海洋海绵毒素 agelasine B 增加细胞内 Ca(2+) 浓度并诱导人乳腺癌细胞 (MCF-7) 凋亡。

The marine sponge toxin agelasine B increases the intracellular Ca(2+) concentration and induces apoptosis in human breast cancer cells (MCF-7).

机构信息

Instituto de Estudios Avanzados (IDEA), Carretera Nacional Hoyo de la Puerta, Sartenejas, Baruta, Caracas, Venezuela.

出版信息

Cancer Chemother Pharmacol. 2012 Jan;69(1):71-83. doi: 10.1007/s00280-011-1677-x. Epub 2011 May 21.

DOI:10.1007/s00280-011-1677-x

PMID:21603866

Abstract

PURPOSE

In search for new drugs derived from natural products for the possible treatment of cancer, we studied the action of agelasine B, a compound purified from a marine sponge Agelas clathrodes.

METHODS

Agelasine B was purified from a marine sponge Agelas clathrodes and assayed for cytotoxicity by MTT on two human breast cancer cells (MCF-7 and SKBr3), on a prostate cancer cells (PC-3) and on human fibroblasts. Changes in the intracellular Ca(2+) concentrations were assessed with FURA 2 and by confocal microscopy. Determination of Ca(2+)-ATPase activity was followed by Pi measurements. Changes in the mitochondria electrochemical potential was followed with Rhodamine 123. Apoptosis and DNA fragmentation were determined by TUNEL experiments.

RESULTS

Upon agelasine B treatment, cell viability of both human breast cancer cell lines was one order of magnitude lower as compared with fibroblasts (IC(50) for MCF-7 = 2.99 μM; SKBr3: IC(50) = 3.22 μM vs. fibroblasts: IC(50) = 32.91 μM), while the IC(50) for PC-3 IC(50) = 6.86 μM. Agelasine B induced a large increase in the intracellular Ca(2+) concentration in MCF-7, SKBr3, and PC-3 cells. By the use of confocal microscopy coupled to a perfusion system, we could observe that this toxin releases Ca(2+) from the endoplasmic reticulum (ER). We also demonstrated that agelasine B produces a potent inhibition of the ER Ca(2+)-ATPase (SERCA), and that this compound induced the fragmentation of DNA. Accordingly, agelasine B reduced the expression of the anti-apoptotic protein Bcl-2 and was able to activate caspase 8, without affecting the activity of caspase 7.

CONCLUSIONS

Agelasine B in MCF-7 cells induce the activation of apoptosis in response to a sustained increase in the Ca(2+) after blocking the SERCA activity. The reproduction of the effects of agelasine B on cell viability and on the Ca(2+) obtained on SKBr3 and PC-3 cancer cells strongly suggests the generality of the mechanism of action of this toxin.

摘要

目的

为了寻找新的天然产物衍生药物来治疗癌症，我们研究了从海洋海绵 Agelas clathrodes 中分离出的化合物 agelasine B 的作用。

方法

从海洋海绵 Agelas clathrodes 中分离出 agelasine B，并通过 MTT 测定法在两种人乳腺癌细胞（MCF-7 和 SKBr3）、前列腺癌细胞（PC-3）和人成纤维细胞上测定其细胞毒性。通过 FURA 2 和共聚焦显微镜评估细胞内 Ca(2+)浓度的变化。通过 Pi 测量来测定 Ca(2+)-ATP 酶活性。通过 Rhodamine 123 监测线粒体电化学势的变化。通过 TUNEL 实验测定细胞凋亡和 DNA 片段化。

结果

与成纤维细胞相比，两种人乳腺癌细胞系的细胞活力在 agelasine B 处理后降低了一个数量级（MCF-7 的 IC(50)=2.99μM；SKBr3：IC(50)=3.22μM 与成纤维细胞：IC(50)=32.91μM），而 PC-3 的 IC(50)=6.86μM。Agelasine B 诱导 MCF-7、SKBr3 和 PC-3 细胞内 Ca(2+)浓度大幅增加。通过与灌注系统耦合的共聚焦显微镜，我们可以观察到该毒素从内质网（ER）中释放 Ca(2+)。我们还证明，agelasine B 对 ER Ca(2+)-ATP 酶（SERCA）产生强烈抑制作用，并且该化合物诱导 DNA 片段化。因此，agelasine B 降低了抗凋亡蛋白 Bcl-2 的表达，并能够激活 caspase 8，而不影响 caspase 7 的活性。