Cytotoxic Activity of Six Samples of Brazilian Propolis on Sea Urchin (Lytechinus variegatus) Eggs.

The cytotoxic activities of extracts of four samples of propolis from the state of Minas Gerais (Southeast Brazil) and two from the state of Paraná (South Brazil) were evaluated using sea urchin (Lytechinus variegatus) eggs. Cytotoxic activity was observed, characterized mainly by the inhibition of the first cleavage of newly fertilized eggs. Methanol extracts at 32 µg mL(-1) of all samples were highly active (97-100%). Extracts were also prepared by successive treatments of the samples with hexane, chloroform, ethyl acetate, and methanol. High activity was observed using the ethyl acetate fractions of all samples, but hexane and chloroform fractions of some samples also had high activity. Based on the chemical composition of the extracts and fractions (published previously), it is hypothesized that the cytotoxic activities observed are due mainly to artepillin C, p-coumaric acid, and kaempferide. The results suggest that caffeoylquinic acids have no cytotoxic activity in sea urchin eggs.

Tumor immune escape mechanisms that operate during metastasis.

Immune cells actively influence, among other factors, each step of tumor development determining the chance of a cancer cell to survive in a threaten microenvironment. Antitumor immune-mediated mechanisms are activated as soon as the first cancer cell is detected and operate both during primary tumor formation and during metastasis. However, when both innate and adaptive immunity becomes impaired, tumor development occurs. In this sense, compelling evidences indicate that tumor cells employ mechanisms that circumvent or thwart the immune response to enhance their own growth. These mechanisms include the secretion of immunosuppressive factors and the induction of distinct regulatory lymphoid or myeloid cells and, as occur with the immune response, they operate both during primary tumor formation and metastasis. Interestingly, cellular and molecular mechanisms of the immune response are important components of the tumor microenvironment and have the ability to promote or suppress tumor progression depending of the context of each cell interaction. In that sense, researchers are focusing their attention in the study of the influence of the tumor microenvironment in tumor growth and metastasis to better understand cancer biology and to formulate novel therapeutic approach. This review will focus on the present knowledge about interaction between immune cells and tumors in the context of metastasis, discussing the participation of different components of innate and adaptive immune response in the process of metastasis formation and dissemination.

Progestin-induced caveolin-1 expression mediates breast cancer cell proliferation.

Progestin regulation of gene expression was assessed in the progestin-dependent murine tumor line C4HD which requires MPA, a synthetic progestin, for in vivo growth and expresses high levels of progesterone receptor (PR). By using suppressive subtractive hybridization, caveolin-1 was identified as a gene whose expression was increased with in vivo MPA treatment. By Northern and Western blot analysis, we further confirmed that caveolin-1 mRNA and protein expression increased in MPA-treated tumors as compared with untreated tumors. When primary cultures of C4HD cells were treated in vitro with MPA, caveolin-1 levels also increased, effect that was abolished by pre-treatment with progestin antagonist RU486. In addition, MPA promoted strong caveolin-1 promoter transcriptional activation both in mouse and human breast cancer cells. We also showed that MPA regulation of caveolin-1 expression involved in activation of two signaling pathways: MAPK and PI-3K. Short-term MPA treatment of C4HD cells led to tyrosine phosphorylation of caveolin-1 protein, where Src was the kinase involved. Additionally, we showed that MPA-induced association of caveolin-1 and PR, which was detected by coimmunoprecipitation and by confocal microscopy. Finally, we proved that MPA-induced proliferation of C4HD cells was inhibited by suppression of caveolin-1 expression with antisense oligodeoxynucleotides to caveolin-1 mRNA. Furthermore, we observed that inhibition of caveolin-1 expression abrogated PR capacity to induced luciferase activity from a progesterone response element-driven reporter plasmid. Comprehensively, our results demonstrated for the first time that caveolin-1 expression is upregulated by progestin in breast cancer. We also demonstrated that caveolin-1 is a downstream effector of MPA that is partially responsible for the stimulation of growth of breast cancer cells.

