RESUMEN
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.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Venenos de Crotálidos/farmacología , Fosfolipasas A2/farmacología , Venenos de Serpiente/enzimología , Secuencia de Aminoácidos , Animales , Bothrops/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Venenos de Crotálidos/química , Venenos de Crotálidos/genética , Daño del ADN/efectos de los fármacos , Femenino , Humanos , Lisina/química , Fosfolipasas A2/química , Fosfolipasas A2/genética , Homología de Secuencia de Aminoácido , Venenos de Serpiente/químicaRESUMEN
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.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Enfermedad de Chagas/etiología , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Actinas/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Línea Celular , Proliferación Celular , Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/parasitología , Citoesqueleto/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Matriz Extracelular , Regulación de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Multimerización de Proteína , Proteínas Protozoarias/farmacología , Receptores CXCR4 , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
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.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Bothrops , Venenos de Crotálidos/enzimología , Metaloproteasas/farmacología , Inhibidores de la Angiogénesis/aislamiento & purificación , Animales , Antineoplásicos/aislamiento & purificación , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Metaloproteasas/aislamiento & purificaciónRESUMEN
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.