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CELLULAR AND MOLECULAR
Department of Pharmaceutical Molecular Biology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (H.F., T.Y., Y.O.); Institute for Chemotherapy, Shizuoka, Japan (M.U., M.I.); and Integrated Proteomics System Project, Pioneer Research on Genome the Frontier, MEXT, c/o Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan (T.S.)
Received February 4, 2004; accepted May 24, 2004.
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Abstract |
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). Apoptosis can be induced by various intracellular signals, including growth factor deprivation (Araki et al., 1990) and activation of cytokine receptor (Laster et al., 1988; Nagata and Golstein, 1995). Several compounds have been reported to cause apoptosis by enhancing or suppressing these signals (Muthukkumar et al., 1995; Yao and Cooper, 1995; Stefanis et al., 1999; Fujino et al., 2002; Suk et al., 2003).
Akt, a serine/threonine kinase, is the cellular homolog of the retroviral oncogene product v-Akt and the major mediator of survival signals (Franke et al., 2003; Tsuruo et al., 2003). After stimulation with growth factors and cytokines, phosphatidylinositol-3 kinase (PI3-kinase) is activated and phosphorylates phosphoinositides. The interaction of the generated phosphatidylinositol 3,4,5-trisphosphate with the pleckstrin homology domain of Akt recruits Akt to the plasma membrane, where it is phosphorylated at two key regulatory sites, Thr308 and Ser473 residues. Phosphorylation at both residues is necessary for full activation of Akt and the subsequent control of biological responses, including apoptosis inhibition by phosphorylating the proapoptotic Bcl-2 family member Bad, and the caspase family member caspase-9 (Datta et al., 1999). Because the Akt signaling pathway is associated with metastasis formation of some tumor cells (Nakanishi et al., 2002), this pathway is a promising new target for developing chemotherapeutic drugs to treat tumors.
Recently reported data indicate that heat shock proteins (HSPs) exert pro- or antiapoptotic function. For example, it has been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins (Garrido et al., 2001). HSPs have been classified into several families according to the molecular size. HSP90 is an abundant and highly conserved protein. In contrast to other HSPs, HSP90 is required for maturation or maintenance of most proteins in vivo (Tsuruo et al., 2003). HSP90 associates with a number of signaling proteins such as v-Src (Whitesell et al., 1994), Raf-1 (Schulte et al., 1997), nitric-oxide synthase (Osawa et al., 2003), ErbB2 (Xu et al., 2002), and Akt (Sato et al., 2000). HSP90 acts as a chaperone for unstable signal transducers and keeps them poised for activation until they are stabilized by conformational changes associated with signal transduction. In Akt, a physical association of HSP90 with Akt protects Akt from protein phosphatase 2A-mediated dephosphorylation to prevent Akt inactivation (Sato et al., 2000). Because Akt is the major mediator of survival signals that protects cells from undergoing apoptosis, the HSP90-Akt binding is important for the regulation of apoptosis induction.
Numerous natural products have been used as pharmacological tools for pharmacological, physiological, and biochemical studies (Ohizumi, 1997). In the course of our survey of pharmacological tools from natural sources, much attention has been given to compounds inducing apoptosis because of their important role in studies of signal transduction (Kugawa et al., 1998). Recently, we have found that several natural products such as halenaquinone (Fujiwara et al., 2001), ophiobolin A (Fujiwara et al., 2000), and saponins (Candra et al., 2001) induce apoptosis in several cell lines. IC101 (Fig. 1), a cyclid depsipeptide, was isolated as antibiotics from Streptomyces sp. MJ202-72F3 (Ueno et al., 1993).
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However, the detailed pharmacological and biochemical properties of this compound have not yet been reported. Here, we present the first evidence that IC101 induces apoptosis and may become a new useful pharmacological tool for studying the molecular mechanism of apoptosis.
