• Biomedical Science Letters
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• v.15 no.1
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• pp.97-99
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• 2009

Geldanamycin is a benzoquinone ansamycin antibiotic that binds to cytosol HSP90 (Heat Shock Protein 90) and changes its biological function. HSP90 is involved in the intracellular important roles for the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis. To identify genes expressed during geldanamycin treatment against neurons of rats (PC12 cells), DNA microarray method was used. We have isolated 2 gene groups (up-or down-regulated genes) which are geldanamycin differentially expressed in neurons. Granzyme B is the gene most significantly increased among 204 up-regulated genes (more than 2 fold over-expression) and Chemokine (C-C motif) ligand 20 is the gene most dramatically decreased among 491 down-regulated genes (more than 2 fold down-expression). The gene increased expression of Cxc110, Cyp11a1, Gadd45a, Gja1, Gpx2, Ifua4, Inpp5e, Sox4, and Stip1 are involved stress-response gene, and Cryab, Dnaja1, Hspa1a, Hspa8, Hspca, Hspcb, Hspd1, Hspd1, and Hsph1 are strongly associated with protein folding. Cell cycle associated genes (Bc13, Brca2, Ccnf, Cdk2, Ddit3, Dusp6, E2f1, Illa, and Junb) and inflammatory response associated genes (Cc12, Cc120, Cxc12, Il23a, Nos2, Nppb, Tgfb1, Tlr2, and Tnt) are down-regulated more than 2 times by geldanamycin treatment. We found that geldanamycin is related to expression of many genes associated with stress response, protein folding, cell cycle, and inflammation by DNA microarray analysis. Further experimental molecular studies will be needed to figure out the exact biological function of various genes described above and the physiological change of neuronal cells by geldanamycin. The resulting data will give the one of the good clues for understanding of geldanamycin under molecular level in the neurons.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.897-900
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• 2008

Various sequences of pH change were applied in a batch bioreactor to investigate pH shock effects on geldanamycin production by Streptomyces hygroscopicus subsp. duamyceticus JCM4427. In the control culture where the pH was not controlled, the maximum geldanamycin concentration was 414 mg/l. With the pHS1 mode of pH shock, that is, an abrupt pH change from pH 6.5 to pH 5.0 and then being maintained at around pH 5.0 afterward, 768 mg/l of geldanamycin was produced. With pHS2, in which the pH was changed sequentially from pH 6.7 to pH 5.0 and then back to pH 6.0, 429 mg/l of geldanamycin was produced. With pHS3 having a sequential pH change from pH 6.0 to pH 4.0 and then back to pH 6.5 followed by the third pH shock to pH 5.5, no geldanamycin production was observed. Considering that the productivity with pHS1 was about two-fold of that of the control culture with no pH control, we concluded that a more sophisticated manipulation of pH would further promote geldanamycin production.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1484-1490
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• 2010

In the Streptomyces hygroscopicus JCM4427 geldanamycin biosynthetic gene cluster, five putative regulatory genes were identified by protein homology searching. Among those genes, gel14, gel17, and gel19 are located downstream of polyketide synthase genes. Gel14 and Gel17 are members of the LAL family of transcriptional regulators, including an ATP/GTP-binding domain at the N-terminus and a DNA-binding helix-turn-helix domain at the C-terminus. Gel19 is a member of the TetR family of transcriptional regulators, which generally act to repress transcription. To verify the biological significance of the putative regulators in geldanamycin production, they were individually characterized by gene disruption, genetic complementation, and transcriptional analyses. All three genes were confirmed as positive regulators of geldanamycin production. Specifically, Gel17 and Gel19 are required for gel14 as well as gelA gene expression.

• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.149-155
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• 2013

Heterologous expression of a putative $K^+/H^+$ antiporter of Streptomyces coelicolor A3(2) (designated as sha4) in E. coli and Streptomyces hygroscopicus JCM4427 showed enhanced tolerance to $K^+$ stress, acidic-pH shock, and/or geldanamycin production under $K^+$ stress. In a series of $K^+$ extrusion experiments with sha4-carrying E. coli deficient in the $K^+/H^+$ antiporter, a restoration of impaired $K^+$ extrusion activity was observed. Based on this, it was concluded that sha4 was a true $K^+/H^+$ antiporter. In different sets of experiments, the sha4-carrying E. coli showed significantly improved tolerances to $K^+$ stresses and acidic-pH shock, whereas sha4-carrying S. hygroscopicus showed an improvement in $K^+$ stress tolerance only. The sha4-carrying S. hygroscopicus showed much higher geldanamycin productivity than the control under $K^+$ stress condition. In another set of experiments with a production medium, the secretion of geldanamycin was also significantly enhanced by the expression of sha4.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.4
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• pp.462-469
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• 2018

