Herbimycin A and Geldanamycin Inhibit Okadaic Acid-Induced Apoptosis and p38 Activation in NRK-52E Renal Epithelial Cells

Davis, Myrtle A.; Carbott, D.

doi:10.1006/taap.1999.8765

Cited by 13 publications

(10 citation statements)

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“…Although OKA is a speci c serine/threonine phosphatase inhibitor, recent studies in our lab have suggested an important role for tyrosine phosphorylation in OKA-induced cytotoxicity (14). We showed that pretreatment of renal epithelial cells with the tyrosine kinase inhibitors herbimycin and geldanamycin abrogated OKA-induced cell rounding and membrane bleb formation, and protected cells from apoptosis (14). Furthermore, microvillar structure appears to be regulated by the phosphorylation status of cytoskeletal proteins.…”

Section: Discussionmentioning

confidence: 76%

“…For example, focal adhesion breakdown, cellular contraction, and cell detachment were induced in broblasts via increases in tyrosine phosphorylation of cytoskeletal proteins, and these effects were enhanced by treatment with low doses of OKA (13). Although OKA is a speci c serine/threonine phosphatase inhibitor, recent studies in our lab have suggested an important role for tyrosine phosphorylation in OKA-induced cytotoxicity (14). We showed that pretreatment of renal epithelial cells with the tyrosine kinase inhibitors herbimycin and geldanamycin abrogated OKA-induced cell rounding and membrane bleb formation, and protected cells from apoptosis (14).…”

Section: Discussionmentioning

confidence: 90%

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Biochemical and Morphological Events During Okadaic Acid-Induced Apoptosis of Tsc2-Null ERC-18 Cell Line

2002

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Several tumor suppressor genes have been shown to regulate cellular susceptibility to proliferation or apoptotic cell death. An essential rst step in studies with the long-range goal of determining the effect of a tumor suppressor gene on cellular susceptibility to apoptosis is careful characterization of the cell's response to an apoptotic stimulus. The goals of this study were to characterize the apoptotic response of a tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene-null cell line, to establish valid biochemical events that can be used as apoptosis markers, and to determine how these events correlate with apoptosis-speci c morphologic changes. For characterization of apoptosis, we treated Tsc2-null renal epithelial tumor cells (ERC-18) with okadaic acid (OKA, 0.1-0.25 l M), and measured the biochemical and morphologic events during the apoptotic response. Electron microscopic and immunocytochemica l evaluation showed an early loss of microvilli and a loss of vinculin and talin staining from focal adhesions within 1 hour. During the rst 2 hours of treatment with 0.25 l M OKA, ERC-18 cells rounded and 50% detached from the culture vessel with minimal membrane bleb formation. Phosphatidylserine externalization, chromatin margination and fragmentation, cytochrome C release, and caspase-3 and -7 cleavage were evident at 6 hours. Maximal membrane bleb formation occurred between 6 and 10 hours. Cells progressed to secondary oncotic necrosis between 10 and 24 hours of OKA treatment. Almost all cells had an oncotic phenotyp e after 24 hours, and 17.5% lost cell membrane integrity. A small subpopulatio n (< 5%) of OKA-treated cells underwent primary oncotic necrosis within 6 hours. Interestingly, the caspase-3 and -7 inhibitor Z-DEVD-FMK did not inhibit or delay OKA-induced apoptosis in these cells. Our results suggest a complex apoptotic model involving 2 or more potentially parallel death pathways. Although caspase-3 and -7 cleavage occurs during apoptosi s in this model, this cleavage may not independentl y regulate cell death in ERC-18 cells. Therefore, measurement of apoptosi s in this model requires analysis of both biochemical and morphologi c events.

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Section: Discussionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 90%

Biochemical and Morphological Events During Okadaic Acid-Induced Apoptosis of Tsc2-Null ERC-18 Cell Line

2002

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“…Hsp90 (in association with Hsp70 and other chaperones) maintains the conformation and activity of MAL/MyD88-and TRAM/TRIF-dependent kinases involved in TNF-a and IL-6 production, such as p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase (Davis and Carbott, 1999;Richter and Buchner, 2001; Beutler et al, 2003;Bode et al, 2003;Yamamoto et al, 2003;Echeverría et al, 2011). Indeed, geldanamycin has been shown to inhibit TNF-a and IL-6 mRNA as well as protein levels in LPS-stimulated macrophages (Vega and De Maio, 2003;Wax et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages

et al. 2015

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Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/ Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 mM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-a, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88-and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-b-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-b knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 mM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-a and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 mM) inhibited LPS-induced prostaglandin E 2 production, cyclooxygenase-2 expression, and nuclear factor kB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.