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Materials and Methods |
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). Proteinase K and RNase A were obtained from Sigma-Aldrich (St. Louis, MO). Fetal calf serum, RPMI 1640 medium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Equitech-Bio, Inc. (Kerrville, TX), Nissui (Tokyo, Japan), and Dojindo (Kumamoto, Japan), respectively. Anti-mouse procaspase-3, anti-mouse PI3-kinase, and anti-mouse HSP90 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse cleaved-caspase-3, anti-mouse phospho-Akt (Ser 473), anti-mouse Akt, anti-mouse phospho-Bad (Ser136), anti-mouse Bad, anti-mouse phosphotyrosine, anti-mouse Raf-1 antibodies, and anti-rabbit IgG antibody horseradish peroxidase-linked were obtained from Cell Signaling Technology Inc. (Beverly, MA). Anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Research Diagnostics (Flandes, NJ). Protein A-Sepharose suspension was purchased from Zymed Laboratories (South San Francisco, CA). [
-32P]ATP (10 mCi/ml) was from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). All other reagents or drugs were of analytical grade. Cell Culture. Mouse lymphocytic leukemia L1210 cells are used for screening of apoptosis-inducing compounds and analysis of apoptotic mechanism. L1210 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum and 4 mM L-glutamine in a humidified atmosphere of 5% CO2 and 95% air.
Cell Viability Assay. Cell viability assay was performed by the MTT method as reported previously (Fujiwara et al., 2000). Briefly, L1210 cells were seeded in 96-well plates at a density of 5 x 104 cells/ml. After 24 h, cells were treated with several concentration of IC101 for 24 h. MTT of 0.5 mg/ml was added to each well, and cells were incubated at 37°C for 4 h. Plates were centrifuged at 170g for 5 min, and medium was replaced with dimethyl sulfoxide. After 15 min, plates were read on the microplate reader (model 450; Bio-Rad, Hercules, CA) at a test wavelength of 595 nm.
Morphological Observations by Using Fluorescence Microscopy. L1210 cells were seeded in 24-well plates at a density of 1 x 104 cells/ml. After 24 h, cells were treated with 100 nM IC101 for 3 h. L1210 cells were fixed with 1% glutaraldehyde, stained with 50 ng/ml propidium iodide for 30 min, rinsed with phosphate-buffered saline (PBS), and visualized by fluorescence microscopy (IMT-2; Olympus, Tokyo, Japan).
DNA Fragmentation Analysis. DNA fragmentation analysis was carried out as described previously (Fujiwara et al., 2000). Shortly, L1210 cells were seeded in six-well plates at a density of 1 x 105 cells/ml. After 24 h, cells were treated with 100 nM IC101 for 24 h. Cells were washed with PBS and resuspended in cell lysis buffer [150 mM NaCl, 15 mM sodium citrate, 10 mM EDTA, 0.005% (w/v) L-lauroyl sarcosine, and 400 µg/ml proteinase K] at 50°C for 2 h. DNA was precipitated in ethanol at 0°C for 1 h, rinsed with 70% ethanol, and dissolved in Tris-EDTA buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.4). DNA preparation was incubated with 10 µg/ml RNase A at 37°C for 1 h. Electrophoresis was carried out in a 2% (w/v) agarose gel at 50 V for 90 min in Tris borate-EDTA buffer (89 mM Tris, 89 mM borate, and 2 mM EDTA). The gel was stained for 45 min with 0.1 µg/ml ethidium bromide and visualized under UV light.
