3A4. Pre-incubation with freshly isolated human granulocytes was not only associated with a myeloperoxidase-dependent degradation of clopidogrel, clopidogrel carboxylate and ticlopidine, but also with dose-dependent cytotoxicity of these compounds starting at 10 μM. In conclusion, both non-metabolized clopidogrel and ticlopidine as well as metabolites of these compounds are toxic towards myeloid progenitor cells. Taking exposure data in humans into account, the myelotoxic element of clopidogrel therapy is likely to be secondary to the formation of metabolites from clopidogrel carboxylate by myeloperoxidase. Concerning ticlopidine, both the parent compound and metabolites formed by myeloperoxidase may be myelotoxic in vivo. The molecular mechanisms of cytotoxicity have to be investigated in further studies.

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Detection of perforin and granzyme B mRNA expressing cells in lichen sclerosus

Granzyme B and perforin messenger RNA (mRNA) expression has been shown to be a specific in vivo activation marker for cytotoxic cells. The aim of this study was to assess the contribution of cell-mediated cytotoxicity in the pathogenesis of lichen sclerosus. In situ hybridization and immunohistochemistry were performed on serial tissue sections of lesional skin biopsies and normal skin as control. Immunohistochemical staining showed that the cellular infiltrate of diseased skin consisted predominantly of T cells (CD3+) and some B cells (CD20+). Among T cells CD4+ and CD8+ cells were found in about equal numbers. In normal skin samples perforin and granzyme B mRNA expressing cells were only rarely found. In contrast, in biopsies from diseased skin a high percentage of infiltrating cells expressed mRNA for perforin and granzyme B. The perforin and granzyme B expressing cells were found in the dermal infiltrate and intraepidermally in close proximity to keratinocytes suggesting in situ activation of these cells. These findings provide evidence that cell-mediated cytotoxicity plays a significant role in tissue destruction in lichen sclerosus.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Cytotoxic diacetylenic spiroketal enol ethers from Plagius flosculosus

Three new diacetylenic spiroketal enol ethers named flosculins A (1), B (2), and C (3), along with five known compounds (4-8) of the same structural class, were isolated from the leaves of Plagius flosculosus. The structures were deduced by extensive 1D and 2D NMR spectroscopy and mass spectrometry. All isolated compounds exhibited significant cytotoxic activity against leukemia cells (Jurkat T and HL-60). Compounds 5-8 induced apoptosis in HL-60 cells with corresponding IC(50) values ranging from 4 to 6 microM.

New cytotoxic saturated and unsaturated cyclohexanones from Anthemis maritima

Two new cyclohexenones (antheminones A and B) and a new cyclohexanone, (antheminone C) along with five known compounds were isolated from the leaves of Anthemis maritima L. The structures were mainly deduced from extensive 1D and 2D NMR spectroscopy and mass spectrometry. The new compounds were tested in vitro for their cytotoxic activity against adherent and non-adherent cancer cell lines. Antheminones A and C exhibited significant antiproliferative activity against leukemia cells with IC(50) values ranging from 3.2 to 14 microM.