The bleb formation of the extracellular pseudopodia; early evidence of microtubule depolymerization by estramustine phosphate in glioma cell; in vitro study

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SummaryEstramustine phosphate (EMP) is an anti-microtubule agent that depolymerizes microtubules and also causes apoptosis of glioma cells. Both of these pharmacological actions have been previously studied within the same cytotoxic range of EMP concentrations. The purpose of this study was to investigate which of these two phenomena occurred before the other. A preliminary MTT assay was done to distinguish non-cytotoxic (0.005-0.1 μM) and cytotoxic (0.5-10 μM) of EMP for BT4C cells. To investigate apoptotic changes, transmission electron microscopy (TEM), DNA laddering, and in situ endo-labeling (TUNEL) method were employed. A chemotaxis assay was used to assess cell motility. Scanning electron microscopy and TEM immunocytochemistry with an anti-β tubulin antibody were applied to detect morphological changes of the microtubules. Suppression of cell motility by cytotoxic doses of EMP (0.5-10 μM) group was attributed by the cyto-reductive effect, relating to apoptosis. At 0.01-0.1 μM (non-cytotoxic doses), EMP did not indue apoptosis. At these concentrations, TEM and immunohistochemistry revealed the formation of blebs on the tip of the pseudopodia that contained abnormally depolymerized microtubules, a finding that was not observed at a low temperature or during cell migration. Cell chemotaxis was significantly inhibited by cytostatic EMP doses (0.05 and 0.1 μM). Bleb formation of the pseudopodia might be evidence of the abnormal disassembly of microtubules by cytostatic EMP concentrations, prior to the induction of apoptosis. In glioma cells EMP probably initiates apoptosis by causing the depolymerization of microtubules. Inhibition of cell motility by cytostatic doses of EMP could be beneficial to support other therapies.

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