The use of gene panels introduces a new dilemma in the genetics field due to the high frequency of variants of uncertain significance (VUS). The objective of this study was to provide evidence that may help in the classification of these germline variants in terms of their clinical impact and association with the disease in question. A total of 52 unrelated women at-risk for HBOC and negative for BRCA1/BRCA2 pathogenic variants were evaluated through a gene panel comprising 14 breast and/or ovarian cancer susceptibility genes. Of the 453 germline variants identified, 15 variants (classes 3, 4, and 5) in the ATM, BRIP1, CHEK2, MRE11A, MUTHY, PALB2, RAD50, and RAD51C genes were evaluated via databases, co-segregation studies and loss of heterozygosity in the tumor. The co-segregation analysis allowed the establishment of an association with the presence of variants and the risk of cancer for variant c.316C>T in the BRIP1 gene. Four variants of uncertain significance showed loss of heterozygosity in the tumor (ATM c.4709T>C; CHEK2 c.1036C>T; PALB2 c.1001A>G, and RAD50 c.281T>C), which is an indication of pathogenicity. Thus, the present study provides novel evidence that favors the association of variants in moderate-risk genes with the development of hereditary breast cancer.
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.
Angiogenesis Inducing Agents/pharmacology , Chondroitin/metabolism , Heparitin Sulfate/metabolism , Neovascularization, Physiologic/drug effects , Plant Lectins/pharmacology , Animals , Capparaceae/metabolism , Cell Movement/drug effects , Chemotactic Factors/pharmacology , Human Umbilical Vein Endothelial Cells , Humans , Male , Mice , Mice, Inbred C57BL , Wound Healing/drug effects
Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin ß1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.
Antineoplastic Agents/pharmacology , Fabaceae/chemistry , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Signal Transduction/drug effects , Trypsin Inhibitors/pharmacology , Antineoplastic Agents/isolation & purification , Cell Adhesion/drug effects , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cortactin/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Integrin beta1/genetics , Integrin beta1/metabolism , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase 14/metabolism , Neoplasm Invasiveness , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , Stomach Neoplasms/pathology , Trypsin Inhibitors/isolation & purification , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism
Supplementary to the efficient inhibition of trypsin, chymotrypsin, plasma kallikrein, and plasmin already described by the EcTI inhibitor from Enterolobium contortisiliquum, it also blocks human neutrophil elastase (K(iapp)=4.3 nM) and prevents phorbol ester (PMA)-stimulated activation of matrix metalloproteinase (MMP)-2 probably via interference with membrane-type 1 (MT1)-MMP. Moreover, plasminogen-induced activation of proMMP-9 and processing of active MMP-2 was also inhibited. Furthermore, the effect of EcTI on the human cancer cell lines HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), K562 and THP-1 (leukemia), as well as on human primary fibroblasts and human mesenchymal stem cells (hMSCs) was studied. EcTI inhibited in a concentration range of 1.0-2.5 µM rather specifically tumor cell viability without targeting primary fibroblasts and hMSCs. Taken together, our data indicate that the polyspecific proteinase inhibitor EcTI prevents proMMP activation and is cytotoxic against tumor cells without affecting normal tissue remodeling fibroblasts or regenerative hMSCs being an important tool in the studies of tumor cell development and dissemination.
Fabaceae/chemistry , Plant Proteins/pharmacology , Protease Inhibitors/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Survival/drug effects , Colorectal Neoplasms/drug therapy , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Fibroblasts/drug effects , Humans , Leukemia/drug therapy , Leukocyte Elastase/antagonists & inhibitors , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mesenchymal Stem Cells/drug effects , Plasminogen/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Trypsin Inhibitors/pharmacology
Seed proteins that inhibit proteinases are classified in families based on amino acid sequence similarity, nature of reactive site and mechanism of action, and are used as tools for investigating proteinases in physiological and pathological events. More recently, the plant Kunitz family of inhibitors with two disulphide bridges was enlarged with members containing variable number of cysteine residues, ranging from no cysteine at all to more than four residues. The characteristic of these proteins, as well the interactions with their target proteinases, are briefly discussed.
Fabaceae/metabolism , Peptides/chemistry , Peptides/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Catalytic Domain
