David Lemus

Survivin expression promotes VEGF-induced tumor angiogenesis via PI3K/Akt enhanced β-catenin/Tcf-Lef dependent transcription

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.

Comparative analysis of the development of the lizard, Liolaemus tenuis tenuis. II. A series of normal postlaying stages in embryonic development

In this work, we have completed a study of the development of the ovoviviparous lizard Liolaemus tenuis tenuis. Ovoviviparity in this lizard is a condition in which eggs are retained within the reproductive duct for about 60 days. During this period the phases of segmentation, gastrulation, neurulation, presomitic, and somitic embryos transpire. During the months of December and January the eggs are laid, and at this time the embryos are comparable to stage 27 Liolaemus gravenhorsti lizard embryos, or to stage 29 Calotes versicolor lizard embryos. Differentiation of the facial region occurs between Days 12 and 42 after egg laying. Limbs develop rapidly between the 8th and 23rd days. By 53 days the appendicular skeleton is completely formed. After 36 days the mesonephros begins to degenerate, and its function is gradually taken over by the developing metanephros. Newborn lizards do not possess an egg caruncle. During the period up to hatching, there is a great increase of liquid within the egg, presumably amniotic fluid. Cracks develop in the leathery shell shortly before hatching and are, perhaps, the first sign of the onset of hatching. Increase of liquid in the egg during postlaying development accounts for its increase in weight and change in shape. Weight of the embryo at hatching does not exceed 32% of the total weight of the egg.

Celecoxib decreases growth and angiogenesis and promotes apoptosis in a tumor cell line resistant to chemotherapy

BackgroundDuring the last few years it has been shown in several laboratories that Celecoxib (Cx), a non-steroidal anti-inflammatory agent (NSAID) normally used for pain and arthritis, mediates antitumor and antiangiogenic effects. However, the effects of this drug on a tumor cell line resistant to chemotherapeutical drugs used in cancer have not been described.Herein we evaluate the angiogenic and antitumor effects of Cx in the development of a drug-resistant mammary adenocarcinoma tumor (TA3-MTXR).ResultsCx reduces angiogenesis in the chick embryonic chorioallantoic membrane assay (CAM), inhibits the growth and microvascular density of the murine TA3-MTXR tumor, reduces microvascular density of tumor metastases, promotes apoptosis and reduces vascular endothelial growth factor (VEGF) production and cell proliferation in the tumor.ConclusionThe antiangiogenic and antitumor Cx effects correlate with its activity on other tumor cell lines, suggesting that Prostaglandins (PGs) and VEGF production are involved. These results open the possibility of using Celecoxib combined with other experimental therapies, ideally aiming to get synergic effects.

Effects of betamethasone, sulindac and quinacrine drugs on the inflammatory neoangiogenesis response induced by polyurethane sponge implanted in mouse

In this study, we showed the effect of the betamethasone, sulindac and quinacrine alone or combined, on the inflammatory angiogenesis promoted by polyurethane sponge on mice. The main finding reported here is that the formation of new blood vessels was strongly inhibited by low concentration of betamethasone, sulindac or quinacrine, whether alone or in combination. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear glucocorticoid receptor (GR) mediated mechanism. This mechanism may occur in endothelial cells as well. Considering that activity of cyclo-oxigenases 1 and 2 is inhibited by sulindac, and that these enzymes are located in the stromal tissue, we propose that the anti-angiogenic effect of these agents may occur via inhibition of both COX isoforms. On the other hand, quinacrine inhibited PLA2 activity, and we propose here that the anti-angiogenic effect occurs via inhibition of the enzyme PLA2. The potentiated effect of the association of betamethasone, sulindac and quinacrine may have some therapeutic benefit in the control of pathological angiogenesis. Further studies are required to validate these propositions.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

Effects of Steroidal and Non Steroidal Drugs on the Neovascularization Response Induced by Tumoral TA3 Supernatant on CAM from Chick Embryo

Angiogenesis, the development of new blood vessels from the existing vascular network, may result as a consequence of the increase or decrease of proangiogenic or antiangiogenic factors, respectively. The tumor itself could up-regulate the production of angiogenic factors. Recently, we established that the steroidal drug betamethasone in low concentration inhibit the neovascularization promoted by TA3 Ts on CAM of chick embryos. We describe here the effects of the non-steroidal drug ketoprofen, alone or in association with betamethasone, on the angiogenesis promoted by TA3 Ts on CAM. The main finding reported here is that the formation of new blood vessels is strongly inhibited by low concentrations of ketoprofen. The association of both drugs produced a synergistic effect, significantly decreasing tumoral supernatant angiogenesis. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear GR mediated mechanism. This may operate as a general mechanism in endothelial cells as well. Considering that the induction of COX 1 and COX2 are inhibited by ketoprofen, and that these enzymes are located in the stromal compartment of the CAM, we propose that its antiangiogenic effect may occur via inhibition of the two COX isoforms. In fact, we found that ketoprofen induced apoptosis in both the stromal fibroblast and endothelial cells. The potentiated effect of the combination of betamethasone and ketoprofen may have some therapeutic projections in the control of pathological angiogenesis.

