A Bifunctional Molecule with Lectin and Protease Inhibitor Activities Isolated from Crataeva tapia Bark Significantly Affects Cocultures of Mesenchymal Stem Cells and Glioblastoma Cells.

Currently available drugs for treatment of glioblastoma, the most aggressive brain tumor, remain inefficient, thus a plethora of natural compounds have already been shown to have antimalignant effects. However, these have not been tested for their impact on tumor cells in their microenvironment-simulated cell models, e.g., mesenchymal stem cells in coculture with glioblastoma cell U87 (GB). Mesenchymal stem cells (MSC) chemotactically infiltrate the glioblastoma microenvironment. Our previous studies have shown that bone-marrow derived MSCs impair U87 growth and invasion via paracrine and cell-cell contact-mediated cross-talk. Here, we report on a plant-derived protein, obtained from Crataeva tapia tree Bark Lectin (CrataBL), having protease inhibitory/lectin activities, and demonstrate its effects on glioblastoma cells U87 alone and their cocultures with MSCs. CrataBL inhibited U87 cell invasion and adhesion. Using a simplified model of the stromal microenvironment, i.e., GB/MSC direct cocultures, we demonstrated that CrataBL, when added in increased concentrations, caused cell cycle arrest and decreased cocultured cells' viability and proliferation, but not invasion. The cocultured cells' phenotypes were affected by CrataBL via a variety of secreted immunomodulatory cytokines, i.e., G-CSF, GM-CSF, IL-6, IL-8, and VEGF. We hypothesize that CrataBL plays a role by boosting the modulatory effects of MSCs on these glioblastoma cell lines and thus the effects of this and other natural lectins and/or inhibitors would certainly be different in the tumor microenvironment compared to tumor cells alone. We have provided clear evidence that it makes much more sense testing these potential therapeutic adjuvants in cocultures, mimicking heterogeneous tumor-stroma interactions with cancer cells in vivo. As such, CrataBL is suggested as a new candidate to approach adjuvant treatment of this deadly tumor.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Zoanthid mucus as new source of useful biologically active proteins.

Palythoa caribaeorum is a very common colonial zoanthid in the coastal reefs of Brazil. It is known for its massive production of mucus, which is traditionally used in folk medicine by fishermen in northeastern Brazil. This study identified biologically active compounds in P. caribaerum mucus. Crude mucus was collected during low tides by the manual scraping of colonies; samples were maintained in an ice bath, homogenized, and centrifuged at 16,000 g for 1 h at 4 °C; the supernatant (mucus) was kept at -80 °C until use. The enzymatic (proteolytic and phospholipase A2), inhibitory (metallo, cysteine and serine proteases), and hemagglutinating (human erythrocyte) activities were determined. The results showed high levels of cysteine and metallo proteases, intermediate levels of phosholipase A2, low levels of trypsin, and no elastase and chymotrypsin like activities. The mucus showed potent inhibitory activity on snake venom metalloproteases and cysteine proteinase papain. In addition, it showed agglutinating activity towards O+, B+, and A+ erythrocyte types. The hemostatic results showed that the mucus prolongs the aPTT and PT, and strongly inhibited platelet aggregation induced by arachidonic acid, collagen, epinephrine, ADP, and thrombin. The antimicrobial activity was tested on 15 strains of bacteria and fungi through the radial diffusion assay in agar, and no activity was observed. Compounds in P. caribaeorum mucus were analyzed for the first time in this study, and our results show potential pharmacological activities in these compounds, which are relevant for use in physiopathological investigations. However, the demonstration of these activities indicates caution in the use of crude mucus in folk medicine. Furthermore, the present or absent activities identified in this mucus suggest that the studied P. caribaeorum colonies were in thermal stress conditions at the time of sample collection; these conditions may precede the bleaching process in zoanthids. Hence, the use of mucus as an indicator of this process should be evaluated in the future.

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells.

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-ß, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 ß-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.