BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation

Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. Methods: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. Results: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. Conclusions: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.(AU)

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody - a potential biomarker of Bothrops jararacussu venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.(AU)

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Abstract Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody a potential biomarker of Bothrops jararacussu venom against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.

Study of irradiated bothropstoxin-1 with60Co gamma rays: immune system behavior

Ionizing radiation has been successfully employed to modify the immunological properties of biomolecules. Very promising results were obtained when crude animal venoms, as well as isolated toxins, were treated with 60Co gamma rays, yielding toxoids with good immunogenicity. The achievement of modified antigens with lower toxicity and preserved or improved immunogenicity can be very useful. Ionizing radiation has already been proven to be a powerful tool to attenuate snake venom toxicity without affecting, and even increasing, their immunogenic properties. However, little is known about the modifications that irradiated molecules undergo and even less about the immunological response that such antigens elicit. In the present work, we investigated the immunological behavior of bothropstoxin-1, a K49 phospholipase, before and after irradiation. Structural modifications of the toxin were analyzed by SDS-PAGE. Isogenic mice were immunized with either the native or the irradiated toxin. The circulating antibodies were isotyped and titrated by ELISA. According to our data, irradiation promoted structural modifications in the toxin characterized by higher molecular weight forms of proteins (aggregates and oligomers). The results also indicated that irradiated toxins were immunogenic and antibodies elicited by them were able to recognize the native toxin in ELISA. These findings suggest that irradiation of toxic proteins can promote significant modifications in their structures; however they still retain many of the original antigenic and immunological properties of native proteins. Also, our data indicate that irradiated proteins induce higher titers of IgG2a and IgG2b, suggesting that Th1 cells are predominantly involved in the immune response.