Immunoblotting. L1210 cells were seeded in six-well plates at a density of 3 x 105 cells/ml. After 24 h, cells were treated with IC101 for indicated time. Cells were washed with PBS and lysed with lysis buffer [20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1.5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM sodium orthovanadate, and 0.2% Triton X-100, pH 7.4]. Lysates were heated in SDS sample buffer (50% glycerol, 4% SDS, 25 mM Tris-HCl, 5% mercaptoethanol, and 0.01% bromphenol blue, pH 6.8), electrophoresed on a 15% (for cleaved-caspase-3 and phospho-Bad), 10% (for phospho-Akt and Raf-1), and 8% (for HSP90) polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After incubation with Tris-buffered saline (10 mM Tris-HCl and 100 mM NaCl, pH 7.4) containing 0.05% Tween 20 and 1% bovine serum albumin for 2 h, the blot was incubated with anti-procaspase-3, anti-cleavedcaspase-3, anti-phospho-Akt, anti-Akt and anti-Raf-1 antibodies (dilution 1:1000), and anti-GAPDH, anti-phospho-Bad, anti-Bad, and anti-phosphotyrosin antibodies (dilution 1:2000) at 4°C overnight, followed by incubation with anti-rabbit IgG antibody HRP-linked in a dilution of 1:2000 for 2 h. The immunoreactive bands were detected with the enhanced chemiluminescence system.
PI3-Kinase and HSP90 Immunoprecipitation. PI3-kinase and HSP90 was immunoprecipitated as described previously (Fujiwara et al., 2001). Briefly, L1210 cells were seeded in six-well plates at a density of 5 x 105 cells/ml. After 24 h, cells were treated with IC101 for 2 h. Cells were washed with PBS and lysed with Nonidet P-40 buffer (50 mM Tris-HCl, 20 mM MgCl2, 150 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM sodium orthovanadate, pH 7.4). Lysates were centrifuged at 10,400g at 4°C for 20 min and incubated with anti-PI3-kinase antibody or anti-HSP90 antibody at 4°C overnight. Subsequently, protein A-Sepharose suspension (50% slurry) was added to each sample and mixed by rotation for 2 h at 4°C. The immune complexes were centrifuged at 1500g at 4°C for 2 min and washed three times with Nonidet P-40 buffer.
PI3-Kinase Activity. Immunoprecipitates with anti-PI3-kinase antibody were resuspended with PBS and then treated with several concentrations of IC101 at 30°C for 10 min. The immunoprecipitate solution (30 µl) was incubated with 50 µl of assay mixture (0.2 mM phosphatidylinositol, 0.375 mM phosphatidylserine, 60 mM Tris-HCl, and 5 mM EGTA, pH 7.4). The reaction was initiated by addition of 20 µl of ATP mixture (25 mM MgCl2, 0.5 mM ATP, and 0.2 mCi/ml [-32P]ATP), allowed to proceed at 30°C for 30 min, and subsequently stopped by addition of 470 µl of the stop solution [methanol/chloroform/6% perchloric acid, 35:15:2 (v/v)]. After 10 min, 150 µl of chloroform, 75 µl of 2% perchloric acid, and 75 µl of 2 M NaCl were added to the reaction mixture. It was mixed and centrifuged for 5 min at room temperature. The lower chloroform phase was collected and applied to the thin layer chromatography (TLC) plate. The TLC was run in a tank equilibrated with chloroform/acetone/methanol/acetic acid/water [40:15:13:12:7 (v/v)]. The radioactivity of [32P]phosphatidylinositol monophosphate on the TLC plate was detected with the molecular imager.
Identification of Proteins by Mass Spectrometry. To identify the phosphorylated protein by mass spectrometry, the IC101-treated cell lysates were immunoprecipitated with anti-phosphotyrosine antibody in a variety of concentrations and electrophoresed on an 8% polyacrylamide gel. After silver staining, protein bands were excised from the gel, in-gel digested with trypsin, and the tryptic digest was analyzed by direct nano-flow liquid chromatography-tandem mass spectrometry (MS) for protein identification (Natsume et al., 2002). The chromatography was performed with a spray tip column (inside diameter, 150 µm x 30 mm) packed with a C18 reversed phase medium (Mightysil-C18, 3 µm; Kanto Chemicals, Tokyo, Japan) using a linear gradient from 0 to 70% acetonitrile in 0.1% formic acid for 35 min at the flow rate of 100 nl/min, and the separated peptides were directly sprayed into a Q-TOF hybrid mass spectrometer equipped with an electrospray source (Q-Tof 2; Micromass, Manchester, UK). Electrospray ionization was carried out at a voltage of 1.5 kV, and MS/MS spectra were automatically acquired in data-dependent mode during the entire run. All MS/MS spectra were converted to text files listing mass values and intensities of fragment ions and processed by the Mascot algorithm (Matrix Science, London, UK) with reference to the nonredundant protein sequence database at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD). The identification was finally confirmed by inspection of the corresponding MS/MS spectrum.