HSP90 regulates various proteins involved in differentiation and cell survival. Levels of HSP90 tend to increase during development of squamous cell carcinoma in the head and neck including the mouth. Thus, many studies have been conducted to treat these cancers through suppression of HSP90. This study investigated the effect of two HSP90 inhibitors, geldanamycin and 17-AAG, on the proliferation, apoptosis, and invasion of human oral squamous cell carcinoma cells. Cell survival and cell cycle analyses, as well as western blot analysis, were performed with oral cancer cell lines, YD-10B and YD-38. After treatment with HSP90 inhibitors, cell proliferation was significantly inhibited. When YD-10B and YD-38 cells were treated with various concentrations of geldanamycin and 17-AAG (0, 0.1, 0.3, 1 and $10{\mu}M$) for 24 hr, the growth of YD-10B cells was markedly reduced compared to that of YD-38 cells. Thereafter, the cells were subjected to flow cytometry, which revealed G2 arrest. These results demonstrated that geldanamycin induced G2 arrest and inhibited cell proliferation through the $p-GSK-3{\beta}$ pathway in YD-10B and YD-38 cells, thus inhibiting cell survival. HSP90 inhibitors are therefore expected to have a therapeutic effect on various cancer cell lines.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1101-1108
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• 2008

Geldanamycin and its analogs are important anticancer agents that inhibit the newly targeted heat-shock protein (Hsp) 90, which is a chaperone protein in eukaryotic cells. To resolve which geldanamycin biosynthetic genes are responsible for particular post-polyketide synthase (PKS) processing steps and in which order the reactions occur, we individually inactivated candidate genes in Streptomyces hygroscopicus subsp. duamyceticus JCM4427 and isolated and elucidated the structures of intermediates from each mutant. The results indicated that gel7 governs at least one of the benzoquinone ring oxidation steps. The gel16 was found to be involved in double-bond formation between C-4 and C-5 of 4,5-dihydrogeldanamycin, which confirmed our previous findings that this double bond is reduced during the post-PKS modification of the polyketide assembly. In addition, pro-geldanamycin, which does not possess a double bond at C-4/5, was purified from the gel7 and gel8 double-gene-inactivated mutant.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1478-1481
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• 2012

Geldanamycin (GM) and its analogs are important anticancer agents that inhibit heat shock protein (Hsp) 90, which is a major chaperone protein in cancer cells. Accordingly, based on interest in obtaining novel natural GM derivatives, the potential of Streptomyces hygroscopicus JCM4427, a GM producer, was explored for novel natural GM derivative(s), resulting in the discovery of new GM analogs as a biosynthetic shunt product and intermediates from its fermentation broth. In this study, the fermentation, isolation, structure determination, and biological activity of the compounds, two new tetracyclic thiazinogeldanamycin (1) and 19-hydroxy-4,5-dihydrogeldanamycin (3), together with the three known 4,5-dihydrothiazinogeldanamycin (2), reblastatin (4), and 17-demethoxy-reblastatin (5), are described.

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.44-54
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• 2019

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be $9.384{\AA}$. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH ${\rightarrow}$ FDR6 ${\rightarrow}$ FDX4 ${\rightarrow}$ Gel16.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.56-60
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• 2016

Two new glucosides (1 and 2) of geldanamycin (GA) analogs were obtained from in vitro glycosylation by UDP-glycosyltransferase (YjiC). Based on spectroscopic (HR-ESI-MS, 1D, and 2D-NMR) analyses, the glucosides were elucidated as 4,5-dihydro-7-O-descarbamoyl-7-hydroxyl GA-7-O-β-D-glucoside (1) and ACDL3172-18-O-β-D-glucoside (2). Furthermore, the water solubility of compounds 1 and 2 was about 215.2 and 90.7 times higher respectively, than that of the substrates. Among compounds 1-4, only 3 showed weak antiproliferative activity against four human tumor cell lines: MDA-MB-231, SMMC7721, HepG2, and SW480 (IC₅₀: 13.6, 15.1, 31.8, and 22.7 μM, respectively).

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.542-550
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• 2018

Heat shock protein 90 (Hsp90) is treated as a molecular therapeutic target for the prevention and treatment of cancer. Geldanamycin (GA) was the first identified natural Hsp90 inhibitor, but hepatotoxicity has limited its clinical application. Nevertheless, a new GA analog (WK-88-1) with the non-benzoquinone skeleton, obtained from genetically engineered Streptomyces hygroscopicus, was found to have anticancer activity against two human breast cancer cell lines. WK-88-1 produced concentration-dependent inhibition of cell proliferation, cell cycle arrest, and apoptosis in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cell lines. Detailed analysis showed that WK-88-1 downregulated some key cell cycle molecules (CDK1 and cyclin B1) and lead to $G_2/M$ cell cycle arrest. Further studies also showed that WK-88-1 could induce human breast cancer cell apoptosis by downregulating Hsp90 client proteins (Akt, p-Akt, IKK, c-Raf, and Bcl-2), decreasing the ATP level, increasing reactive oxygen species production, and lowering the mitochondrial membrane potential. Meanwhile, we discovered that WK-88-1 significantly decreased the levels of Her-2 and $ER-{\alpha}$ in MCF-7 cells but not in MDA-MB-231 cells. In addition, WK-88-1 significantly increased caspase-3, -8, and -9 activities and the cleavage of PARP in a concentration-dependent manner (with the exception of caspase-3 and PARP in MCF-7 cells). Taken together, our preliminary results suggest that WK-88-1 has the potential to play a role in breast cancer therapy.