Bloqueo del receptor del factor de crecimiento semejante a la insulina tipo I utilizando oligodeoxinucleótidos antisentido en cáncer de mama experimental / Type I insulin-like growth factor receptor antisense strategies in experimental breast cancer

We addressed the effect of targeting type I insulin-like growth factor receptor (IGF-IR), with antisense strategies in in vivo growth of breast cancer cells. We used C4HD tumors from an experimental model of hormonal carcinogenesis in which medroxyprogesterone acetate induced mammary adenocarcinomas in Balb/c mice. Intratumor or systemic administration of phosphorothiolated antisense oligodeoxynucleotides (AS[S]ODN) to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. The antitumor effect was specific since inhibition of tumor growth was dose-dependent and no effect was observed in mice treated with sense S[S]ODN. Tumors from AS[S]ODN-treated mice showed a decrease in IGF-IR expression and in insulin receptor substrate-1 tyrosine phosphorylation. Activation of PI-3K/Akt, p42/p44 MAPK and ErbB-2 was abolished in tumors treated with AS[S]ODN. Progesterone receptor expression or activity remained invariable. This is the first demonstration that breast cancer growth can be inhibited by direct in vivo administration of IGF-IR AS[S]ODN.

Evaluamos el efecto del bloqueo de la expresión del receptor del factor de crecimiento semejante a lainsulina tipo I (IGF-IR) sobre el crecimiento in vivo de cáncer de mama empleando una estrategia“antisentido”. Utilizamos el adenocarcinoma mamario murino progestágeno-dependiente C4HD. La administración intratumoral o sistémica de oligodeoxinucleótidos antisentido fosfotiolados al ARNm del IGF-IR (AS[S]ODN) inhibió el crecimiento tumoral. El efecto antitumoral fue específico debido a su dosis-dependencia y a la faltade efecto en ratones tratados con el S[S]ODN “sentido”. Los tumores obtenidos de ratones tratados con AS[S]ODN mostraron: disminución en la expresión de IGF-IR y en la fosforilación del sustrato del receptor de insulina-1, inhibición de la activación de PI-3K/Akt, p42/p44MAPK y ErbB-2, mientras que la expresión y activación del receptor de progesterona no se afectó. Es la primera demostración que el

Bloqueo del receptor del factor de crecimiento semejante a la insulina tipo I utilizando oligodeoxinucleótidos antisentido en cáncer de mama experimental / Type I insulin-like growth factor receptor antisense strategies in experimental breast cancer

We addressed the effect of targeting type I insulin-like growth factor receptor (IGF-IR), with antisense strategies in in vivo growth of breast cancer cells. We used C4HD tumors from an experimental model of hormonal carcinogenesis in which medroxyprogesterone acetate induced mammary adenocarcinomas in Balb/c mice. Intratumor or systemic administration of phosphorothiolated antisense oligodeoxynucleotides (AS[S]ODN) to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. The antitumor effect was specific since inhibition of tumor growth was dose-dependent and no effect was observed in mice treated with sense S[S]ODN. Tumors from AS[S]ODN-treated mice showed a decrease in IGF-IR expression and in insulin receptor substrate-1 tyrosine phosphorylation. Activation of PI-3K/Akt, p42/p44 MAPK and ErbB-2 was abolished in tumors treated with AS[S]ODN. Progesterone receptor expression or activity remained invariable. This is the first demonstration that breast cancer growth can be inhibited by direct in vivo administration of IGF-IR AS[S]ODN.(AU)

Evaluamos el efecto del bloqueo de la expresión del receptor del factor de crecimiento semejante a lainsulina tipo I (IGF-IR) sobre el crecimiento in vivo de cáncer de mama empleando una estrategia¶antisentido÷. Utilizamos el adenocarcinoma mamario murino progestágeno-dependiente C4HD. La administración intratumoral o sistémica de oligodeoxinucleótidos antisentido fosfotiolados al ARNm del IGF-IR (AS[S]ODN) inhibió el crecimiento tumoral. El efecto antitumoral fue específico debido a su dosis-dependencia y a la faltade efecto en ratones tratados con el S[S]ODN ¶sentido÷. Los tumores obtenidos de ratones tratados con AS[S]ODN mostraron: disminución en la expresión de IGF-IR y en la fosforilación del sustrato del receptor de insulina-1, inhibición de la activación de PI-3K/Akt, p42/p44MAPK y ErbB-2, mientras que la expresión y activación del receptor de progesterona no se afectó. Es la primera demostración que el(AU)

Flavonoids of Lavoisiera, Microlicia and Trembleya (Melastomataceae) and their taxonomic meaning.

