ascochlorin and Osteosarcoma

Ascochlorin, a prenylphenol antitumor antibiotic, profoundly increases the expression of endogenous p53 by increasing protein stability in the human osteosarcoma cells and human colon cancer cells. Ascochlorin also increases DNA binding activity to the p53 consensus sequence in nuclear extract and enhances transcription of p53 downstream targets. Ascochlorin specifically induces p53 phosphorylation at ser 392 without affecting ser 15 or 20, whereas DNA damaging agents typically phosphorylate these serines. Moreover, ascochlorin does not induce phosphorylation of ATM and CHK1, an established substrate of ATR that is activated by genotoxins, nor does it increase DNA strand break, as confirmed by comet assay. The structure-activity relationship suggests that p53 activation by ascochlorin is related to inhibition of mitochondrial respiration, which is further supported by the observation that respiratory inhibitors activate p53 in a manner similar to ascochlorin. These results suggest that ascochlorin, through the inhibition of mitochondrial respiration, activates p53 through a mechanism distinct from genotoxins.

Topics: Adenosine Triphosphate; Alkenes; Antibiotics, Antineoplastic; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Bone Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Respiration; Checkpoint Kinase 1; Comet Assay; DNA Breaks, Double-Stranded; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique; Humans; Mitochondria; Osteosarcoma; Phenols; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine; Sesquiterpenes; Structure-Activity Relationship; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Ascochlorin is a prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. Ascochlorin reduces serum cholesterol and triglyceride levels, suppresses hypertension and tumor development, and ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ascochlorin regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the proteome of the human osteosarcoma cells U2OS in response to ascochlorin. In addition, we established the first two-dimensional map of the U2OS proteome. The U2OS cell proteomes with and without treatment with ascochlorin were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and bioinformatics. The largest differences in expression were observed for the epidermal growth factor receptor (4-fold decrease), ribulose-5-phosphate-epimerase (13-fold decrease), ATP-dependent RNA helicase (8-fold decrease), and kelch-like ECH-associated protein 1 (6-fold decrease). The abundance of heterogeneous nuclear ribonucleoprotein L and minichromosome maintenance protein 7 increased 12- and 8.2-fold, respectively. In addition, Erk 2 was increased 3-fold in U2OS cells treated with ascochlorin. The expression of some selected proteins was confirmed by western blotting, zymography and RT-PCR analysis.

Topics: Alkenes; Antineoplastic Agents; Biomarkers, Tumor; Blotting, Western; Bone Neoplasms; Cell Line, Tumor; Electrophoresis, Gel, Two-Dimensional; Humans; Neoplasm Proteins; Osteosarcoma; Phenols; Proteome; Proteomics; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Nuclear receptor family proteins are structurally related transcription factors activated by specific lipophilic compounds. Because they are activated by a variety of hormonal molecules, including retinoic acid, vitamin D, and steroid hormones, they are assumed to be promising targets for clinical drugs. We previously found that one ascochlorin (1) derivative, 4-O-carboxymethyl-ascochlorin (2), is a potent agonist of peroxisome proliferator activated receptor gamma (PPARgamma). Here, we synthesized derivatives of 1, designated as a lead compound, to create new modulators of nuclear hormone receptors. Two derivatives, 4-O-carboxymethyl-2-O-methylascochlorin (9) and 4-O-isonicotinoyl-2-O-methylascochlorin (10), showed improved agonistic activity for PPARgamma and induced differentiation of a progenitor cell line, C3H10T1/2. We also found that 1, dehydroascofuranon (29), and a 2,4-O-diacetyl-1-carboxylic acid derivative of 1 (5) specifically activated estrogen receptors, PPARalpha, and an androgen receptor. All of the derivatives (1-29) activated the pregnane X receptor. These results suggest that the chemical structure of 1 is useful in designing novel modulators of nuclear receptors.

Topics: Alkenes; Animals; Cell Differentiation; Cells, Cultured; Fibroblasts; Furans; Genes, Reporter; Genetic Vectors; Glycolates; Humans; Inhibitory Concentration 50; Ligands; Mice; Models, Molecular; Osteosarcoma; Phenols; Plasmids; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Transfection