Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia , Via Campi 287, I-41125 Modena, Italy.
Chem Res Toxicol. 2011 Aug 15;24(8):1283-96. doi: 10.1021/tx2001866. Epub 2011 Jul 18.
The cytolytic action of palytoxin (PlTX) was recognized long ago, but its features have remained largely undetermined. We used biochemical, morphological, physiological, and physical tools, to study the cytolytic response in MCF-7 cells, as our model system. Cytolysis represented a stereotyped response induced by the addition of isotonic phosphate buffer (PBS) to cells that had been exposed to PlTX, after toxin removal and under optimal and suboptimal experimental conditions. Cytolysis was sensitive to osmolytes present during cell exposure to PlTX but not in the course of the lytic phase. Fluorescence microscopy showed that PlTX caused cell rounding and rearrangement of the actin cytoskeleton. Atomic force microscopy (AFM) was used to monitor PlTX effects in real time, and we found that morphological and mechanical properties of MCF-7 cells did not change during toxin exposure, but increased cell height and decreased stiffness at its surface were observed when PBS was added to PlTX-treated cells. The presence of an osmolyte during PlTX treatment prevented the detection of changes in morphological and mechanical properties caused by PBS addition to toxin-treated cells, as detected by AFM. By patch-clamp technique, we confirmed that PlTX action involved the transformation of the Na(+),K(+)-ATPase into a channel and found that cell membrane capacitance was not changed by PlTX, indicating that the membrane surface area was not greatly affected in our model system. Overall, our findings show that the cytolytic response triggered by PlTX in MCF-7 cells includes a first phase, which is toxin-dependent and osmolyte-sensitive, priming cells to lytic events taking place in a separate phase, which does not require the presence of the toxin and is osmolyte-insensitive but is accompanied by marked reorganization of actin-based cytoskeleton and altered mechanical properties at the cell's surface. A model of the two-step process of PlTX-induced cytolysis is presented.
海兔毒素（PlTX）的细胞溶解作用很早就被认识到了，但它的特性在很大程度上仍未确定。我们使用生化、形态、生理和物理工具，以 MCF-7 细胞作为我们的模型系统，研究细胞溶解反应。在毒素去除后，在最佳和次优的实验条件下，向已经暴露于 PlTX 的细胞中加入等渗磷酸盐缓冲液（PBS），会引发典型的细胞溶解反应。细胞溶解对细胞暴露于 PlTX 期间存在的渗透物敏感，但在溶胞阶段则不敏感。荧光显微镜显示 PlTX 导致细胞圆化和肌动蛋白细胞骨架的重排。原子力显微镜（AFM）用于实时监测 PlTX 的作用，我们发现 MCF-7 细胞的形态和机械性能在毒素暴露期间没有变化，但当向 PlTX 处理过的细胞中加入 PBS 时，观察到细胞高度增加和表面刚度降低。在 PlTX 处理期间存在渗透物可防止通过 AFM 检测到向 PlTX 处理过的细胞中加入 PBS 引起的形态和机械性能变化。通过膜片钳技术，我们证实 PlTX 作用涉及将 Na(+),K(+)-ATP 酶转化为通道，并发现细胞膜电容未被 PlTX 改变，这表明在我们的模型系统中，膜表面积没有受到很大影响。总的来说，我们的研究结果表明，PlTX 在 MCF-7 细胞中引发的细胞溶解反应包括一个第一阶段，该阶段依赖于毒素且对渗透物敏感，使细胞为发生在独立阶段的溶胞事件做好准备，该阶段不需要毒素存在且对渗透物不敏感，但伴随着肌动蛋白细胞骨架的显著重排和细胞表面机械性能的改变。提出了 PlTX 诱导的细胞溶解的两步过程模型。
