Yoshida D, Noha M, Watanabe K, Bergenheim T, Henriksson R, Teramoto A
Department of Neurosurgery, Nippon Medical School, Tokyo, Japan.
J Neurooncol. 2001 Mar;52(1):37-47. doi: 10.1023/a:1010653613588.
Estramustine 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 microM) and cytotoxic (0.5-10 microM) 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-beta tubulin antibody were applied to detect morphological changes of the microtubules. Suppression of cell motility by cytotoxic doses of EMP (0.5-10 microM) group was attributed by the cyto-reductive effect, relating to apoptosis. At 0.01-0.1 microM (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 microM). 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.
磷酸雌莫司汀（EMP）是一种抗微管药物，可使微管解聚并导致胶质瘤细胞凋亡。此前，已在相同的EMP细胞毒性浓度范围内对这两种药理作用进行了研究。本研究的目的是调查这两种现象中哪一种先发生。进行了初步的MTT试验，以区分EMP对BT4C细胞的非细胞毒性（0.005 - 0.1微摩尔）和细胞毒性（0.5 - 10微摩尔）。为了研究凋亡变化，采用了透射电子显微镜（TEM）、DNA梯状条带分析和原位末端标记（TUNEL）法。使用趋化性试验评估细胞运动性。应用扫描电子显微镜和抗β微管蛋白抗体的TEM免疫细胞化学来检测微管的形态变化。细胞毒性剂量的EMP（0.5 - 10微摩尔）组对细胞运动性的抑制归因于细胞还原作用，与凋亡有关。在0.01 - 0.1微摩尔（非细胞毒性剂量）时，EMP不会诱导凋亡。在这些浓度下，TEM和免疫组织化学显示伪足尖端形成了含有异常解聚微管的泡状结构，这一发现在低温或细胞迁移过程中未观察到。细胞趋化性被细胞生长抑制剂量的EMP（0.05和0.1微摩尔）显著抑制。伪足的泡状结构形成可能是细胞生长抑制剂量的EMP在诱导凋亡之前导致微管异常解聚的证据。在胶质瘤细胞中，EMP可能通过引起微管解聚来启动凋亡。细胞生长抑制剂量的EMP对细胞运动性的抑制可能有助于支持其他治疗。
