ABSTRACT
PURPOSE: Various proapoptotic agents are currently being explored to improve the outcome of radiotherapy. We have evaluated whether APO010-a novel recombinant ligand of the Fas/CD95 death receptor-enhanced the cytotoxic effect of radiation on lymphoid and solid tumor cell types. EXPERIMENTAL DESIGN: A Bcl-2-overexpressing T-leukemic cell line (Jurkat), a colon carcinoma cell line (HCT116), and a mesothelioma cell line were used as model systems in vitro and in a subcutaneous transplant setting in immunodeficient mice. Sensitivity to single and combined treatment was read out by apoptosis hallmarks and clonogenic survival in vitro, and by tumor growth delay using bioluminescence and palpation in vivo. RESULTS: Whereas the three cell lines resisted apoptosis induction by irradiation and APO010 alone, combined treatment greatly enhanced their apoptotic response. In clonogenic survival assays, APO010 reduced the outgrowth of Jurkat-Bcl-2 and HCT116 cells and sensitized the mesothelioma cell line to radiation. In vivo, systemic treatment with APO010 alone caused tumor growth delay in Jurkat-Bcl-2 and HCT116 cells. However, APO010 did not improve the efficacy of radiotherapy in any of the model systems at the selected single dose, which had moderate and reversible systemic toxicity. CONCLUSIONS: Although APO010 and radiation had a clear combined cytotoxic effect on tumor cells in vitro, a combined therapeutic effect was not achieved on the same cells subcutaneously grafted in mice, at APO010 doses approximating the maximally tolerable level. These findings suggest that it will be difficult to identify a therapeutic window for this combined modality approach in a clinical setting.
Subject(s)
Adiponectin/pharmacology , Fas Ligand Protein/pharmacology , Neoplasms/therapy , Recombinant Fusion Proteins/pharmacology , Adiponectin/toxicity , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Body Weight/drug effects , Body Weight/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Combined Modality Therapy , Fas Ligand Protein/toxicity , HCT116 Cells , Humans , Jurkat Cells , Mice , Mice, Inbred BALB C , Xenograft Model Antitumor AssaysABSTRACT
Malignant mesothelioma is a devastating disease that has been associated with loss of Neurofibromatosis type 2 (NF2) and genetic lesions affecting RB and P53 pathways. We introduced similar lesions in the mesothelial lining of the thoracic cavity of mice. Mesothelioma developed at high incidence in Nf2;Ink4a/Arf and Nf2;p53 conditional knockout mice with median survival times of approximately 30 and 20 weeks, respectively. Murine mesothelioma closely mimicked human malignant mesothelioma. Conditional Nf2;Ink4a/Arf mice showed increased pleural invasion compared to conditional Nf2;p53 mice. Interestingly, upon Ink4a loss in the latter mice median survival was significantly reduced and all tumors were highly invasive, suggesting that Ink4a loss substantially contributes to the poor clinical outcome of malignant mesothelioma.
Subject(s)
Cell Transformation, Neoplastic/pathology , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Epithelial Cells/metabolism , Mesothelioma/pathology , Neurofibromatosis 2/metabolism , Thoracic Cavity/metabolism , Thoracic Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Adenoviridae/genetics , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Epithelial Cells/pathology , Epithelioid Cells/metabolism , Epithelioid Cells/pathology , Genetic Vectors , Genotype , Immunohistochemistry , Integrases/genetics , Integrases/metabolism , Loss of Heterozygosity , Luminescent Measurements , Mesothelioma/genetics , Mesothelioma/metabolism , Mice , Mice, Knockout , Mixed Tumor, Malignant/metabolism , Mixed Tumor, Malignant/pathology , Neoplasm Invasiveness , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neurofibromatosis 2/genetics , Phenotype , Recombination, Genetic , Sarcoma/metabolism , Sarcoma/pathology , Thoracic Cavity/pathology , Thoracic Neoplasms/genetics , Thoracic Neoplasms/metabolism , Time Factors , Tumor Suppressor Protein p53/geneticsABSTRACT
Most of the locally advanced and metastatic prostate carcinomas (PCs) treated with antiandrogenic therapy eventually become refractory to this treatment. Locally produced factors may control prostate tumor biology after androgen withdrawal. Adrenomedullin (AM) is expressed in the prostate and could control cell growth in androgen-independent conditions. AM needs to be amidated by the enzyme peptidylglycine alpha-amidating monooxygenase (PAM) to become fully active. The objective of the present study was to analyze whether the expression of preproadrenomedullin (preproAM) and PAM in PC is regulated by androgens. For this purpose, human in vitro and in vivo PC models were grown in the presence or absence of androgens, and the expression of AM and PAM was examined by immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. Furthermore, immunohistochemical analysis of AM in clinical specimens was performed to test if its expression is related to Gleason score and antiandrogenic therapy. In PC cell lines and xenografts, mRNA and protein AM levels were similar in the presence or absence of androgens. PAM expression seemed to be induced by androgen-withdrawal. Our results in clinical samples showed no relationship between AM expression and Gleason score or antiandrogenic treatment. In conclusion, our results demonstrate that preproAM and PAM expression in the human prostate is androgen-independent. In addition, we also report for the first time the expression of a novel PAM transcript in PC, which has not been previously described in other tissues.
Subject(s)
Mixed Function Oxygenases/genetics , Multienzyme Complexes/genetics , Peptides/genetics , Prostate/metabolism , Prostatic Neoplasms/metabolism , Adolescent , Adrenomedullin , Adult , Androgens/deficiency , Androgens/metabolism , Base Sequence , Blotting, Western , Humans , Immunohistochemistry , Male , Mixed Function Oxygenases/biosynthesis , Molecular Sequence Data , Multienzyme Complexes/biosynthesis , Peptides/metabolismABSTRACT
BACKGROUND: Neuroendocrine (NE) cells are androgen-independent cells and secrete growth-modulating peptide hormones via a regulated secretory pathway (RSP). We studied NE differentiation after long-term androgen withdrawal in the androgen-dependent human prostate cancer xenograft PC-310. METHODS: Tumor-bearing nude mice were killed at 0, 2, 5, 7, 14, 21, 47, 84, and 154 days after castration. The half-life of the PC-310 tumor was 10 days, with a stable residual tumor volume of 30--40% after 21 days and longer periods of androgen deprivation. RESULTS: Proliferative activity and prostate-specific antigen serum levels decreased to zero after castration, whereas cell-cycle arrest was manifested by increased p27(kip1) expression. A temporary downregulation of androgen receptor (AR) expression was noted after androgen deprivation. The expression of chromogranin A, secretogranin III, and secretogranin V (7B2) increased 5 days after castration and later. Subsequently, pro-hormone convertase 1 and peptidyl alpha--amidating monooxygenase as well as vascular endothelial growth factor were expressed from 7 days after castration on. Finally, such growth factors as gastrin-releasing peptide and serotonin were expressed in a small part of the NE cells 21 days after castration, but strong expression was induced late during androgen deprivation, that is, 84 and 154 days after castration, respectively. CONCLUSIONS: Androgen deprivation of the NE-differentiated PC-310 model induced the formation of NE-differentiated AR(minus sign) and non-NE AR(+) tumor residues. The NE-differentiated cells actively produced growth factors via an RSP that may lead to hormone-refractory disease. The dormant non-NE AR(+) tumor cells were shown to remain androgen sensitive even after long-term androgen deprivation. In the PC-310 xenograft, time-dependent NE differentiation and subsequent maturation were induced after androgen depletion. The androgen-dependent PC-310 xenograft model constitutes an excellent model for studying the role of NE cells in the progression of clinical prostate cancer.
