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

ABSTRACT Background: 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.

【摘要】背景：乳腺癌是全球女性中发病率与死亡率均位居首位的肿瘤。鉴于蛇毒对多种肿瘤类型已被证实具有细胞毒性，本研究评估了贾拉库苏矛头蝮（Bothrops jararacussu）来源的BthTX-I对MCF7、SKBR3及MDAMB231三种乳腺癌细胞系的作用。方法：采用MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）比色法检测BthTX-I的细胞毒性；通过低渗荧光溶液法、膜联蛋白V-异硫氰酸荧光素（annexin-V-FITC）/碘化丙啶（propidium iodide）染色法以及凋亡/自噬相关蛋白表达水平检测细胞死亡情况；采用流式细胞术，以抗CD24-FITC抗体、抗CD44-别藻蓝蛋白（APC）抗体联合碘化丙啶对肿瘤干细胞（Cancer Stem Cells, CSCs）进行定量分析。结果：浓度为102 µg/mL的BthTX-I可在所有受试细胞系中诱导细胞死亡。该毒素可通过剂量依赖方式诱导MCF7、SKBR3及MDAMB231细胞发生凋亡，亚二倍体核数量增加的结果证实了这一点。在MCF7细胞中，半胱天冬氨酸蛋白酶3前体（pro-caspase 3）、半胱天冬氨酸蛋白酶8前体（pro-caspase 8）及Beclin-1蛋白的表达水平均有所升高，而抗凋亡蛋白B细胞淋巴瘤-2（Bcl-2）的表达水平则被下调。BthTX-I可通过上调CD24受体的表达改变MDAMB231细胞中肿瘤干细胞的染色模式，进而介导细胞死亡。结论：BthTX-I可在所有受试乳腺癌细胞系中诱导细胞凋亡与自噬，同时可降低肿瘤干细胞亚群比例，使其成为一种颇具潜力的乳腺癌治疗候选方案。