HSP90-ATP Binding Assay. HSP90-ATP binding assay was performed as described by Langer et al. (2002) with modifications. Immunoprecipitates with anti-HSP90 antibody were suspended with 30 µl of incubation buffer (50 mM Tris-HCl, 2.5 mM MgCl2, 5 mM NaCl, 2.5 mM Na2HPO4, 25 mM KCl, 0.5 mM CaCl2, 2 mM dithiothreitol, and 0.5 mM PMSF, pH 7.4) and treated with IC101 at 37°C for 10 min. The reaction was initiated by addition of several concentrations of [-32P]ATP (10 mCi/ml), allowed to proceed at 37°C for 20 min, and stopped by addition of 30 µl of SDS sample buffer and heating at 60°C for 15 min. After 15 min, 20 µl of each sample was electrophoresed on an 8% polyacrylamide gel. The gel was dried, and the radioactivity of [32P] HSP90 was detected with the molecular imager.
Statistical Analysis. The data are expressed as means ± S.E.M. Statistical comparisons were made by using Student's t test. P < 0.05 was considered to be significant.
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Results |
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Apoptotic Morphological Change and DNA Fragmentation in IC101-Treated L1210 Cells. To clearly characterize the mode of cell death, it was examined whether typical apoptotic characteristics were shown by IC101 in L1210 cells. IC101 at a concentration of 100 nM shrank cell soma (Fig. 3A, c). IC101-treated L1210 cells contained one or more compact spheres of condensed chromatin in their nuclei (Fig. 3A, d), in contrast to the chromatin uniformly dispersed in the nuclei of untreated L1210 cells (Fig. 3A, a and b). Moreover, DNA isolated from 100 nM IC101-treated cells showed typical apoptotic DNA ladders (bands of 360 and 540 bp, etc.; Fig. 3B, lane 2), whereas DNA from untreated cells did not show a DNA ladder (Fig. 3B, lane 1).
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Caspase-3 Activation by IC101. Caspase-3 is an effecter caspase that is cleaved by other caspases to propagate the death signal. IC101-treated L1210 cells showed a degradation of procaspase-3 and a marked augmentation of expression of cleaved caspase-3 fragment in a concentration-dependent manner without affecting the expression of GAPDH protein (Fig. 4), suggesting activation of caspase-3 by IC101.
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Regulation of PI3-Kinase-Independent Akt and Bad Dephosphorylation by IC101. Akt, a major survival signal molecule, is phosphorylated to be activated in a PI3-kinase cascade-dependent manner. In IC101-treated L1210 cells, a concentration-dependent dephosphorylation of Akt was shown (Fig. 5A). Moreover, Bad, a downstream target of Akt, also was dephosphorylated by IC101 in a concentration-dependent manner (Fig. 5B). But PI3-kinase was not affected by IC101 even at concentration of 100 nM (Fig. 5C).
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Effect of HSP90 phosphorylation by IC101. To find the other proteins that interacted with Akt, we performed immunoblot analysis with an anti-phosphotyrosine antibody. In IC101-treated L1210 cells, a concentration-dependent dephosphorylation of 90-kDa protein was observed (Fig. 6). To identify this protein, the immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained phosphorylated protein was processed to generate the tryptic peptides and then analyzed by direct nano-flow LC-MS for protein identification. Using this procedure, we identified nine peptides that could be assigned to a protein, HSP90 (Table 1). When immunoblot analysis of immunoprecipitates with anti-HSP90 antibody was performed by using anti-phosphotyrosine antibody, consistently, it was observed that IC101 induced the HSP90 tyrosine dephosphorylation (Fig. 7). From these results, we identified the tyrosine-phosphorylated 90-kDa protein as HSP90.
