17-(dimethylaminoethylamino)-17-demethoxygeldanamycin and Pancreatic-Neoplasms

Topics: Adenocarcinoma; Apoptosis; Benzoquinones; Cell Line, Tumor; Deoxycytidine; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Fluorouracil; Gemcitabine; HSP90 Heat-Shock Proteins; Humans; Inhibitory Concentration 50; Lactams, Macrocyclic; Molecular Targeted Therapy; Neoplasm Proteins; Pancreatic Neoplasms; Recurrence

Pancreatic endocrine tumors (PET) represent a heterogenous group of neoplasms. Although surgical resection is considered a safe and effective treatment for many PET, therapeutic options for inoperable and progressive PET are limited. The expression of heat-shock protein (HSP) 90 was investigated in 120 clinically and pathomorphologically well-characterized PET from 84 patients using immunohistochemistry. In addition, in 19 snap-frozen PET and in three healthy pancreatic tissues, we performed immunoblot analyses, and in 15 snap-frozen PET and in three healthy pancreatic tissues, we investigated the expression of HSP90 isoforms by means of semiquantitative RT-PCR. Functional tests were conducted using the human pancreas carcinoid cell line BON and the mouse insulinoma cell line β-TC-3. HSP90 was expressed in 95% of the PET patients. The transcript levels of the HSP90 isoforms HSP90α, HSP90β, glucose-related protein 94, and TNF receptor-associated protein 1 were significantly increased in PET compared with non-neoplastic pancreatic tissues. The treatment of the cell lines BON and β-TC-3 with the HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin resulted in significant, dose-dependent reduction of cell viability, cell cycle arrest, and increased apoptosis. Furthermore, HSP90 inhibition induced the degradation and inactivation of several oncogenetic HSP90 client proteins in a time- and dose-dependent manner. HSP90 inhibitors increased the therapeutic effects of doxorubicin and 5-fluorucacil in BON and β-TC-3 cells. HSP90 is expressed in the vast majority of PET and its inhibition reveals significant treatment effects in vitro. Thus, HSP90 qualifies as a promising new target.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Apoptosis; Benzoquinones; Cell Line, Tumor; Cell Survival; Doxorubicin; Female; Fluorouracil; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Male; Mice; Middle Aged; Multiple Endocrine Neoplasia Type 1; Pancreatic Neoplasms; RNA, Messenger; Young Adult

Inhibitors of heat-shock protein 90 (Hsp90) may interfere with oncogenic signaling pathways, including Erk, Akt, and hypoxia-inducible factor-1alpha (HIF-1alpha). Because insulin-like growth factor-I receptor (IGF-IR) and signal transducer and activator of transcription 3 (STAT3) signaling pathways are implicated in the progression of pancreatic cancer, we hypothesized that blocking Hsp90 with geldanamycin derivates [17-allylamino-geldanamycin (17-AAG), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)] would impair IGF-I- and interleukin-6-mediated signaling and thus reduce pancreatic tumor growth and angiogenesis in vivo.. Human pancreatic cancer cells (HPAF-II, L3.6pl) were used for experiments. Changes in signaling pathway activation upon Hsp90 blockade were investigated by Western blotting. Effects of Hsp90 inhibition (17-AAG) on vascular endothelial growth factor were determined by ELISA and real-time PCR. Effects of 17-DMAG (25 mg/kg; thrice a week; i.p.) on tumor growth and vascularization were investigated in a s.c. xenograft model and in an orthotopic model of pancreatic cancer.. 17-AAG inhibited IGF-IR signaling by down-regulating IGF-IRbeta and directly impairing IGF-IR phosphorylation. Hypoxia- and IL-6-mediated activation of HIF-1alpha or STAT3/STAT5 were substantially inhibited by 17-AAG. Moreover, a novel IL-6/STAT3/HIF-1alpha autocrine loop was effectively disrupted by Hsp90 blockade. In vivo, 17-DMAG significantly reduced s.c. tumor growth and diminished STAT3 phosphorylation and IGF-IRbeta expression in tumor tissues. In an orthotopic model, pancreatic tumor growth and vascularization were both significantly reduced upon Hsp90 inhibition, as reflected by final tumor weights and CD31 staining, respectively.. Blocking Hsp90 disrupts IGF-I and IL-6-induced proangiogenic signaling cascades by targeting IGF-IR and STAT3 in pancreatic cancer, leading to significant growth-inhibitory effects. Therefore, we suggest that Hsp90 inhibitors could prove to be valuable in the treatment of pancreatic cancer.