Histochemical detection of sugar residues in lizard teeth (Liolaemus gravenhorsti): a lectin-binding study

The structural diversity of the many oligosaccharide chains of surface glycoconjugates renders them likely candidates for modulators of cell-interactions, cellular movements, differentiation, and cellular recognition. A selection of different lectins was used to investigate the appearance of cellular distribution and changes in sugar residues during tooth development in the polyphyodont lizard, Liolaemus gravenhorsti. Lectins from three groups were used: (1) N-acetylgalactosamine specificity: BS-1, PNA, RCA-120; (2) N-acetylglucosamine specificity: ECA; and (3) fucose specificity: UEA 1 and LTA.. Digital images were processed using Scion Image. Grayscale graphics in each image were obtained. The lectins used showed a strong, wide distribution of the L-fucose and N-acetylgalactosamine at the cell surface and in the cytoplasm of multinucleate odontoclast cell, while mononuclear odontoclast cells showed no binding, suggesting some roles that the residues sugar might play in the resorption of dentine or with multinucleation of odontoclast after the attachment to the dentine surface in this polyphyodont species. Further studies must be planned to determine the specific identities of these glycoconjugates,and to elucidate the roles played by these sugar residues in the complex processes related to odontogenesis in polyphyodont species.

Human Survivin and Trypanosoma cruzi Calreticulin Act in Synergy against a Murine Melanoma In Vivo

Immune-based anti-tumor or anti-angiogenic therapies hold considerable promise for the treatment of cancer. The first approach seeks to activate tumor antigen-specific T lymphocytes while, the second, delays tumor growth by interfering with blood supply. Tumor Associated Antigens are often employed to target tumors with therapeutic drugs, but some are also essential for tumor viability. Survivin (Surv) is a member of the inhibitor of apoptosis protein family that is considered a Tumor Associated Antigen important for cancer cell viability and proliferation. On the other hand, Trypanosoma cruzi (the agent of Chagas’ disease) calreticulin (TcCRT) displays remarkable anti-angiogenic properties. Because these molecules are associated with different tumor targets, we reasoned that immunization with a Surv-encoding plasmid (pSurv) and concomitant TcCRT administration should generate a stronger anti-tumor response than application of either treatment separately. To evaluate this possibility, C57BL/6 mice were immunized with pSurv and challenged with an isogenic melanoma cell line that had been pre-incubated with recombinant TcCRT (rTcCRT). Following tumor cell inoculation, mice were injected with additional doses of rTcCRT. For the combined regimen we observed in mice that: i). Tumor growth was impaired, ii). Humoral anti-rTcCRT immunity was induced and, iii). In vitro rTcCRT bound to melanocytes, thereby promoting the incorporation of human C1q and subsequent macrophage phagocytosis of tumor cells. These observations are interpreted to reflect the consequence of the following sequence of events: rTcCRT anti-angiogenic activity leads to stress in tumor cells. Murine CRT is then translocated to the external membrane where, together with rTcCRT, complement C1 is captured, thus promoting tumor phagocytosis. Presentation of the Tumor Associated Antigen Surv induces the adaptive anti-tumor immunity and, independently, mediates anti-endothelial cell immunity leading to an important delay in tumor growth.

Effect of sulfated β-cyclodextrin, a water soluble cycloamylose, on the promotion and/or inhibition of angiogenesis

Previous studies have reported that sulfated β-cyclodextrin, a naturally occurring cycloamylose built up from six to eight glucopyranose units, when administered alone promotes angiogenesis, but administered with an angiostatic steroid inhibits angiogenesis in the cick embryo bioassay. In our experiments sulfated β-cyclodextrin has been shown to posses many properties unrelated to its classical functions in the promotion and inhibition of angiogenesis that were not previously described. We studied the angiogenic and angiostatic properties of β-cyclodextrin in a subcutaneosus plastic sponge model in mice. We realized two set of experiments. In each set mice were randomized into five groups (n= 5 mice). The first group was treated with sulfated β-cyclodextrin (200 ng), the second group was treated with sulfated β-cyclodextrin (2000 ng), the third group received unsubstituted β-cyclodextrin (2000 ng), the fourth group was treated with sulfated β-cyclodextrin (20 000 ng) and the last group was used as a control group. In all groups compounds were administered intraperitonally 4 days after subcutaneous implantation of a sterile polyvinyl sponge on day 0, controls were not treated. Cyclodextrin administered alone at low drug concentration (200 ng) promoted angiogenesis and increased the development of venules in the sponge matrix. However, cyclodextrin administered at high drug concentration (2000 and 20 000 ng) reduced the vessel index in the sponge and areas of microhemorraghes were observed. From our results we propose that β-cyclodextrin contains both a promoter and an inhibitor of angiogenesis and that the activation of both is drug concentration dependent.

Antiangiogenic effect of betamethasone on the chick cam stimulated by TA3 tumor supernatant

Tumor growth is the result of combined cell proliferation overwhelming cell death and neoangiogenesis. This report shows CAM angiogenesis promoted by TA3 tumor supernatant with or without low dosis of betamethasone (Minimal antiangiogenic concentration: beta-MAAC). Methylcellulose discs instilled with 10 microliters of beta-MAAC (0.08 microgram/ml), 10 microliters of tumor supernatant (TA3ts), 5 microliters beta-MAAC + 5 microliters TA3ts, and 10 microliters of PBS as control were implanted in host chick eggs. On day 12, the grafts were removed, photographed and fixed. Sections were stained in parallel, one and three with hematoxylin-eosin, and section two by the Tunel method. The number of vessels was evaluated in a microscopic field of the CAM (2250 micron 2). The results show that beta-MAAC produced a significant inhibition of neovascularization in comparison to that observed in controls (P < 0.0025; Student t-Test). Discs instilled with TA3ts produced an intense stimulation of angiogenesis in contrast, when discs were instilled with 5 microliters of beta-MAAC + 5 microliters of TA3ts the angiogenesis was significantly inhibited (P < 0.001). The results show that effective antiangiogenic doses of betamethasone are in the range of 10(-7) M, (probably a genomic mediated action) and that this effect of low concentration may have clinical applications.