Novel quinazoline HMJ-30 induces U-2 OS human osteogenic sarcoma cell apoptosis through induction of oxidative stress and up-regulation of ATM/p53 signaling pathway

J Orthop Res. 2011 Sep;29(9):1448-56. doi: 10.1002/jor.21398. Epub 2011 Mar 21.

Abstract

Human osteogenic sarcoma is the most common primary bone tumor. Despite of the success of frontline therapy, about 40% of patients have disease progression and further therapy is palliative and toxic. In this study, we developed a novel quinazoline HMJ-30 to investigate the cell growth inhibition and apoptotic responses in U-2 OS human osteogenic sarcoma cells. Our results demonstrated that HMJ-30 significantly reduced cell viabilities of U-2 OS, HOS, and 143B cells in a dose-dependent manner, but it exhibited low cytotoxicity in normal hFOB cells. HMJ-30 induced DNA damage and apoptosis in U-2 OS cells as revealed by morphologic changes, comet assay and DAPI staining. Immuno-staining, colorimetric assays, and Western blotting analyses indicated that activities of caspase-8, caspase-9, and caspase-3 and the levels of Bcl-2 family-related proteins (Bcl-2, Mcl-1, Bax, BAD, and t-Bid) were altered in HMJ-30-treated U-2 OS cells. Pretreatment of cells with caspase-8, -9, and -3 specific inhibitors significantly reduced the cell growth inhibition. HMJ-30-induced apoptosis was mediated through both death-receptor and mitochondria-dependent apoptotic pathways in U-2 OS cells. HMJ-30 induced early phosphorylation of p53(Ser18) was through the activation of ataxia telangiectasia mutated (ATM) in U-2 OS cells. The cell growth inhibition by HMJ-30 was substantially attenuated either by the pre-incubation of U-2 OS cells with N-acetylcysteine (NAC, an antioxidant) and caffeine (an ATM kinase inhibitor) or by p53 knockdown via RNAi. In conclusion, ROS dependent-ATM/p53 signaling pathway is involved in HMJ-30-induced apoptosis in U-2 OS cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Apoptosis / drug effects*
  • Ataxia Telangiectasia Mutated Proteins
  • Caspases / metabolism
  • Cell Cycle Proteins / biosynthesis*
  • Cell Cycle Proteins / genetics
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Comet Assay
  • DNA Damage
  • DNA, Neoplasm / drug effects
  • DNA-Binding Proteins / biosynthesis*
  • DNA-Binding Proteins / genetics
  • Humans
  • Osteosarcoma / drug therapy*
  • Osteosarcoma / metabolism
  • Osteosarcoma / pathology
  • Oxidative Stress / drug effects*
  • Protein-Serine-Threonine Kinases / biosynthesis*
  • Protein-Serine-Threonine Kinases / genetics
  • Quinazolines / pharmacology*
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / drug effects
  • Tumor Stem Cell Assay
  • Tumor Suppressor Protein p53 / biosynthesis*
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Proteins / biosynthesis*
  • Tumor Suppressor Proteins / genetics
  • Up-Regulation / drug effects

Substances

  • 6-fluoro-2-(3-fluorophenyl)-4-(cyanoanilino)quinazoline
  • Antineoplastic Agents
  • Cell Cycle Proteins
  • DNA, Neoplasm
  • DNA-Binding Proteins
  • Quinazolines
  • Reactive Oxygen Species
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases
  • Caspases