ABSTRACT
Osteosarcoma is the most common primary malignancy of the bone and is characterized by local invasion and distant metastasis. Over the past 20 years, long-term outcomes have reached a plateau even with aggressive therapy. Overexpression of insulin-like growth factor 1 receptor (IGF1R) is associated with tumor proliferation, invasion and migration in osteosarcoma. In the present study, our group developed a novel quinazoline derivative, 6-fluoro2-(3-fluorophenyl)-4-(cyanoanilino)quinazoline (HMJ30), in order to disrupt IGF1R signaling and tumor invasiveness in osteosarcoma U2 OS cells. Molecular modeling, immune-precipitation, western blotting and phosphorylated protein kinase sandwich ELISA assays were used to confirm this hypothesis. The results demonstrated that HMJ30 selectively targeted the ATP-binding site of IGF1R and inhibited its downstream phosphoinositide 3-kinase/protein kinase B, Ras/mitogen-activated protein kinase, and IκK/nuclear factor-κB signaling pathways in U2 OS cells. HMJ30 inhibited U2 OS cell invasion and migration and downregulated protein levels and activities of matrix metalloproteinase (MMP)2 and MMP-9. An increase in protein levels of tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2 was also observed. Furthermore, HMJ30 caused U2 OS cells to aggregate and form tight clusters, and these cells were flattened, less elongated and displayed cobblestone-like shapes. There was an increase in epithelial markers and a decrease in mesenchymal markers, indicating that the cells underwent the reverse epithelial-mesenchymal transition (EMT) process. Overall, these results demonstrated the potential molecular mechanisms underlying the effects of HMJ30 on invasiveness and EMT in U2 OS cells, suggesting that this compound deserves further investigation as a potential anti-osteosarcoma drug.
ABSTRACT
The aim of the present study was to explore the effect of 6-fluoro-2-(3-fluorophenyl)-4-(cyanoanilino) quinazoline (HMJ-30) on the anti-angiogenic properties and apoptosis-related mechanism of human umbilical vein endothelial cells (HUVECs). In this study, HMJ-30 dose- and time-dependently inhibited the viability of HUVECs. We also found that HMJ-30 enhanced disruption of tube-like structures and suppressed cell migration in HUVECs after vascular endothelial growth factor (VEGF) induction. HMJ-30 was also observed to inhibit vessel branching and sprouting in chicken chorioallantoic membrane (CAM). Microsprouting induced by VEGF in the rat aortic ring and blood vessel formation in a mouse Matrigel plug were individually suppressed by HMJ-30. In an in vitro study, HMJ-30 induced the apoptotic death of HUVECs as indicated by DNA fragmentation and promoted reactive oxygen species (ROS) production as determined by flow cytometric assay. In addition, extrinsic caspase signaling (caspase-8 and -3) was activated in the HMJ-30-treated HUVECs and their inhibitors were applied to assess the signal transduction. We investigated the upstream of the death receptor pathway and further observed that the levels of death receptor 5 (DR5) and phosphorylated c-Jun N-terminal kinase (JNK) signals were upregulated in HUVECs following HMJ-30 challenge, which was confirmed by a JNK-specific inhibitor (SP600125). Hence, HMJ-30-induced endothelial cell apoptosis involved the ROS/JNK-regulated DR5 pathway. In summary, HMJ-30 may provide a potential therapeutic effect for the anti-vascular targeting of angiogenesis during cancer treatment.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Endothelial Cells/drug effects , MAP Kinase Signaling System/drug effects , Neovascularization, Physiologic/drug effects , Quinazolines/administration & dosage , Angiogenesis Inhibitors/pharmacology , Animals , Anthracenes/pharmacology , Aorta/cytology , Aorta/drug effects , Apoptosis/drug effects , Cell Movement/drug effects , Cells, Cultured , Chick Embryo , DNA/analysis , DNA/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Male , Mice , Quinazolines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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.