ascochlorin and Bone-Neoplasms

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