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Effect of IC101 on Raf-1 Degradation. Raf-1 exists in a native heterocomplex with HSP90 in several cell lines. Schulte et al. (1997) have been reported that geldanamycin, a selective HSP90 inhibitor, prevents HSP90-Raf-1 binding and leads to a rapid decrease of Raf-1. To evaluate whether HSP90 was affected by IC101, the degradation of Raf-1 protein was examined by immunoblot analysis. As shown in Fig. 8, a concentration-dependent degradation of Raf-1 was observed by IC101 treatment, indicating that this compound interfered with the function of HSP90 like geldanamycin.
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Inhibition of HSP90-ATP Binding by IC101. HSP90 has been reported to have an ATP binding pocket and several HSP90 inhibitors have been demonstrated to bind to this site and inhibited the ATP binding (Soti et al., 2002). In our experiment, it was shown that IC101 inhibited the HSP90-ATP binding in a noncompetitive manner (Fig. 9).
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Inhibition of HSP90-Akt Binding by IC101. Because HSP90 has been known to interact with Akt to stabilize it, immunoblot analysis of immunoprecipitates with anti-HSP90 antibody was performed by using anti-Akt antibody. It was demonstrate that HSP90 was interacted with Akt in L1210 cells and that the HSP90-Akt binding was inhibited by IC101 in a concentration-dependent manner (Fig. 10, top) without affecting the amount of immunoprecipitated HSP90 (Fig. 10, middle). When cell extracts were immunoprecipitated with anti-HSP90 antibody and thereafter the supernatants were subjected to immunoblot analysis using anti-Akt antibody, consistently, the amount of Akt remaining in supernatants seemed to increase in a manner dependent on IC101 concentration (Fig. 10, bottom).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Because it has recently been reported that cancer chemotherapeutics exert part of their pharmacological effect by triggering apoptotic cell death (Shrivastava et al., 2000), apoptosis-inducing compounds in tumor cells have become useful as lead compounds for the development of anticancer drugs. In the present study, IC101 in the range of 1 nM to 1 µM induced cell death in a concentration-dependent manner with an EC50 value of 20 nM (Fig. 2). This compound caused cell shrinkage and chromatin condensation, which were the characteristic features of apoptosis. Moreover, annexin V assay was done to determine the proportion of cells in the early stages of apoptosis or undergoing necrosis. Treatment of L1210 cells with 100 nM IC101 for 3 h increased the proportion of cells in the early stage of apoptosis (our unpublished observations). IC101 at 100 nM also caused apoptotic DNA fragmentation. Furthermore, degradation of procaspase-3 and up-regulation of cleaved caspase-3 expression were caused by IC101 in a concentration-dependent manner. These results demonstrate that IC101 causes the death of L1210 cells through an apoptotic mechanism.
The highly conserved HSPs accumulate in cells exposed to heat and a variety of other stressful stimuli (Jolly and Morimoto, 2000). HSPs inducible in response to these stresses seem to function at key regulatory points in the control of apoptosis (Creagh et al., 2000; Klettner and Herdegen, 2003; Parcellier et al., 2003). Among them, HSP90 has been reported to interact with a variety of cellular proteins, including an antiapoptotic proteins Akt (Bucci et al., 2002), Raf-1 (Schulte et al., 1997), and Bcr-Abl (An et al., 2000), to acquire a mature conformation that is a prerequisite for proper protein function. Moreover, HSP90 possesses a nucleotide binding site that acts as an ATPase domain which regulates its conformation and function (Morishima et al., 2000). Therefore, HSP90 acts as a key molecule in the antiapoptotic mechanism. In the present study, it was shown that IC101 caused a concentration-dependent dephosphorylation of 90-kDa protein (Fig. 6). By direct nano-flow LC-MS and immunoblt analysis of HSP90 immunoprecipitate using anti-phosphotyrosine antibody, this phosphorylated protein was identified as HSP90 (Table 1; Fig. 7). IC101 also caused the degradation of Raf-1, a major HSP90-binding protein (Fig. 8). Furthermore, IC101 inhibited the HSP90-ATP binding in a noncompetitive manner (Fig. 9). Thus, it is very possible to interpret that IC101 directly affects the binding of ATP to HSP90 to inhibit the function of this protein.
