Journal Article

Animals; Anoikis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bioware; Caspase 3; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Humans; L-Lactate Dehydrogenase; MDA-MB-231-D3H2LN cells; Membrane Proteins; Mice; Neoplasm Metastasis; Neoplasm Proteins; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Anoikis, a Bax-dependent apoptosis triggered by detachment from the extracellular matrix, is often dysfunctional in metastatic cancer cells. Using wild-type and c-Src-transformed NIH3T3 cells as a model, we identified Mcl-1 degradation and Bim up-regulation as a critical determinant of anoikis initiation. Detachment rapidly degraded Mcl-1 via a GSK-3beta-dependent proteasomal pathway and transcriptionally up-regulated Bim expression. Mcl-1 degradation in the presence of Bim was sufficient to induce anoikis. By analyzing nonmetastatic Saos-2 and metastatic derivative LM7 cells, we confirmed that dysregulation of Mcl-1 degradation and Bim induction during detachment contributes to decreased anoikis sensitivity of metastatic cells. Furthermore, knockdown of Mcl-1 or pharmacologic inhibition of the phosphoinositide-3-kinase/Akt and mitogen-activated protein kinase pathways that suppress Mcl-1 degradation and Bim expression could markedly sensitize metastatic breast cancer cells to anoikis and prevent metastases in vivo. Therefore, Mcl-1 degradation primes the cell for Bax activation and anoikis, which can be blocked by oncogenic signaling in metastatic cells.

Journal Article

Animals; Bioware; Carcinoma; Estradiol; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; PC-3M-luc; Phosphorylation; Prostatic Neoplasms; Radiation-Sensitizing Agents; Subcutaneous Tissue; Transplantation, Heterotopic; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

2-Methoxyestradiol (2ME2) is an endogenous estradiol metabolite that inhibits microtubule polymerization, tumor growth, and angiogenesis. Because prostate cancer is often treated with radiotherapy, and 2ME2 has shown efficacy as a single agent against human prostate carcinoma, we evaluated 2ME2 as a potential radiosensitizer in prostate cancer models. A dose-dependent decrease in mitogen-activated protein kinase phosphorylation was observed in human PC3 prostate cancer cells treated with 2ME2 for 18 h. This decrease correlated with in vitro radiosensitization measured by clonogenic assays, and these effects were blocked by the expression of constitutively active MEK. Male nude mice with subcutaneous PC3 xenografts in the hind leg were treated with 2ME2 (75 mg/kg) p.o. for 5 days, and 2 Gy radiation fractions were delivered each day at 4 h after drug treatment. A statistically significant super-additive effect between radiation and 2ME2 was observed in this subcutaneous model, using analysis of within-animal slopes. A PC-3M orthotopic model was also used, with bioluminescence imaging as an end point. PC-3M cells stably expressing the luciferase gene were surgically implanted into the prostates of male nude mice. Mice were given oral doses of 2ME2 (75 mg/kg), with radiation fractions (3 Gy) delivered 4 h later. Mice were then imaged weekly for 4 to 5 weeks with a Xenogen system. A significant super-additive effect was also observed in the orthotopic model. These data show that 2ME2 is an effective radiosensitizing agent against human prostate cancer xenografts, and that the mechanism may involve a decrease in mitogen-activated protein kinase phosphorylation by 2ME2.

Journal Article

Androgens; Animals; Bioware; Cell Transformation, Neoplastic; Disease Models, Animal; Genes, Reporter; Humans; Image Processing, Computer-Assisted; In Situ Hybridization; Luciferases, Firefly; Luminescent Measurements; Male; Mice; Mice, Transgenic; PC-3M-luc; Promoter Regions, Genetic; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

Journal Article

Biomarkers; imaging; fluorescence; cancer; fluorescence mediated tomography; in vivo imaging

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Journal Article

MMPSense, IVIS, Acrylamides/pharmacology/*therapeutic use; Animals; Arthritis, Experimental/*drug therapy/metabolism/pathology; Cartilage/*metabolism/pathology; Fibroblasts/metabolism/pathology; Humans; Inflammation/metabolism/pathology; Leukocytes/*drug effects/metabolism/pathology; Mice; Nicotinamide Phosphoribosyltransferase/*antagonists & inhibitors; Piperidines/pharmacology/*therapeutic use

OBJECTIVE: To assess the ability of pre-B cell colony-enhancing factor (PBEF) to regulate inflammation and degradative processes in inflammatory arthritis, using the small molecule inhibitor APO866 in human fibroblasts in vitro and in murine collagen-induced arthritis (CIA). METHODS: Enzyme-linked immunosorbent assays were used to examine regulation of expression of metalloproteinases and chemokines in human fibroblasts. The role of PBEF was further examined using APO866 in mice with CIA, with effects on disease activity assessed using radiography, histology, in vivo imaging, and quantitative polymerase chain reaction (qPCR). RESULTS: In vitro activation of human fibroblasts with PBEF promoted expression of matrix metalloproteinase 3 (MMP-3), CCL2, and CXCL8, an effect inhibited by APO866. In mice with CIA, early intervention with APO866 inhibited synovial inflammation, including chemokine-directed leukocyte infiltration, and reduced a systemic marker of inflammation, serum hyaluronic acid. APO866 blockade led to reduced expression of MMP-3 and MMP-13 in joint extracts and to a reduction in a systemic marker of cartilage erosion, serum cartilage oligomeric matrix protein. Radiologic images revealed that APO866 protected against bone erosion, while qPCR demonstrated inhibition of RANKL expression. In mice with established disease, APO866 reduced synovial inflammation and cartilage destruction, and halted bone erosion. In addition, APO866 reduced the activity of MMP-3, CCL2, and RANKL in vivo, and inhibited production of CCL2 and RANKL in synovial explants from arthritic mice, a result that was reversed with nicotinamide mononucleotide. CONCLUSION: These findings confirm PBEF to be an important regulator of inflammation, cartilage catabolism, and bone erosion, and highlight APO866 as a promising therapeutic agent for targeting PBEF activity in inflammatory arthritis.