ABSTRACT
Integrin-linked kinase (ILK), a highly conserved intracellular protein of serine/threonine protein kinase activities, which is associated with the integrin and growth factor receptor signaling pathway, is involved in the regulation of cell proliferation, apoptosis, differentiation, migration and epithelial-mesenchymal transition (EMT). Findings of a previous study showed that ILK overexpression was strongly correlated with a more aggressive tumor phenotype, recurrence and poor survival for oral squamous cell carcinoma (OSCC) patients, as well as some EMT markers. In order to investigate the underlying mechanisms involved, a lentivirus-mediated short hairpin RNA (shRNA) was employed to downregulate ILK. The results showed that the knockdown of ILK inhibited cell growth, adhesion and invasion ability in vitro, and OSCC cells deficient of ILK were blocked in the S phase and underwent apoptosis. Additionally, ILK shRNA inhibited EMT by impairing the expression of Snail, Slug and Twist2 and enhacning E-cadherin expression. ILK shRNA suppressed the phosphorylation of downstream signaling targets Akt and GSk-3ß. In addition, the knockdown of ILK inhibited tumor growth, invasion and metastasis of xenograft tumors in vivo. These results suggested that ILK is a promising therapeutic target for the treatment of OSCC.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/therapy , Mouth Neoplasms/metabolism , Mouth Neoplasms/therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , RNA, Small Interfering/genetics , Carcinoma, Squamous Cell/genetics , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Genetic Vectors/administration & dosage , Humans , In Vitro Techniques , Lentivirus/genetics , Mouth Neoplasms/genetics , Neoplasm Transplantation , Xenograft Model Antitumor AssaysABSTRACT
His296 of Zymomonas mobilis levansucrase (EC 2.4.1.10) is crucial for the catalysis of the transfructosylation reaction. The three-dimensional structures of levansucrases revealed the His296 is involved in the substrate recognition and binding. In this study, nine mutants were created by site-directed mutagenesis, in which His296 was substituted with amino acids of different polarity, charge and length. The substitutions of His296 with Arg or Trp retained partial hydrolysis and transfructosylation activities. The positively charged Lys substitution resulted in a 2.5-fold increase of sucrose hydrolysis. Substitutions with short (Cys or Ser), negatively charged (Glu) or polar (Tyr) amino acids virtually abolished both the activities. Analysis of transfructosylation products indicated that the mutants synthesized different oligosaccharides, suggesting that amino acid substitutions of His296 strongly affected both the enzyme activity and transfructosylation products.