Jackisch C, Hahm H A, Tombal B, McCloskey D, Butash K, Davidson N E, Denmeade S R
The Johns Hopkins Oncology Center, Baltimore, Maryland 21231-1001, USA.
Clin Cancer Res. 2000 Jul;6(7):2844-50.
Thapsigargin (TG), a highly specific inhibitor of the sarcoplasmic reticulum and endoplasmic reticulum Ca2+-ATPase pump, can induce apoptosis in a variety of epithelial and lymphoid cell types. In prostate cancer cell lines, TG induces an initial 5- to 10-fold elevation of intracellular calcium ([Ca2+]i) within a few minutes of exposure. With prolonged exposure times (i.e., 12-36 h) a second elevation of [Ca2+]i to >10 microM is observed. In this study, the human breast carcinoma cell lines MCF-7 and MDA MB 468 cells were used to determine the temporal relationship between TG-induced elevation of [Ca2+]i and activation of programmed cell death. Using a microinjection method that allows for long-term analysis of [Ca2+]i changes, we found that after TG exposure, calcium measurements in these cells demonstrated an initial rise (>4-fold) in [Ca2+]i that occurred within minutes and returned to baseline within a few hours. With prolonged TG exposure, the cells underwent a second elevation (>5 microM) of [Ca2+]i occurring stochastically between 12 and 36 h after the initial exposure to TG. Both of the cell lines were growth-inhibited by 100 nM TG after only 1 h of exposure, but clonogenic ability in the MCF-7 cells was significantly reduced only after 48 h of exposure. The induction of apoptosis by TG was demonstrated by morphological changes typical for programmed cell death and DNA fragmentation (both high molecular weight and oligonucleosomal-sized fragments were detected) after 48 h of treatment. TG induction of apoptosis in these breast cancer cells occurred subsequent to the secondary rise in [Ca2+]i, which confirmed that this secondary rise in [Ca2+]i is not prostate cancer-specific. The secondary rise in [Ca2+]i to micromolar levels may directly activate the endonucleases responsible for DNA fragmentation that occurs as part of the apoptotic process. These studies indicate that TG is an active agent in vitro against breast cancer cells. Inactive prodrug analogues of TG are currently being developed that can be activated by tissue-specific proteases, and further pursuit of this strategy as a potential treatment for breast cancer is warranted.
毒胡萝卜素（TG）是肌浆网和内质网Ca2 + -ATP酶泵的高度特异性抑制剂，可诱导多种上皮和淋巴细胞类型的细胞凋亡。在前列腺癌细胞系中，TG在暴露后几分钟内可诱导细胞内钙（[Ca2 + ]i）最初升高5至10倍。随着暴露时间延长（即12 - 36小时），可观察到[Ca2 + ]i第二次升高至>10微摩尔。在本研究中，使用人乳腺癌细胞系MCF - 7和MDA MB 468细胞来确定TG诱导的[Ca2 + ]i升高与程序性细胞死亡激活之间的时间关系。使用一种允许对[Ca2 + ]i变化进行长期分析的显微注射方法，我们发现TG暴露后，这些细胞中的钙测量显示[Ca2 + ]i最初在数分钟内升高（>4倍），并在数小时内恢复到基线。随着TG暴露时间延长，细胞在最初暴露于TG后12至36小时之间随机出现[Ca2 + ]i的第二次升高（>5微摩尔）。两种细胞系在仅暴露1小时后就受到100 nM TG的生长抑制，但仅在暴露48小时后MCF - 7细胞的克隆形成能力才显著降低。在处理48小时后，通过程序性细胞死亡典型的形态变化和DNA片段化（检测到高分子量和寡核小体大小的片段）证明了TG诱导凋亡。TG在这些乳腺癌细胞中诱导凋亡发生在[Ca2 + ]i的二次升高之后，这证实了[Ca2 + ]i的这种二次升高并非前列腺癌所特有。[Ca2 + ]i升高至微摩尔水平可能直接激活负责作为凋亡过程一部分发生的DNA片段化的核酸内切酶。这些研究表明TG在体外是一种抗乳腺癌细胞的活性剂。目前正在开发可被组织特异性蛋白酶激活的TG无活性前药类似物，因此进一步探索这一策略作为乳腺癌的潜在治疗方法是有必要的。
