ABSTRACT
Herein we evaluated the genotoxic effects of BnSP-6, a Lys-49 phospholipase A2 (PLA2) from Bothrops pauloensis, on breast cancer cells. BnSP-6 was able to induce a higher cytotoxic and genotoxic activity in MDA-MB-231 cells, when compared to MCF10A (a non-tumorigenic breast cell line), suggesting that this protein presented a possible preference for cancer cells. BnSP-6 inhibited MDA-MB-231 proliferation at 24, 48 and 72â¯h. In addition, BnSP-6 induced significant increase in the percentage of TUNEL-positive cells, a marker of DNA damage. To obtain novel insight into the direct DNA damage interference in MDA-MB-231 survival and proliferation, we evaluated cell cycle progression. BnSP-6 produced a significant decrease in 2N (G1) and an increase in the G2/M phase and this capacity is likely related to the modulation of expression of progression cell cycle-associated genes (CCND1, CCNE1, CDC25A, CHEK2, E2F1, CDH-1 and NF-kB). Taken together, these results indicate that BnSP-6 induces DNA damage in breast cancer cells and is an attractive model for developing innovative therapeutic agents against breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Cell Proliferation/drug effects , Crotalid Venoms/pharmacology , Phospholipases A2/pharmacology , Snake Venoms/enzymology , Amino Acid Sequence , Animals , Bothrops/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Crotalid Venoms/chemistry , Crotalid Venoms/genetics , DNA Damage/drug effects , Female , Humans , Lysine/chemistry , Phospholipases A2/chemistry , Phospholipases A2/genetics , Sequence Homology, Amino Acid , Snake Venoms/chemistryABSTRACT
Chronic chagasic cardiomyopathy (CCC) is arguably the most important form of the Chagas Disease, caused by the intracellular protozoan Trypanosoma cruzi; it is estimated that 10-30% of chronic patients develop this clinical manifestation. The most common and severe form of CCC can be related to ventricular abnormalities, such as heart failure, arrhythmias, heart blocks, thromboembolic events and sudden death. Therefore, in this study, we proposed to evaluate the anti-angiogenic activity of a recombinant protein from T. cruzi named P21 (rP21) and the potential impact of the native protein on CCC. Our data suggest that the anti-angiogenic activity of rP21 depends on the protein's direct interaction with the CXCR4 receptor. This capacity is likely related to the modulation of the expression of actin and angiogenesis-associated genes. Thus, our results indicate that T. cruzi P21 is an attractive target for the development of innovative therapeutic agents against CCC.
Subject(s)
Angiogenesis Inhibitors/metabolism , Chagas Disease/etiology , Protozoan Proteins/metabolism , Trypanosoma cruzi/metabolism , Actins/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Cell Line , Cell Proliferation , Chagas Disease/metabolism , Chagas Disease/parasitology , Cytoskeleton/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Extracellular Matrix , Gene Expression Regulation , Humans , Mice , Models, Biological , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Protein Multimerization , Protozoan Proteins/pharmacology , Receptors, CXCR4 , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacologyABSTRACT
Breast cancer is a highly malignant carcinoma and remains the second leading cause of mortality among women. The antitumor effects of metalloproteinases and disintegrins from snake venom on various types of cancer cells have been investigated. In this study, we evaluated the antitumor and antiangiogenic effects on MDA-MB-231 human breast cancer cells and endothelial cells induced by Bothropoidin, a disintegrin-like metalloproteinase isolated from Bothrops pauloensis snake venom. At 24h after treatment at 100µg/mL, Bothropoidin exerted a moderate cytotoxic effect of 30% on MDA-MB-231 versus 10% cytotoxicity against MCF10A (a non-tumorigenic breast cell line), a significant difference that suggests a possible preference by this protein for targets in cancer cells. Early and late apoptosis of MDA-MB-231 was observed after Bothropoidin treatment (10µg/mL and 40µg/mL). Furthermore, this toxin inhibited not only the adhesion of MDA-MB-231 cells in a dose-dependent manner but also cell migration by approximately 45%. In addition, Bothropoidin decreased endothelial cells viability and adhesion in Matrigel and inhibited in vitro angiogenesis in Matrigel stimulated by bFGF, showing significantly fewer formed vessels. The results demonstrated that Bothropoidin has potent in vitro antitumor and antiangiogenic effect and represents a biotechnological tool for elucidating the antitumor effect of disintegrins-like metalloproteinases in cancer cells.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Bothrops , Crotalid Venoms/enzymology , Metalloproteases/pharmacology , Angiogenesis Inhibitors/isolation & purification , Animals , Antineoplastic Agents/isolation & purification , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Humans , Metalloproteases/isolation & purificationABSTRACT
Chronic chagasic cardiomyopathy (CCC) is arguably the most important form of the Chagas Disease, caused by the intracellular protozoan Trypanosoma cruzi; it is estimated that 10-30% of chronic patients develop this clinical manifestation. The most common and severe form of CCC can be related to ventricular abnormalities, such as heart failure, arrhythmias, heart blocks, thromboembolic events and sudden death. Therefore, in this study, we proposed to evaluate the anti-angiogenic activity of a recombinant protein from T. cruzi named P21 (rP21) and the potential impact of the native protein on CCC. Our data suggest that the anti-angiogenic activity of rP21 depends on the protein's direct interaction with the CXCR4 receptor. This capacity is likely related to the modulation of the expression of actin and angiogenesis-associated genes. Thus, our results indicate that T. cruzi P21 is an attractive target for the development of innovative therapeutic agents against CCC.
ABSTRACT
In the present work, we demonstrate some biochemical and functional properties of a new PI snake venom metalloproteinase (SVMP) isolated from Bothrops pauloensis snake venom (BpMP-II), in addition we evaluated its capacity to inhibit endothelial cell adhesion and in vitro angiogenesis. BpMP-II was purified after a combination of three chromatography steps and showed molecular mass of 23,000 Da determined by MALDI-TOF, an isoelectric point of 6.1 and the sequence of some fragments obtained by MS/MS (MALDI TOF\TOF) presented high structural similarity with other PI-SVMPs. BpMP-II showed proteolytic activity against azocasein, was able to degrade bovine fibrinogen and was inhibited by EDTA, 1.10 phenantroline and ß-mercaptoethanol. BpMP-II did not induce local hemorrhage in the dorsal region of mice even at high doses and did not affect plasma creatine kinase (CK) levels when administered intramuscularly into the gastrocnemius muscle of mice. Moreover, this metalloproteinase decreased tEnd cells viability at concentrations higher than 20 µg/mL. With sub-toxic doses this metalloproteinase affected tEnd cell adhesion and was also able to inhibit in vitro angiogenesis. BpMP-II showed very important functional properties suggesting considerable therapeutic potential for this class of protein.
