Hepatocyte nuclear factor-1alpha (HNF1a) is one of the key transcription factors of the HNF family, which plays a critical role in hepatocyte differentiation. Substantial evidence has suggested that down-regulation of HNF1a may contribute to the development of hepatocellular carcinoma (HCC). Herein, human cancer cells and tumor-associated fibroblasts (TAFs) were isolated from human HCC tissues, respectively. A recombinant adenovirus carrying the HNF1a gene (AdHNF1a) was constructed to determine its effect on HCC in vitro and in vivo. Our results demonstrated that HCC cells and HCC tissues revealed reduced expression of HNF1a. Forced reexpression of HNF1a significantly suppressed the proliferation of HCC cells and TAFs and inhibited the clonogenic growth of hepatoma cells in vitro. In parallel, HNF1a overexpression reestablished the expression of certain liver-specific genes and microRNA 192 and 194 levels, with a resultant increase in p21 levels and induction of G 2 /M arrest. Additionally, AdHNF1a inhibited the expression of cluster of differentiation 133 and epithelial cell adhesion molecule and the signal pathways of the mammalian target of rapamycin and transforming growth factor beta/Smads. Furthermore, HNF1a abolished the tumorigenicity of hepatoma cells in vivo. Most interestingly, intratumoral injection of AdHNF1a significantly inhibited the growth of subcutaneous HCC xenografts in nude mice. Systemic delivery of AdHNF1a could eradicate the orthotopic liver HCC nodules in nonobese diabetic/severe combined immunodeficiency mice. Conclusion: These results suggest that the potent inhibitive effect of HNF1a on HCC is attained by inducing the differentiation of hepatoma cells into mature hepatocytes and G 2 /M arrest. HNF1a might represent a novel, promising therapeutic agent for human HCC treatment. Our findings also encourage the evaluation of differentiation therapy for tumors of organs other than liver using their corresponding differentiation-determining transcription factor.
Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC50 ϭ 26.1 Ϯ 0.2 and 27.5 Ϯ 0.5 M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC 50 ϭ 32.3 Ϯ 0.9 and 31.6 Ϯ 1.3 M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg 2ϩ -ATPase activity of the unphosphorylated (IC 50 ϭ 12.6 Ϯ 1.6 and 4.3 Ϯ 0.5 M for gizzard and bovine stomach, respectively) and phosphorylated (IC 50 ϭ 15.0 Ϯ 0.6 M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg 2ϩ -ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC 50 ϭ 14.4 Ϯ 1.6 M) and subfragment 1 (IC50 ϭ 5.5 Ϯ 0.4 M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy CELL MIGRATION IS DRIVEN through complex processes, such as extension of the leading membrane edge, with formation of adhesive contacts and stress fibers (26, 52). Myosin II is widely believed to be one of the main components producing the forces required in this process (12). However, the details of the regulatory mechanism of myosin II in cell migration remain to be established. The general understanding is that cell migration is regulated not only by pathways of signal transduction involving myosin light chain (MLC) phosphorylation, but also by MLC phosphorylation-independent pathways (2,18,28,30). The exact role of myosin II in cell migration and the mechanism whereby myosin II contributes force generation for cell migration remain unanswered.Blebbistatin was recently identified as a specific inhibitor of myosin II-dependent cell processes (49). Its membrane-permeable characteristic and effect on various myosin II isoforms make this agent an invaluable tool in research of myosin II-involved cellular events, including cell motility (5, 23), cell shape maintenance (50), muscle contraction (7, 15), and cytokinesis (49). Blebbistatin has been shown to preferentially bind to the myosin-ADP-phosphate complex with high affinity and prevent phosphate release (24, 37), resulting in inhibition of the actin-myosin interaction. Blebbistatin also inhibited the ability of skeletal muscle and nonmuscle myosin II to move actin f...
The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration.
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