Inhibition of osteosarcoma cell growth by geraniin in vivo

• Published in: European journal of cancer (1990) 2016-12, Vol.69, p.S37-S37
• Main Authors: Tatsuda, D, Momose, I, Ohba, S.I, Umezawa, Y, Kawada, M, Shibasaki, M
• Format: Article
• Language: English
• Subjects: Activation; Apoptosis; Assaying; Binding; Biocompatibility; Biomedical materials; Bone cancer; Cancer; Cell cycle; Cells; Chemical agents; Colonies; Degradation; Docking; Doxorubicin; Drug development; Energy measurement; Energy transfer; Fluorescence; Gastric cancer; Glioblastoma; Hematology, Oncology and Palliative Medicine; Implantation; Inhibition; Iridium; Lysates; MDM2 protein; Natural products; Osteosarcoma; p53 Protein; Poly(ADP-ribose) polymerase; Proteasomes; Radiation; Radiation damage; Sarcoma; Studies; Surgical implants; Tumor cells; Tumors; Ubiquitin; Ubiquitin-protein ligase; Ultraviolet radiation; Xenografts; Xenotransplantation
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/S0959-8049(16)32691-0

Background: p53 is a regulated transcription factor that induces cell cycle arrest or apoptosis in response to damage by UV and IR radiation, and chemical agents. In unstressed cells, Mdm2. an ubiquitin E3 ligase, binds to p53 and suppresses p53 activity. Mdm2 also ubiquitinates p53 and induces degradation of p53 by 26S proteasomes. Inhibition ol the p53-Mdm2 interaction, however, leads to cell cycle arrest and apoptosis. Because p53 regulates the cell cycle and apoptosis in tumor cells, Inhibition of the p53-Mdm2 interaction is an attractive target for the development of anti-cancer drugs. We established a cell-free assay to screen natural products that could inhibit the p53-Mdm2 interaction. This screening led to the identification of geraniin, an ellagilannins. Material and Methods: A time-resolved fluorescence energy transfer assay was used to measure inhibition of the p53-Mdm2 interaction. Human glioblastoma LNZTA3 cells with tetracycline-regulated p53 expression, human gastric cancer MKN45 cells (p53 wild-type), and MKN7 cells (p53 mutant-type) were incubated with geraniin for 14 days, and cell viability was measured by colony formation assay. Human osteosarcoma SJSA-1-p53 wild-type) tumor-bearing mice were treated with geraniin or/and doxorubicin twice per week lor 3 weeks. Tumor weights were measured 4 weeks after implantation. Results: The time-resolved fluorescence energy transfer assay revealed that geraniin inhibited the p53-Mdm2 interaction. To investigate the binding mode of geraniin, we created a structural model of geraniin bound to Mdm2. The docking study suggested that geraniin bound to Mdm2 In the p53-binding pocket and mimicked the interaction of the p53 peptide to a high degree. The colony formation assay revealed that geraniin suppressed p53-dependent growth in human glioblastoma and human gastric cancer cells. To confirm activation of the p53-dependent apoptosis pathway by geraniin, we prepared lysates of LNZTA3 cells (presence and absence of p53) treated with geraniin and performed immunoblot analysis. Geraniin led to the accumulation of p53. Mdm2. p21, and PUMA, and promoted the degradation of PARP in LNZTA3 cells-presence of p53). In a xenograft model, geraniin suppressed osteosarcoma SJSA-1 tumor growth. The combination of geraniin and doxorubicin showed higher inhibition of osteosarcoma growth than each single agent in vivo. Conclusions: Geraniin suppressed p53-dependent growth by inhibiting the p53-Mdm2 interaction in vitro. Th