Leaves of species of three closely related genera of Melastomataceae, Lavoisiera, Microlicia and Trembleya were analyzed for their flavonoid composition. A total of 116 compounds were obtained, comprising 69 flavonol and 47 flavone glycosides. The common occurrence of flavones, including 6-oxygenated derivatives, characterize Lavoisiera (which often yielded methoxylated flavonols and flavones). Flavonols predominate in species of Microlicia. The flavonoid patterns of Trembleya are rather confusing, some species being akin to Lavoisiera, others to Microlicia. UPGMA analysis using the flavonoid aglycones as characters and the samples analyzed as OTUs gives no complete resolution for the three genera, but provides clusters combining exclusively or preferentially species of either Lavoisiera and Microlicia. Species of Trembleya emerge from the phenogram interspersed among species of the other genera. The data suggest the recognition of Lavoisiera and Microlicia and the lumping of species of Trembleya with either of these genera. If greater flavone diversity is viewed as indicative of further evolutionary advancement, shrubby habits in Microliceae (Lavoisiera and Trembleya) should be regarded as derived from herbaceous ones (Microlicia).

Mechanisms of cell cycle arrest in response to TGF-beta in progestin-dependent and -independent growth of mammary tumors.

TGF-beta1 modulation of cell cycle components was assessed in an experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary tumors in Balb/c mice. TGF-beta1 inhibited both MPA-induced proliferation of progestin-dependent C4HD epithelial cells and proliferation of the progestin-independent variant cell type C4HI, arresting cells in G(1) phase of the cell cycle. Progestin-independent 60 epithelial cells evidenced reduced response to TGF-beta1 antiproliferative effects. TGF-beta1 inhibition of cyclins D1 and A expression and up-regulation of p21(CIP1) levels were the common findings in all three cell types. In addition, a significant content reduction of cyclin D1/cdk4 and cyclin A/cdk2 complexes was found after TGF-beta1 inhibition of MPA-dependent and -independent proliferation. TGF-beta1 inhibited cyclin D2 expression and up-regulated p27(KIP1) levels only when acting as inhibitor of MPA-induced proliferation of C4HD cells. Regulation of these two cell cycle components resulted in decreased cyclin D2/cdk2 complex and in increased p27(KIP1) association with cdk2 in C4HD cells treated with TGF-beta1. These two molecular mechanisms, unobserved in progestin-independent growth of C4HI or 60 cells, were associated with a significantly higher degree of inhibition of cdk2 kinase activity in C4HD cells compared to that found in TGF-beta-treated C4HI or 60 cells. Reduced sensitivity of 60 cells to the growth-inhibitory effects of TGF-beta1 correlated with significantly lower levels of p15(INK4B), p21(CIP1), and p27(KIP1) expressed in these cells, compared to the levels present in C4HD or C4HI cells, and correlated as well with lack of expression of p16(INK4). Thus, common targets were found to exist in TGF-beta1 inhibitory action on breast cancer cells, but regulation of specific targets was found when TGF-beta1-inhibited proliferation driven by the progesterone receptor.

Activation of ErbB-2 via a hierarchical interaction between ErbB-2 and type I insulin-like growth factor receptor in mammary tumor cells.

