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

Patrícia Heloise Alves Bezerra,Fabíola Attié De Castro,Beatriz Tinoco Franceschi,Maria Regina Torqueti,Suely Vilela Sampaio,Adélia Cristina O Cintra,Isadora Marques Ferreira,Francine Bianchini,Luciana Ambrósio

doi:10.1590/1678-9199-jvatitd-2019-0010

Patrícia Heloise Alves Bezerra, Fabíola Attié De Castro + Show 7 more

Open Access

https://doi.org/10.1590/1678-9199-jvatitd-2019-0010

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Abstract

ABSTRACTBackground: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.

Highlights

Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide
The MCF7, SKBR3 (HER-2-enriched), and MDAMB231 breast cancer cell lines were purchased from Rio de Janeiro Cell Bank (BCRJ, Rio de Janeiro, RJ, Brazil) and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% glutamine, 1% antibiotic/antimycotic solution, and incubated at 37 oC under 5% CO2
The cell lines were treated with bothropstoxin I (BthTX-I) diluted in estrogen-free RPMI 1640 medium supplemented with charcoal stripped fetal bovine serum (CS-FBS) with increasing concentrations of the toxin (12, 25, 51, 102, 204, 409 μg/mL)

Summary

Introduction

Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. The ineffectiveness and high toxicity of chemotherapeutic drugs, and the fact that they are associated with tumor resistance have limited breast cancer therapy and promoted a high demand for novel antitumor agents [4]. This has led to research using animal venoms and toxins, that have already demonstrated promising cytotoxic activity against many tumor types such as breast cancer, colorectal cancer, lung adenocarcinoma, melanoma, promyelocytic leukemia [5], chronic myeloid leukemia (CML) [6,7], and myeloproliferative neoplasm [8], both in vitro and in vivo [5]

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