It has been demonstrated that HSP90 binds to Akt to prevent the protein phosphatase 2A-mediated Akt dephosphorylation and that geldanamycin, a selective HSP90 inhibitor, does not inhibit the HSP90-binding to Akt (Sato et al., 2000). In addition, Langer et al. (2002) have reported that novobiocin, another HSP90 inhibitor, binds to the C-terminal ATP-binding site of HSP90, whereas geldanamycin binds to the N-terminal ATP-binding site. In this study, it was shown that like geldanamycin, IC101 induced PI3-kinase-independent Akt dephosphorylation in a concentration-dependent manner. To strengthen the hypothesis that IC101 induced apoptosis by Akt dephosphorylation in L1210 cells, immunoblotting using anti-phospho Bad antibody was performed. IC101 caused a concentration-dependent Bad dephosphorylation like Akt. But interestingly, the immunoblot analysis in the present study demonstrated that unlike geldanamycin, IC101 inhibited the HSP90-Akt binding in a concentration-dependent manner (Fig. 10). In addition, like IC101, novobiocin was observed to induce HSP90 dephosphorylation and the inhibition of the HSP90-Akt binding at a concentration of 1 mM in L1210 cells (our unpublished observations). These findings indicate that IC101 inhibits HSP90 functions by a similar mechanism to that of novobiocin, but much more potently than this compound. The fact that IC101 causes the tyrosine dephosphorylation of HSP90 was demonstrated by the present immunoblot and immunoprecipitation analyses (Figs. 6 and 7; Table. 1). It also was observed that the inhibitory effect of IC101 on HSP90-Akt binding was partially suppressed by 1 mM sodium orthovanadate, a tyrosine phosphatase inhibitor (our unpublished observation). Treatment with 1 mM sodium orthovanadate also inhibited IC101-induced cell death and Akt dephosphorylation (our unpublished observation). These facts evidently indicate that HSP90 tyrosine phosphorylation is required for the HSP90-Akt binding and that IC101 inhibits the HSP90 tyrosine phosphorylation to cause Akt dephosphorylation and apoptosis in L1210 cells. Moreover, this compound also caused the inhibition of the HSP90-Akt binding and cell death in PC12D cells (our unpublished observation), further strengthening our idea that IC101 induces cell death by releasing Akt from HSP90.
In conclusion, these results suggest that IC101 inhibits HSP90 activity via tyrosine dephosphorylation of this protein to prevent the Akt binding, which accounts for the Akt dephosphorylation, to induce apoptosis in L1210 cells. IC101 may be a valuable pharmacological tool for clarifying the HSP90 functions, including the antiapoptotic action. Moreover, IC101 is an antibiotic that is possible to secure quantitatively like other antibiotics such as staurosporine. HSP90 is also a greatly important target for development of novel anticancer drugs (Maloney and Workman, 2002; Neckers, 2002). Because IC101 has more potent HSP90 inhibitory effect than other HSP90 inhibitor, this compound also may be a new therapeutic lead compound of cancer chemotherapy.
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Acknowledgements |
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Footnotes |
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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.