The present study focused on interactions between signaling pathways activated by progestins and by type I and II receptor tyrosine kinases (RTKs) in mammary tumors. An experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice was used. MPA-stimulated proliferation, both in vivo and in vitro, of progestin-dependent tumors induced up-regulation of ErbB-2 protein levels and tyrosine phosphorylation of this receptor. Combinations of antisense oligodeoxynucleotides (ASODNs) directed to ErbB-2 mRNA with ASODNs directed to the insulin-like growth factor-I receptor (IGF-IR) were used to study the effect of the simultaneous block of these receptors on the MPA-induced proliferation of epithelial cells from the progestin-dependent C4HD line. Neither synergistic nor additive effects on the inhibition of MPA-induced proliferation of C4HD cells were observed as a result of the combination of these ASODNs. Suppression of IGF-IR expression by ASODNs resulted in complete abrogation of MPA-induced phosphorylation of ErbB-2 in C4HD cells, whereas blockage of ErbB-2 did not affect IGF-IR phosphorylation. These results show the existence of a hierarchical interaction between IGF-IR and ErbB-2, by means of which IGF-IR directs ErbB-2 phosphorylation. We demonstrated, for the first time, that this hierarchical interaction involves physical association of both receptors, resulting in the formation of a heteromeric complex. Furthermore, confocal laser microscopy experiments demonstrated that MPA was able to induce co-localization of ErbB-2 and IGF-IR. This hetero-oligomer was also found in MCF-7 human breast cancer cells in which association of IGF-IR and ErbB-2 was induced by heregulin and IGF-I. Oncogene (2001) 20, 34 - 47.

Foliar flavonoids of Annonaceae from Brazil: taxonomic significance.

Foliar flavonoids of 31 species of the Annonaceae native to Brazil, amounting to 76 compounds, were isolated and identified. All phenols found were glycosides of either flavones (apigenin, scutellarein, hispidulin and luteolin) or flavonols (kaempferol, rhamnocitrin, 6-hydroxyrhamnocitrin, quercetin, isorhamnetin and rhamnetin), with the latter predominating. Some members of the tribe Bocageeae are distinctive for accumulating 6-oxygenated flavones and flavonols, in addition to 7-O-methylated flavonols, a feature possibly linked to the assumed advanced condition of the tribe within the family. Members of Duguetia stand out for the apparent absence of quercetin glycosides. Anaxagorea dolichocharpa seemingly lacks flavones and flavonols entirely. A UPGMA analysis based on the distribution of flavonoids does not group the analyzed species according to the available tribal division of the Annonaceae. However, several taxonomically meaningful groupings emerged through the multivariate analysis.

Differential expression of and responsiveness to transforming growth factor-beta (TGF-beta) isoforms in hormone-dependent and independent lines of mouse mammary tumors.

Transforming growth factor-beta2 (TGF-beta2) and -beta3 mRNA expressions were studied in ductal hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. MPA treatment of HD tumors induced a significant decrease in TGF-beta2 and -beta3 mRNA levels. Progression to an HI phenotype of ductal tumors was associated with reduced TGF-beta2 and -beta3 expressions, as compared with their HD counterparts. Exogenously added TGF-beta1, -beta2, and -beta3 (1 ng/ml) inhibited the proliferation of primary cultures of epithelial cells from ductal HD and HI tumors. In addition, TGF-beta expression and effects were studied in the other type of MPA-induced mammary tumors, which are of lobular origin and lack steroid hormone receptors and evidence an HI behavior. These lobular HI lines showed TGF-beta2 levels similar to those found in HD lines growing in MPA-treated mice. In contrast, TGF-beta3 mRNA levels were 12- to 20-fold higher than in HD tumors. Primary cultures of lobular HI epithelial cells required either TGF-beta concentrations of 10 ng/ml to show an inhibitory response, or were completely resistant to TGF-beta inhibition. Studies of the molecular mechanisms involved in reduction or loss of TGF-beta responsiveness in lobular HI tumors showed that cell surface type II TGF-beta receptor levels were lower in these tumors than those present in HD tumors. Our results support the hypothesis that TGF-beta could play a role as an autocrine growth inhibitor in HD and HI ductal tumors. Autonomous growth of lobular HI tumors could be favored by undetectable or low TGF-beta1 and -beta2 expressions and by reduced or lost sensitivity of epithelial cells to TGF-beta's antiproliferative effects. However, the extremely high levels of TGF-beta3 expression in lobular HI tumors, in spite of reduced sensitivity to TGF-beta3 inhibitory growth effect in tumor epithelial cells, suggest a net positive role for TGF-beta3 in these tumors.