Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser(428). This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser(15)) and Chk1 (Ser(296)) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo. (c) 2007 Elsevier Inc. All rights reserved.

Substituted titanocenes induce caspase-dependent apoptosis in human epidermoid carcinoma cells in vitro and exhibit antitumour activity in vivo

Titanocene compounds are a novel series of agents that exhibit cytotoxic effects in a variety of human cancer cells in vitro and in vivo. In this study, the antiproliferative activity of two titanocenes (Titanocenes X and Y) was evaluated in human epidermoid cancer cells in vitro. Titanocenes X and Y induce apoptotic cell death in epidermoid cancer cells, with IC50 values that are comparable to cisplatin. Characterisation of the cell death pathway induced by titanocene compounds in A431 cells revealed that apoptosis is preceded by cell cycle arrest and the inhibition of cell proliferation. The induction of apoptosis is dependent on the activation of caspase-3 and -7 but not caspase-8. Furthermore, the antitumour activity of Titanocene Y was tested in an A431 xenograft model of epidermoid cancer. Results indicate that Titanocene Y significantly reduced the growth of A431 xenografts with an antitumour effect similar to cisplatin. These results suggest that titanocenes represent a novel series of promising antitumour agents.

In vivo effects of TGF beta 1 on the the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGF beta) wanes in the milk of lactating rat, an increase in TGF beta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGF beta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGF beta 1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGF beta 1 while T beta RII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p < 0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p < 0.05). Importantly, TGF beta 1 inhibited cell proliferation (p < 0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p < 0.05). Also, TGF beta 1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p < 0.001), and by morphological features at 12 h (p < 0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGF beta 1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TG beta 1 in gastric growth during postnatal development, (C) 2007 Elsevier B.V. All rights reserved.

Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. Methods: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. Results: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. Conclusion: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.

Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis

p53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATWATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. The levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling. (c) 2008 Elsevier B.V. All rights reserved.

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair(NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gamma H2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase II alpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. (C) 2009 Elsevier B.V. All rights reserved.

Arginine vasopressin controls p27(KiP1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated beta-galactosidase (SA-beta Gal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP`s deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187. (C) 2011 Elsevier B.V. All rights reserved.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Indução de morte celular em células de adenocarcinoma cervical humano (HeLa) pela lectina da esponja Cinachyrella apion (CaL)

Cancer is a term used to represent a set of more than 100 diseases, including malignant tumors from different locations. The malignancies are the second leading cause of death in the population, representing approximately 17% of deaths of known cause. Strategies that induce differentiation have had limited success in the treatment of established cancers. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated due to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death via apoptosis in tumor cells. The antiproliferative activity of CaL was tested against cell lines, with the highest inhibition of tumor growth for HeLa, reducing cell growth at a dose dependent manner, with a concentration of 10 μg/mL. The hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. The results showed the lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase, with accumulation of cells of approximately 57% in this phase, and acting as both dependent and/or independent of caspases pathway. These results suggest that CaL has the potential to be used as drug treatment against cancer.

Bioprospecção de extratos metanólicos de macroalgas marinhas do litoral do Rio Grande do Norte: avaliação das atividades anticoagulante, antioxidante e antiproliferativa