Address correspondence to: Dr. Y. Ohizumi, Department of Pharmaceutical Molecular Biology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. E-mail: ohizumi{at}mail.pharm.tohoku.ac.jp
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
An WG, Schulte TW, and Neckers LM (2000) The heat shock protein 90 antagonist geldanamycin alters chaperone association with p210bcr-abl and v-src proteins before their degradation by the proteasome. Cell Growth Differ 11: 355-360.
Araki S, Shimada Y, Kaji K, and Hayashi H (1990) Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation. Biochem Biophys Res Commun 168: 1194-1200.[CrossRef][Medline]
Bucci M, Roviezzo F, Cicala C, Pinto A, and Cirino G (2002) 17-beta-Oestradiol-induced vasorelaxation in vitro is mediated by eNOS through hsp90 and akt/pkb dependent mechanism. Br J Pharmacol 135: 1695-1700.[CrossRef][Medline]
Candra E, Matsunaga K, Fujiwara H, Mimaki Y, Sashida Y, Yamakuni T, and Ohizumi Y (2001) Two steroidal saponins from Camassia cusickii induce L1210 cell death through the apoptotic mechanism. Can J Physiol Pharmacol 79: 953-958.[CrossRef][Medline]
Creagh EM, Sheehan D, and Cotter TG (2000) Heat shock proteins-modulators of apoptosis in tumor cells. Leukemia 14: 1161-1173.[CrossRef][Medline]
Datta SR, Brunet A, and Greenberg ME (1999) Cellular survival: a play in three Akts. Genes Dev 13: 2905-2927.
Fujino M, Li XK, Kitazawa Y, Guo L, Kawasaki M, Funeshima N, Amano T, and Suzuki S (2002) Distinct pathways of apoptosis triggered by FTY720, etoposide and anti-Fas antibody in human T-lymphoma cell line (Jurkat cells). J Pharmacol Exp Ther 300: 939-945.
Fujiwara H, Matsunaga K, Kumagai H, Ishizuka M, and Ohizumi Y (2000) Ophiobolin A, a novel apoptosis-inducing agent from fungus strain f-7438. Pharm Pharmacol Commun 6: 427-431.
Fujiwara H, Matsunaga K, Saito M, Hagiya S, Furukawa K, Nakamura H, and Ohizumi Y (2001) Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells. Eur J Pharmacol 413: 37-45.[CrossRef][Medline]
Franke TF, Hornik CP, Segev L, Shostak GA, and Sugimoto C (2003) PI3K/Akt and apoptosis: size matters. Oncogene 22: 8983-8998.[CrossRef][Medline]
Garrido C, Gurbuxani S, Ravagnan L, and Kroemer G (2001) Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 286: 433-442.[CrossRef][Medline]
Jolly C and Morimoto RI (2000) Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst 92: 1564-1572.
Kerr JF, Wyllie AH, and Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257.[Medline]
Klettner A and Herdegen T (2003) The immunophilin-ligands FK506 and V-10,367 mediate neuroprotection by the heat shock response. Br J Pharmacol 138: 1004-1012.[CrossRef][Medline]
Kugawa F, Arae K, Ueno A, and Aoki M (1998) Buprenorphine hydrochloride induces apoptosis in NG108-15 nerve cells. Eur J Pharmacol 347: 105-112.[CrossRef][Medline]
Langer T, Schlatter H, and Fasold H (2002) Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its auto-phosphorylation. Cell Biol Int 26: 653-657.[CrossRef][Medline]
Laster SM, Wood JG, and Gooding LR (1988) Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol 141: 2629-2634.[Abstract]
Maloney A and Workman P (2002) HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biol Ther 2: 3-24.[CrossRef][Medline]
Morishima Y, Murphy PJ, Li DP, Sanchez ER, and Pratt WB (2000) Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket. J Biol Chem 275: 18054-18060.
Muthukkumar S, Nair P, Sells SF, Maddiwar NG, Jacob RJ, and Rangnekar VM (1995) Role of EGR-1 in thapsigargin-inducible apoptosis in the melanoma cell line A375-C6. Mol Cell Biol 15: 6262-6272.[Abstract]
Nagata S and Golstein P (1995) The Fas death factor. Science (Wash DC) 267: 1449-1456.
