novobiocin and Prostatic-Neoplasms

The 90-kDa heat-shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Inhibiting Hsp90 consequently is an attractive strategy for cancer therapy as the concomitant degradation of multiple oncoproteins may lead to effective antineoplastic agents. Here we report a novel C-terminal Hsp90 inhibitor, designated KU675, that exhibits potent antiproliferative and cytotoxic activity along with client protein degradation without induction of the heat-shock response in both androgen-dependent and -independent prostate cancer cell lines. In addition, KU675 demonstrates direct inhibition of Hsp90 complexes as measured by the inhibition of luciferase refolding in prostate cancer cells. In direct binding studies, the internal fluorescence signal of KU675 was used to determine the binding affinity of KU675 to recombinant Hsp90α, Hsp90β, and Hsc70 proteins. The binding affinity (Kd) for Hsp90α was determined to be 191 μM, whereas the Kd for Hsp90β was 726 μM, demonstrating a preference for Hsp90α. Western blot experiments with four different prostate cancer cell lines treated with KU675 supported this selectivity by inducing the degradation of Hsp90α -: dependent client proteins. KU675 also displayed binding to Hsc70 with a Kd value at 76.3 μM, which was supported in cellular by lower levels of Hsc70-specific client proteins on Western blot analyses. Overall, these findings suggest that KU675 is an Hsp90 C-terminal inhibitor, as well as a dual inhibitor of Hsc70, and may have potential use for the treatment of cancer.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Coumarins; Gene Expression Regulation, Neoplastic; HSC70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Male; Novobiocin; Phenylurea Compounds; Prostatic Neoplasms; Protein Binding

The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.. PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.. KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.. Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Growth Inhibitors; HSP90 Heat-Shock Proteins; Humans; Male; Neoplasm Proteins; Novobiocin; Prostatic Neoplasms; Protein Binding; Rats

Hsp90 is important in the folding, maturation and stabilization of pro-tumorigenic client proteins and represents a viable drug target for the design of chemotherapies. Previously, we reported the development of novobiocin analogues designed to inhibit the C-terminal portion of Hsp90, which demonstrated the ability to decrease client protein expression. We now report the characterization of the novel novobiocin analogue, F-4, which demonstrates improved cytotoxicity in prostate cancer cell lines compared to the N-terminal inhibitor, 17-AAG.. LNCaP and PC-3 cells were treated with 17-AAG or F-4 in anti-proliferative, apoptosis, cell cycle and cytotoxicity assays. Western blot and prostate specific antigen (PSA) ELISAs were used to determine client protein degradation, induction of Hsp90 and to assess the functional status of the androgen receptor (AR) in response to F-4 treatment. Surface plasmon resonance (SPR) was also used to determine the binding properties of F-4 to Hsp90.. F-4 demonstrated improved potency and efficacy compared to novobiocin in anti-proliferative assays and decreased expression of client proteins. PSA secretion was inhibited in a dose-dependent manner that paralleled a decrease in AR expression. The binding of F-4 to Hsp90 was determined to be saturable with a binding affinity (K(d)) of 100 microM. In addition, superior efficacy was demonstrated by F-4 compared to 17-AAG in experiments measuring cytotoxicity and apoptosis.. These data reveal distinct modes of action for N-terminal and C-terminal Hsp90 inhibitors, which may offer unique therapeutic benefits for the treatment of prostate cancer.

Topics: Amino Acid Sequence; Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Growth Inhibitors; HSP90 Heat-Shock Proteins; Humans; Male; Novobiocin; Prostatic Neoplasms; Spodoptera

Malignantly transformed stem cells represent a potential common nidus for the primary cancer and the recurrent cancer that arises after treatment failure. Putative prostate stem cells and prostate tumor stem cells in benign and malignant human prostate tissue, in primary human prostate xenografts, and in the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, are defined by expression of breast cancer resistance protein (BCRP), a marker of pluripotent hematopoietic, muscle, and neural stem cells, and by an absence of androgen receptor (AR) protein. Inhibition of BCRP-mediated efflux of dihydrotestosterone by novobiocin or fumitremorgin C in a rat prostate progenitor cell line that expresses BCRP and AR mRNAs, but minimal AR protein, results in stabilization and nuclear translocation of AR protein, providing a mechanism for lack of AR protein in BCRP-expressing stem cells. In both benign and malignant human prostate tissue, the rare epithelial cells that express BCRP and lack AR protein are localized in the basal cell compartment, survive androgen deprivation, and maintain proliferative potential in the hypoxic, androgen-deprived prostate. Putative prostate tumor stem cells that express BCRP but not AR protein in TRAMP are the source of a BCRP-negative and AR-negative, Foxa2- and SV40Tag-expressing, transit amplifying compartment that progresses to the poorly differentiated carcinomas that arise rapidly after castration. Therefore, BCRP expression isolates prostate stem/tumor stem cells from the prostate tissue microenvironment through constitutive efflux of androgen, protecting the putative tumor stem cells from androgen deprivation, hypoxia, or adjuvant chemotherapy, and providing the nidus for recurrent prostate cancer.

Topics: Androgens; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Cell Nucleus; Humans; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Proteins; Neoplastic Stem Cells; Novobiocin; Prostate; Prostatic Neoplasms; Protein Processing, Post-Translational; Rats; Receptors, Androgen; RNA, Messenger; Transplantation, Heterologous