Seaweeds are organisms known to exhibit a variety of biomolecules with pharmacological properties. The coast of Rio Grande do Norte has over 100 species of seaweeds, most of them not yet explored for their pharmacological potential. Sugars and phenolic compounds are the most studied of these being assigned a range of biological properties, such as anticoagulant , antiinflammatory, antitumor and antioxidant activities. In this work, we obtained methanolic extracts from thirteen seaweeds of the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; D. mertensis; Sargassum filipendula; Spatoglossum schröederi; Acanthophora specifera; Botryocladia occidentalis; Caulerpa cupresoides; C. racemosa; C. prolifera; C. sertularioides e Codium isthmocladum). They were evaluated as anticoagulant and antioxidant drugs, as well as antiproliferative drugs against the tumor cell line HeLa. None of the methanolic extracts showed anticoagulant activity, but when they were evaluated as antioxidant drugs all of extracts showed antioxidant activity in all tests performed (total antioxidant capacity, sequestration of superoxide and hydroxyl radicals, ferric chelation and reductase activity), especially the algae D. mentrualis, D. cilliolata and C. prolifera, who had the greatest potential to donate electrons.In addition, the ability of iron ions chelation appears as the main antioxidant mechanism of the methanolic extracts of these seaweeds mainly for the extract of the C. racemosa seaweed, which reached almost 100% activity. In the MTT assay, all extracts showed inhibitory activity at different levels againts HeLa cells. Moreover, D. cilliolata (MEDC) and D. menstrualis (MEDM) extracts showed specific activity to this cell line, not inhibiting the viability of 3T3 normal cell line, so they were chosen for detailing the antiproliferative mechanism of action. Using flow cytometry, fluorescence microscopy and in vitro assays we demonstrated that MEDC and MEDM induced apoptosis in HeLa cells by activation of caspases 3 and 9 and yet, MEDC induces cell cycle arrest in S phase. Together, these results showed that the methanolic extracts of brown seaweed D. menstrualis and D. cilliolata may contain agents with potential use in combatting cells from human uterine adenocarcinoma. This study also points to the need for more in-depth research on phytochemical and biological context to enable the purification of biologically active products of these extracts

Cytotoxic constituents of propolis inducing anticancer effects: a review

Objectives Propolis is a honeybee product used extensively in traditional medicine for its antioxidant, anti-inflammatory, immunomodulatory and anticancer effects. Propolis exhibits a broad spectrum of biological activities because it is a complex mixture of natural substances. In this review, the antitumour effects of propolis extracts and its constituents (e. g. flavonoids, terpenes and caffeic acid phenethyl ester) are discussed.Key findings The effect of propolis on experimental carcinogenesis is discussed, as well as its possible mechanisms of action against tumours, involving apoptosis, cell cycle arrest and interference on metabolic pathways. Propolis seems to be efficient against different tumour cells both in vitro and in vivo, which suggests its potential in the development of new anticancer drugs.Summary Propolis extracts may be important economically and would allow a relatively inexpensive cancer treatment. Preclinical investigations are needed to further elucidate the benefits of propolis and its antitumour properties.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Synthesis, characterization, and biological activity of a new palladium(II) complex with deoxyalliin

Synthesis, characterization, and biological activity of a new water-soluble Pd(II)-deoxyalliin (S-allyl-L-cysteine) complex are described in this article. Elemental and thermal analysis for the complex are consistent with the formula [Pd(C6H10NO2S)2]. 13C NMR, 1H NMR, and IR spectroscopy show coordination of the ligand to Pd(II) through S and N atoms in a square planar geometry. Final residue of the thermal treatment was identified as a mixture of PdO and metallic Pd. Antiproliferative assays using aqueous solutions of the complex against HeLa and TM5 tumor cells showed a pronounced activity of the complex even at low concentrations. After incubation for 24 h, the complex induced cytotoxic effect over HeLa cells when used at concentrations higher than 0.40 mmol/L. At lower concentrations, the complex was nontoxic, indicating its action is probably due to cell cycle arrest, rather than cell death. In agreement with these results, the flow cytometric analysis indicated that after incubation for 24 h at low concentrations of the complex cells are arrested in G0/G1. © 2005 NRC Canada.

Antitumor properties of Ganoderma lucidum polysaccharides and terpenoids

Ganoderma lucidum is an edible medicinal mushroom with immunomodulatory and antitumor properties, which are mainly attributed to polysaccharides and triterpenes that can be isolated from mycelia, fruiting bodies and spores. G. lucidum has been us d in a powdered form, as a medicinal beverage and a nutraceutical food (usually dried). In the present review we report some historical facts and the experimental evidence that polysaccharides and triterpenes obtained from this mushroom present potential antitumor activity. Direct effects on tumor cells include induction of apoptosis and interference in the cell cycle, whereas indirect effects are based on the modulation of immune response, usually impaired by cancer cells. Data indicate that G. lucidum can be used as a complementary tool for treatment of cancer patients. © by São Paulo State University.