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 addition of isotonic phosphate buffer (PBS) to cells that had been exposed to PlTX, after toxin removal and under optimal and sub-optimal 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, that is toxin-dependent and osmolyte-sensitive, priming cells to lytic events taking place in a separate phase, that 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.
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|Data di pubblicazione:||2011|
|Titolo:||Palytoxin induces cell lysis by priming a two-step process in MCF-7 cells|
|Autori:||S. Prandi; G.L. Sala; M. Bellocci; A. Alessandrini; P. Facci; A. Bigiani; G.P. Rossini|
|Digital Object Identifier (DOI):||10.1021/tx2001866|
|Appare nelle tipologie:||Articolo su rivista|
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