Topics: Animals; Benzoquinones; Cell Line, Tumor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-6; Lactams, Macrocyclic; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Pancreatic Neoplasms; Receptor, IGF Type 1; Signal Transduction; STAT3 Transcription Factor

An accurate, sensitive, robust and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin hydrochloride (17-DMAG) in human plasma has been developed and validated. Plasma samples were prepared by liquid/liquid extraction with ethyl acetate. The chromatographic separation was achieved within 9 min on a Synergy Polar column with a linear gradient and a mobile phase consisting of methanol and 0.1% formic acid in water. Detection of 17-DMAG and the internal standard (IS), olomoucine, was achieved by MS/MS with electrospray ionisation in positive ion mode. The calibration curve, ranging from 1.89 to 1890 nM, was linear r > 0.994 using a 1/y2 weighted linear regression. The assay showed no significant interferences from endogenous compounds. The lower limit of quantitation (LLOQ) was 1.89 nM, using 250 microL of plasma, with inter-assay precision (%RSD) and accuracy (%RE) values of 11.6% and -5.8%, respectively. Intra-assay precision ranged from 7.8-13.6%. The method described here is being used to evaluate the pharmacokinetic profiles of 17-DMAG given as a once weekly infusion in patients with advanced solid tumours.

Topics: Antineoplastic Agents; Area Under Curve; Benzoquinones; Chromatography, High Pressure Liquid; Half-Life; Humans; Kinetin; Lactams, Macrocyclic; Pancreatic Neoplasms; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Inhibitors of the molecular chaperone Hsp90 have been shown to enhance tumor cell radiosensitivity. To begin to address the mechanism responsible, we have determined the effect of the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) on the DNA damage response to radiation. Exposure of MiaPaCa tumor cells to 17DMAG, which results in radiosensitization, inhibited the repair of DNA double-strand breaks according to gammaH2AX foci dispersal and the neutral comet assay. This repair inhibition was associated with reduced DNA-PK catalytic subunit (DNA-PKcs) phosphorylation after irradiation and a disruption of DNA-PKcs/ErbB1 interaction. These data suggest that the previously established 17DMAG-mediated reduction in ErbB1 activity reduces its interaction with DNA-PKcs and thus accounts for the attenuation of radiation-induced DNA-PK activation. 17DMAG was also found to abrogate the activation of the G(2)- and S-phase cell cycle checkpoints. Associated with these events was a reduction in radiation-induced ataxia-telangiectasia mutated (ATM) activation and foci formation in 17DMAG-treated cells. Although no interaction between ATM and Hsp90 was detected, Hsp90 was found to interact with the MRE11/Rad50/NBS1 (MRN) complex. 17DMAG exposure reduced the ability of the MRN components to form nuclear foci after irradiation. Moreover, 17DMAG exposure reduced the interaction between NBS1 and ATM, although no degradation of the MRN complex was detected. These results suggest that the diminished radiation-induced activation of ATM in 17DMAG-treated cells was the result of a compromise in the function of the MRN complex. These data indicate that Hsp90 can contribute to the DNA damage response to radiation affecting both DNA repair and cell cycle checkpoint activation.

Topics: Benzoquinones; Cell Cycle; Cell Line, Tumor; DNA Damage; DNA Repair; DNA-Activated Protein Kinase; DNA, Neoplasm; Enzyme Activation; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Pancreatic Neoplasms; Radiation-Sensitizing Agents

The ability to identify tumors that are susceptible to a given molecularly targeted radiosensitizer would be of clinical benefit. Towards this end, we have investigated the effects of a representative Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), on the radiosensitivity of a panel of human tumor cell lines. 17DMAG was previously shown to enhance the radiosensitivity of a number of human cell lines, which correlated with the loss of ErbB2. We now report on cell lines in which 17DMAG induced the degradation of ErbB2, yet had no effect on radiosensitivity. In a comparison of ErbB family members, ErbB3 protein was only detectable in cells resistant to 17DMAG-induced radiosensitization. To determine whether ErbB3 plays a casual role in this resistance, short interfering RNA (siRNA) was used to knockdown ErbB3 in the resistant cell line AsPC1. Whereas individual treatments with siRNA to ErbB3 or 17DMAG had no effect on radiosensitivity, the combination, which reduced both ErbB2 and ErbB3, resulted in a significant enhancement in AsPC1 radiosensitivity. In contrast to siRNA to ErbB3 or 17DMAG treatments only, AsPC1 cell exposure to the combination also resulted in a decrease in ErbB1 kinase activity. These results indicate that ErbB3 expression predicts for tumor cell susceptibility to and suggests that the loss of ErbB1 signaling activity is necessary for 17DMAG-induced radiosensitization. However, for cell lines sensitized by 17DMAG, treatment with siRNA to ErbB2, which reduced ErbB1 activity, had no effect on radiosensitivity. These results suggest that, whereas the loss of ErbB1 signaling may be necessary for 17DMAG-induced radiosensitization, it is not sufficient.

Topics: Benzoquinones; Cell Line, Tumor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Male; Pancreatic Neoplasms; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinones; Radiation Tolerance; Radiation-Sensitizing Agents; raf Kinases; Receptor, ErbB-2; Receptor, ErbB-3; RNA, Small Interfering