C15-methoxyphenylated 18-deoxy-herbimycin A analogues, their in vitro anticancer activity and heat shock protein 90 binding affinity.

Benzoquinone ansamycins are important leads for the discovery of novel inhibitors of heat shock protein 90 (Hsp90), a promising target of cancer chemotherapeutics. Intrinsic hepatotoxicity caused by the benzoquinone moiety appeared to be a serious limitation to the development of these compounds. To solve this problem by rational structure optimization, a short series of C18-deoxy analogues of herbimycin A were designed based on putative interactions between the compound and the protein. Chemical synthesis of the target molecules were attempted by following the established synthetic route to the natural product, but resulted in the isolation of four serendipitous C15 phenylated final products. In vitro antiproliferative activity and Hsp90 binding affinity of the compounds were determined, suggesting the C18-oxygen of herbimycin A is removable and bulky lipophilic groups can be accommodated at C15 without loss of activity.

17-Allylamino-17-demethoxygeldanamycin and Herbimycin A Induce Cell Death by Modulating ß-Catenin and PI3K/AKT Signaling in FRO Anaplastic Thyroid Carcinoma Cells.

AIM: The aim of the present study was to evaluate the effect of heat-shock protein 90 (HSP90) inhibitors, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and herbimycin A (HMA) on survival of anaplastic thyroid carcinoma (ATC) cells. MATERIALS AND METHODS: Antitumor activities of 17-AAG and HMA were investigated in FRO ATC cells. RESULTS: In FRO ATC cells, 17-AAG and HMA caused cell death with concomitant changes in the expression of HSP90 client proteins, increased ß-catenin protein levels, and inhibited PI3K/AKT signaling. The inactivation of ß-catenin by ß-catenin siRNA transfection and the activation of PI3K/AKT signaling by p110α plasmid transfection abrogated cell death caused by 17-AAG and HMA. CONCLUSION: 17-AAG and HMA have cytotoxic activities accompanied by regulation of HSP90 client proteins, and cytotoxicity is associated with overexpression of ß-catenin and suppression of PI3K/AKT signaling in FRO ATC cells.

Herbimycin A inhibits cell growth with reversal of epithelial-mesenchymal transition in anaplastic thyroid carcinoma cells.

We aimed to elucidate the effect of herbimycin A (HMA), a heat shock protein 90 inhibitor, on cell growth and epithelial-mesenchymal transition (EMT) in anaplastic thyroid carcinoma (ATC) cells. HMA inhibited cell growth and migration concomitantly with increase of E-cadherin as well as decrease of N-cadherin and vimentin. Moreover, HMA upregulated p21 and p27, while it downregulated p53 and Akt. In HMA-treated condition, knockdown of E-cadherin and overexpression of p53 increased N-cadherin and vimentin, and mitigated the inhibitory effects of HMA on cell growth and migration. Furthermore, knockdown of p21 and p27 ameliorated inhibition of cell growth and reversal of EMT. In addition, the activation of Akt attenuated growth inhibition, cell death and EMT reversal. Therefore, we propose that HMA suppresses cell growth, and reverses EMT in conjunction with the activation of E-cadherin, p21 and p27 and the inactivation of p53 and PI3K/Akt signaling in ATC cells.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90 percent of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Signal Transduction Factors on the Modulation of Radiosusceptibility in K562 Cells / 대한방사선종양학회지

PURPOSE: The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes the induction of apoptosis via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A (HMA) coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosis of p210bcr/abl protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the induction of a number of transcription factors and the differential gene expression in this model were investigated. MATERIALS AND METHODS: K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 MeV Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with 0.25 microM of HMA and 25 microM of genistein, and the expressions and the activities of abl kinase, MAPK family, NF-kB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. RESULTS: The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either. In association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF-kB activity and the TK1 expression and activity. CONCLUSION: The effects of HMA and genistein on the radiosensitivity of the K562 cells were not related to the bcr-abl kinase activity. In this study, another signaling pathway, besides the MAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the optional radiosensitization or radioprotection.

Regulatory Mechanism of Radiation-induced Cancer Cell Death by the Change of Cell Cycle / 대한방사선종양학회지

PURPOSE: In our previous study, we have shown the main cell death pattern induced by irradiation or protein tyrosine kinase (PTK) inhibitors in K562 human myelogenous leukemic cell line. Death of the cells treated with irradiation alone was characterized by mitotic catastrophe and typical radiation-induced apoptosis was accelerated by herbimycin A (HMA). Both types of cell death were inhibited by genistein. In this study, we investigated the effects of HMA and genistein on cell cycle regulation and its correlation with the alterations of radiation-induced cell death. MATERIALS AND METHODS: K562 cells in exponential growth phase were used for this study. The cells were irradiated with 10 Gy using 6 MeV Linac (200-300 cGy/min). Immediately after irradiation, cells were treated with 250 nM of HMA or 25 microM of genistein. The distributions of cell cycle, the expressions of cell cycle-related protein, the activities of cyclin-dependent kinase, and the yield of senescence and differentiation were analyzed. RESULTS: X-irradiated cells were arrested in the G2 phase of the cell cycle but unlike the p53-positive cells, they were not able to sustain the cell cycle arrest. An accumulation of cells in G2 phase of first cell-cycle post-treatment and an increase of cyclin B1 were correlated with spontaneous, premature, chromosome condensation and mitotic catastrophe. HMA induced rapid G2 checkpoint abrogation and concomitant p53-independent G1 accumulation. HMA-induced cell cycle modifications correlated with the increase of cdc2 kinase activity, the decrease of the expressions of cyclins E and A and of CDK2 kinase activity, and the enhancement of radiation-induced apoptosis. Genistein maintained cells that were arrested in the G2-phase, decreased the expressions of cyclin B1 and cdc25C and cdc2 kinase activity, increased the expression of p16, and sustained senescence and megakaryocytic differentiation. CONCLUSION: The effects of HMA and genistein on the radiation-induced cell death of K562 cells were closely related to the cell cycle regulatory activities. In this study, we present a unique and reproducible model in which for investigating the mechanisms of various, radiation-induced, cancer cell death patterns. Further evaluation by using this model will provide a potent target for a new strategy of radiotherapy.

Effects of herbimycin A on proliferation and nitric oxide production stimulated by interleukin 1 in chondrocytes / 第二军医大学学报

Objective: To study the effects of herbimycin A on proliferation and NO production stimulated by IL 1 in chondrocytes. Methods: IL 1 alone or in combination with herbimycin A was administered.Chondrocytes proliferation was detected by crystal violet dying method and NO production was detected by Griess method. Results: IL 1 inhibited chondrocytes proliferation. Herbimycin A dose dependently reversed the effect. Also herbimycin A could inhibit NO production induced by IL 1. Conclusion: The reversing effect of herbimycin A on chondrocytes proliferation may be mediated by NO.