ABSTRACT
OBJECTIVE: Therapeutic effects of focal adhesion kinase (FAK) inhibition using a small molecule inhibitor was evaluated in apolipoprotein E (apoE) knockout (KO) and low-density lipoprotein receptor (LDLr) KO mouse atherosclerosis models. RESULTS: The prevention trial consisted of an 8-week treatment with an FAK inhibitor concurrent treatment with a high fat (HF)/high cholesterol (HC) diet. The intervention trial consisted of 6- and 8-week treatment after 6- and 8-week pre-loading, respectively, of a HF/HC diet in apoE KO and LDLr KO mice, respectively. The inhibitor was admixed with a HF/HC diet and mice were given free access to the admixture. The FAK inhibitor exhibited marked inhibition against the development of the atherosclerosis in both of prevention and intervention trials at a dose of 0.03% without showing any remarkable toxic properties in biochemical examinations. These results indicated that FAK inhibition might be a possible candidate for novel therapeutic targets against atherosclerosis.
Subject(s)
Apolipoproteins E/deficiency , Atherosclerosis/drug therapy , Atherosclerosis/prevention & control , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Receptors, LDL/deficiency , Animals , Diet, High-Fat , Disease Models, Animal , Male , Mice , Mice, KnockoutABSTRACT
An activating mutation of Fms-like tyrosine kinase 3 (FLT3) is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring FLT3 internal tandem duplication (FLT3-ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L-expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either FLT3-ITD or FLT3-D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with FLT3 mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations.
Subject(s)
Amides/therapeutic use , Antineoplastic Agents/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Mutation , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/genetics , Amides/pharmacokinetics , Amides/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Molecular Docking Simulation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , fms-Like Tyrosine Kinase 3/chemistryABSTRACT
Focal adhesion kinase (FAK) is shown to be frequently correlated with malignancy of the tumor and poor prognosis of the diseases.Because FAK resides immediately downstream of the interaction of cell surface adhesion molecules and extracellular matricies, it is considered to be critical to regulate several cellular processes including growth, differentiation, adhesion, motility and apoptosis. However, the studies on the role of FAK related to cell proliferation have been limited even in vitro. Here, in order to validate the role of FAK in in vivo tumor formation and proliferation, we employed direct intratumoral injection of short hairpin RNA (shRNA) targeting FAK with cationic liposome. Using shRNAs targeting FAK selected from the constructed shRNA library for FAK and by optimization of in vivo delivery conditions, we demonstrated different patterns of the association of FAK inhibition with in vivo tumor formation/proliferation inhibition in two models, PC3M heterotopic xenograft and 4T1 orthotopic syngraft models. These observations indicated that the roles of FAK in tumorigenesis are different among the tumor species. In addition, we showed that ERK is the critical MAP kinase in the signaling pathway down stream of FAK in in vivo proliferation of 4T1 tumor cells.
Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/genetics , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , RNA Interference , Animals , Blotting, Western , Cell Line, Tumor , Female , Focal Adhesion Protein-Tyrosine Kinases/metabolism , HeLa Cells , Humans , Injections , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms, Experimental/therapy , Plasmids/administration & dosage , Plasmids/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/therapy , RNA, Small Interfering/genetics , Tumor Burden , Xenograft Model Antitumor Assays/methodsABSTRACT
We investigated the anti-angiogenic effects of a matrix metalloproteinase inhibitor, (MMI), so called MMI270, against B16-BL6 melanoma through the inhibition of the migrating and invasive abilities of hepatic sinusoidal endothelial (HSE) cells, as well as the formation of tube-like structures by HSE cells. MMI270, at the concentration of 12.5 micrograms/ml, significantly inhibited the migration and invasion of HSE cells, in addition to tube formation by approximately 40%. Furthermore, the enzymatic degradation of metalloproteinases MMP-9 and MMP-2 produced by HSE cells was inhibited by treatment with 1 microgram/ml of MMI270, showing 30% and 100% of inhibition in comparison to the control, respectively. The intraperitoneal administration of MMI270 (200 mg/kg, twice daily for 8 days) after the implantation of B16-BL6 melanoma cells into mice reduced the number of vessels towards the established primary tumor on the dorsal side of mice. These results suggest that MMI270 might be useful as an anti-tumor angiogenic drug.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Hydroxamic Acids , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/pharmacology , Pyrazines , Animals , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured , Endothelium/cytology , Endothelium/drug effects , Endothelium/enzymology , Female , Liver/cytology , Liver/drug effects , Liver/enzymology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Melanoma, Experimental/blood supply , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/drug therapy , SulfonamidesABSTRACT
This study was designed to establish an intrahepatic metastasis model to investigate the biology and therapy of hepatocellular carcinoma (HCC) in mice. A fragment of mouse HCC tumor CBO140C12 was orthotopically implanted into the mouse liver. The number of intrahepatic metastatic colonies and the volume of the implanted tumor increased in a time-dependent manner. At 28 days after fragment implantation, all mice showed intrahepatic metastasis. Intravenous administrations of cisplatin and doxorubicin at 7 and 21 days after the implantation significantly suppressed the growth of the primary tumor nodule, but tended to inhibit intrahepatic metastasis. However, a marked decrease of body weight was observed during the experiment. On the other hand, an inhibitor of matrix metalloproteinases (MMPs), ONO-4817, decreased the gelatinase activity of MMP-9 secreted by CBO140C12 cells, and significantly reduced the number of colonies of intrahepatic metastasis when administered orally. Our established model, which is focused on intrahepatic metastasis, is suitable for evaluating the therapeutic effect of HCC and for analyzing intrahepatic metastasis, because this model reflects the clinical features of HCC and all the steps of tumor metastasis.