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“…Since geldanamycin is known to induce apoptosis and some but not all cell types (40,49,53) and, as mentioned above, caspase activity seems involved in ErbB-2 degradation induced by this drug, we tested other known inducers of apoptosis for their capacity to provoke ErbB-2 degradation and formation of the 23-kDa fragment. Of the various inducers of apoptosis tested, two compounds (staurosporin and curcumin) were found to promote formation of a 23-kDa fragment.…”

Section: Discussionmentioning

confidence: 99%

Caspase-dependent Cleavage of ErbB-2 by Geldanamycin and Staurosporin

Tikhomirov

Carpenter

2001

Journal of Biological Chemistry

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The geldanamycin-induced degradation of ErbB-2 produces a 23-kDa carboxyl-terminal fragment, which has been isolated and subjected to amino-terminal microsequencing. The obtained sequence indicates that the amino terminus of this fragment corresponds to Gly-1126 of ErbB-2. Analysis of the residues immediately before Gly-1126 suggests that cleavage may involve caspase activity. Site-directed mutagenesis of Asp-1125 in ErbB-2 prevents geldanamycin-provoked formation of the 23-kDa fragment, consistent with the requirement of this residue for caspase-dependent cleavage in known substrates. Also, the addition of the pan-caspase inhibitor Z-VAD-FMK blocks formation of the 23-kDa ErbB-2 fragment in cells exposed to geldanamycin. Interestingly, staurosporin and curcumin are also shown to provoke the degradation of ErbB-2 with formation of the 23-kDa carboxyl-terminal fragment. The generation of this fragment by staurosporin or curcumin is likewise blocked by caspase inhibition. Caspase inhibition does not prevent accelerated degradation of the 185-kDa native ErbB-2 in geldanamycin-treated cells but does significantly prevent staurosporin-stimulated metabolic loss of ErbB-2.ErbB-2, a Type I transmembrane receptor tyrosine kinase, functions as a co-receptor by dimerizing with ligand-occupied members of the ErbB family, the EGF receptor, ErbB-3, or ErbB-4 (1-3). This heterodimerization event is considered to alter the signaling capacity and cellular responses provoked by homodimers of occupied ErbB receptors. No ligand has been identified that directly interacts with the ectodomain of ErbB-2.ErbB-2 was originally identified as the transforming oncogene neu, which contains a point mutation in the transmembrane domain that is responsible for its oncogenic potential (4). Overexpression of ErbB-2 also produces a transformed phenotype in experimental systems (5-7). Importantly, ErbB-2 is frequently overexpressed in carcinomas, particularly mammary and ovarian carcinomas, and is associated with a poor prognosis (8 -10). ErbB-2 antibodies (11, 12) and agents such as interferon (13) or tyrosine kinase inhibitors (14) decrease the growth of ErbB-2-expressing tumor cells and also reduce the cellular level of ErbB-2 (15). In many of these instances the decreased growth provoked by the loss of ErbB-2 is due to increased apoptosis. In contrast, the overexpression of ErbB-2 can prevent the induction of apoptosis (15). Hence, the growth-controlling activity of ErbB-2 is related to structural changes or alterations in its level of expression.The benzoquinoid anasamycin antibiotic geldanamycin was first isolated and described as an inhibitor of tyrosine kinase activity (16). Subsequently, geldanamycin was shown to possess tumoricidal activity toward cell lines that overexpress ErbB-2 (17). Currently geldanamycin derivatives designed to reduce toxicity are in clinical trials for certain cancer patients (18). The addition of geldanamycin to cells results in an increased rate of degradation of several protein kinases, including ErbB-2 (17), S...

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Herbimycin A and Geldanamycin Inhibit Okadaic Acid-Induced Apoptosis and p38 Activation in NRK-52E Renal Epithelial Cells

Cited by 13 publications

References 42 publications

Biochemical and Morphological Events During Okadaic Acid-Induced Apoptosis of Tsc2-Null ERC-18 Cell Line

Biochemical and Morphological Events During Okadaic Acid-Induced Apoptosis of Tsc2-Null ERC-18 Cell Line

Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages

Caspase-dependent Cleavage of ErbB-2 by Geldanamycin and Staurosporin

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