Nakanishi K, Sakamoto M, Yasuda J, Takamura M, Fujita N, Tsuruo T, Toda S, and Hirohashi S (2002) Critical involvement of the phosphatidylinositol 3-kinase/Akt pathway in anchorage-independent growth and hematogenous intrahepatic metastasis of liver cancer. Cancer Res 62: 2971-2975.
Natsume T, Yamauchi Y, Nakayama H, Shinkawa T, Yanagida M, Takahashi N, and Isobe T (2002) A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics. Anal Chem 74: 4725-4733.[Medline]
Neckers L (2002) Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol Med 8: 55-61.
Ohizumi Y (1997) Application of physiologically active substances isolated from natural resources to pharmacological studies. Jpn J Pharmacol 73: 263-289.[Medline]
Osawa Y, Lowe ER, Everett AC, Dunbar AY, and Billecke SS (2003) Proteolytic degradation of nitric oxide synthase: effect of inhibitors and role of hsp90-based chaperones. J Pharmacol Exp Ther 304: 493-497.
Parcellier A, Gurbuxani S, Schmitt E, Solary E, and Garrido C (2003) Heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways. Biochem Biophys Res Commun 304: 505-512.[CrossRef][Medline]
Sato S, Fujita N, and Tsuruo T (2000) Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA 97: 10832-10837.
Schulte TW, An WG, and Neckers LM (1997) Geldanamycin-induced destabilization of Raf-1 involves the proteasome. Biochem Biophys Res Commun 239: 655-659.[CrossRef][Medline]
Shrivastava P, Sodhi A, and Ranjan P (2000) Anticancer drug-induced apoptosis in human monocytic leukemic cell line U937 requires activation of endonuclease. Anticancer Drugs 11: 39-48.[CrossRef][Medline]
Solary E, Droin N, Bettaieb A, Corco L, Dimanche-Boitrel MT, and Garrido C (2000) Positive and negative regulation of apoptotic pathways by cytotoxic agents in hematological malignancies. Leukemia 14: 1833-1849.[CrossRef][Medline]
Soti C, Racz A, and Csermely P (2002) A Nucleotide-dependent molecular switch controls ATP binding at the C-terminal domain of Hsp90. N-terminal nucleotide binding unmasks a C-terminal binding pocket. J Biol Chem 277: 7066-7075.
Stefanis L, Park DS, Friedman WJ, and Grene LA (1999) Caspase-dependent and -independent death of camptothecin-treated embryonic cortical neurons. J Neurosci 19: 6235-6247.
Suk K, Lee H, Kang SS, Cho GJ, and Choi WS (2003) Flavonoid baicalein attenuates activation-induced cell death of brain microglia. J Pharmacol Exp Ther 305: 638-645.
Tsuruo T, Naito M, Tomida A, Fujita N, Mashima T, Sakamoto H, and Haga N (2003) Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal. Cancer Sci 94: 15-21.[CrossRef][Medline]
Ueno M, Amemiya M, Someno T, Masuda T, Iinuma H, Naganawa H, Hamada M, Ishizuka M, and Takeuchi T (1993) IC101, extracellular matrix antagonist produced by Streptomyces sp. MJ202-72F3. Production, isolation, structure determination and biological activity. J Antibiot (Tokyo) 46: 1658-1665.[Medline]
Whitesell L, Mimnaugh EG, De Costa B, Myers CE, and Neckers LM (1994) Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci USA 91: 8324-8328.
Xu W, Mimnaugh EG, Kim JS, Trepel JB, and Neckers LM (2002) Hsp90, not Grp94, regulates the intracellular trafficking and stability of nascent ErbB2. Cell Stress Chaperones 7: 91-96.[CrossRef][Medline]
Yao R and Cooper GM (1995) Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science (Wash DC) 267: 2003-2006.
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