Taylor Jack, Jaiswal Ritu, Bebawy Mary
Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney, NSW. Australia.
Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney, P.O. Box: 123 Broadway, NSW 2007. Australia.
Curr Cancer Drug Targets. 2017;17(5):486-494. doi: 10.2174/1568009616666161026165736.
Multidrug resistance in cancer (MDR) occurs when tumours become crossresistant to a range of different anticancer agents. One mechanism by which MDR can be acquired is through cell to cell communication pathways. Membrane-derived microparticles (MPs) are emerging as important signaling molecules in this process. MPs are released from most eukaryotic cells and transfer functional proteins and nucleic acids to recipient cells conferring deleterious traits within the cancer cell population including MDR, metastasis, and angiogenesis. MP formation is known to be dependent on calpain, an intracellular cysteine protease which acts to cleave the cytoskeleton underlying the plasma membrane, resulting in cellular surface blebbing Objective: To establish the role of calpain in vesiculation in malignant and non-malignant cells by 1) comparing membrane vesiculation at rest and following the release of intracellular calcium, and 2) comparing vesiculation in the presence and absence of calpain inhibitor II (ALLM).
This study examines the differences in vesiculation between malignant and non-malignant cells using high-resolution Atomic Force Microscopy (AFM). HBEC, MBE-F, MCF-7, and MCF- 7/Dx cells were analysed at rest and following treatment with calcium ionophore A23187 for 18 hours. Vesiculation of calcium activated and resting malignant and non-malignant cells was also assessed after 18 hour treatment of calpain inhibitor II (ALLM).
We demonstrate that malignant MCF-7 and MCF-7/Dx cells have an intrinsically higher degree of vesiculation at rest when compared to non-malignant human brain endothelial cells (HBEC) and human mammary epithelial cells (MBE-F). Cellular activation with the calcium ionophore A23187 resulted in an increase in vesiculation in all cell types. We show that calpain-mediated MP biogenesis is the dominant pathway at rest in malignant cells as vesiculation was shown to be inhibited with calpain inhibitor II (ALLM).
These results suggest that differences in the biogenic pathways exist in malignant and non-malignant cells and have important implications in defining novel strategies to selectively target malignant cells for the circumvention of deleterious traits acquired through intercellular exchange of extracellular vesicles.
癌症中的多药耐药（MDR）发生于肿瘤细胞对一系列不同抗癌药物产生交叉耐药时。获得性MDR的一种机制是通过细胞间通讯途径。膜衍生微粒（MPs）正成为这一过程中的重要信号分子。MPs从大多数真核细胞释放，并将功能性蛋白质和核酸转移至受体细胞，赋予癌细胞群体有害特性，包括MDR、转移和血管生成。已知MP形成依赖于钙蛋白酶，这是一种细胞内半胱氨酸蛋白酶，作用是切割质膜下方的细胞骨架，导致细胞表面形成泡状突起。目的：通过1）比较静息状态下及细胞内钙释放后的膜泡形成情况，以及2）比较存在和不存在钙蛋白酶抑制剂II（ALLM）时的膜泡形成情况，来确定钙蛋白酶在恶性和非恶性细胞的囊泡化过程中的作用。
本研究使用高分辨率原子力显微镜（AFM）检查恶性和非恶性细胞之间膜泡形成的差异。对静息状态下以及用钙离子载体A23187处理18小时后的人支气管上皮细胞（HBEC）、永生化人乳腺上皮细胞（MBE-F）、人乳腺癌细胞系MCF-7和MCF-7/Dx细胞进行分析。在用钙蛋白酶抑制剂II（ALLM）处理18小时后，还评估了钙激活和静息状态下的恶性和非恶性细胞的膜泡形成情况。
我们证明，与非恶性的人脑血管内皮细胞（HBEC）和人乳腺上皮细胞（MBE-F）相比，恶性的MCF-7和MCF-7/Dx细胞在静息状态下具有更高程度的膜泡形成。用钙离子载体A23187激活细胞会导致所有细胞类型的膜泡形成增加。我们表明，在恶性细胞中，钙蛋白酶介导的MP生物合成是静息状态下的主要途径，因为钙蛋白酶抑制剂II（ALLM）可抑制膜泡形成。
这些结果表明，恶性和非恶性细胞的生物合成途径存在差异，这对于确定选择性靶向恶性细胞以规避通过细胞外囊泡的细胞间交换获得的有害特性的新策略具有重要意义。
