Abstract
To find novel pharmacological tools useful for analyzing the molecular mechanism of apoptosis from natural resources, in the present study, we examined the activity of IC101, a cyclic depsipeptide isolated from Streptomyces sp. MJ202-72F3, to induce apoptosis in the L1210 cell line. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that IC101 caused a concentration-dependent cell death with a 50% effective concentration value of 20 nM. Cell shrinkage, chromatin condensation, a typical DNA ladder pattern, and up-regulation of cleaved caspase-3 expression, which were biochemical characteristics of apoptosis, were induced by IC101. It also was observed that IC101 caused a concentration-dependent dephosphorylation of Akt and Bad without affecting phosphatidylinositol-3 kinase, an upstream molecule of Akt. IC101 dephosphorylated the 90-kDa protein, as assayed by immunblotting of the cell extract by using anti-phosphotyrosine antibody. To identify the 90-kDa protein, immunoprecipitation and direct nano-flow liquid chromatography-tandem mass spectrometry (LC-MS) were performed to demonstrate that this protein was heat shock protein 90 (HSP90). Consistently, it was observed that IC101 induced the HSP90 tyrosine dephosphorylation by immunoblot analysis of immunoprecipitates with anti-HSP90 antibody using anti-phosphotyrosine antibody. IC101 caused the degradation of Raf-1, which formed a complex with HSP90. The HSP90-ATP binding also was inhibited by IC101 in a noncompetitive manner. An interaction of HSP90 with Akt was shown to be inhibited by IC101 in a concentration-dependent manner. These results suggest that IC101 dephosphorylates Akt through an inhibition of HSP90 functions, resulting in the interaction with Akt to induce apoptotic cell death of L1210 cells.
Footnotes
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doi:10.1124/jpet.104.065979.
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ABBREVIATIONS: bp, base pair(s); PI3-kinase, phosphatidylinositol 3-kinase; HSP, heat shock protein; IC101, cyclic depsipeptide isolated from Streptomyces sp. MJ202-72F3; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phophate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMSF, phenylmethylsulfonyl fluoride; TLC, thin layer chromatography; LC-MS, liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry.
- Received February 4, 2004.
- Accepted May 24, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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