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The Type 1 Insulin-Like Growth Factor Receptor in Hepatocellular Carcinoma Cell Lines Zhengyu Wei1, Ph.D., Tiziana DeAngelis2, M.S., Reginald Hurtt1, B.S., Renato Baserga2, M.D., and Cataldo Doria1, M.D., Ph.D. 1 Division of Transplantation, Department of Surgery; 2 Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107 Methods: A + Mahlavu HepG2 Hep3B PLC Huh7 C-Met β-actin B UU UU A CUCUCUCGGAGGACACUUU AG IGF-1R shRNA Fig. 2. Western blots showing basal expression levels of c-Met (A) and IGF-1R (B) in HCC cells. C Lysate IP:IRS-1 C-Met C Lysate D BT20 BT20 IRS-1 + R600 1. Human HCC cells have higher expression levels of IGF-1R, IRS1, and c-Met. shRNA β-actin C-Met + R600 Ctl IGF-1R β subunit IP:IRS-1 4. Cell viability was detected by MTT method. 48 well plate (2x104 cells/well) was used and 5 mg/ml MTT was added. Summary: Huh7 Ctl shRNA BT20 R600 BT20 0 20 40 60 R600 Huh7 Mahlavu IRS-1 D A G B Mahlavu 2. pLL3.7 lentiviral vector (Rubinson, D.A, et al. 2003,Nature Genetics), was used to construct the human IGF-1R shRNA. Tansfection and transduction were performed according to the manufacturer’s instructions. 4 Ctl IGF-IR shRNA 3 Fig. 3. Immunoprecipitation of c-Met by IRS-1. R600 (overexpression of IGF-1) cells were used for this experiment. BT20 (no IRS-1 expression) was used as negative control. Cells were starved for 24h before stimulated with IGF-1 (20 ng/ml) for 20, 40, and 60 min (C) or HGF (40 ng/ml) for 30 min . Anti-IRS-1 antibody was used to pull down c-Met from total lysate. Immunoblot was performed against c-Met and IRS1. 2. IRS1 interacts with c-Met. The role of IGF-1R in human HCC should be revisited. 3. Lentivirus-mediated shRNA delivery system targeting IGF-1R showed significant suppressive effects on HCC cell growth, promising a potential therapeutic treatment for human HCC. 2 1 0 0 Fig. 1. Schematic diagram of IGF-1R signaling pathway A 5’-GAGAGAGCCUCCUGUGAAA 1. Western blot and immunoprecipitation (IP) were done according to standard protocol. 3. Analysis of colony formation in soft agar was performed on 1,000 cells in each 6-well plate. Colonies (>125um in diameter) were counted 3 weeks after seeding. C Only HepG2 and PLC expressed reasonable levels of IGF-IR, roughly between 10 and 15x103 receptor/cell. The other 3 cell lines expressed levels below 5X103 IGF-IRs/cell (Fig. 2B). All cell lines expressed high levels of the docking protein of the IGFIR, the insulin receptor substrate-1 (IRS-1) (data not shown) which activates the PI3-K signaling pathway. IRS-1, however, also interacts with c-met, the receptor for the hepatocyte growth factor (HGF), which is also present in substantial amounts in the liver (Fig. 2A). IP results showed that IRS1 interacted with c-Met under both HGF and IGF-1 treatments (Fig. 3). Colony formation in soft agar was not strictly correlated with the expression level of IGF-1R (Fig. 2B and Fig. 5), indicating other factors may also involved in the determination of anchorage-independent growth of HCC cells. Lentivirus-mediated shRNA targeting IGF-1R in HCC cells showed significant knockdown of IGF-1R expression (Fig. 4C). Down-regulated IGF-1R inhibited human HCC cell growth (Fig. 4D). 1 2 3 Day Fig. 4. Inhibitory effect on HCC cell growth by a lentiviral vector exprssing IGF-1R shRNA. A. Creation of an shRNA-expressing lentivius vector targeting IGF-1R. B. Transduction efficiencies. C. Functional silencing of IGF-1R in HCC cells. D. Reduced cell viability in Mahlavu cells. Similar result was obtained with Huh7 (not shown). Number of colony The type 1 insulin-like growth factor receptor (IGFIR) plays a major role in growth, differentiation and transformation of cells. Many different types of cancer cells have elevated expression of this receptor. IGF-1R signaling pathway is activated mainly through IRS1 (to Akt) and Erk (Fig. 1). Antibodies to the IGF-IR are now in phase 1 and 2 clinical trials. The liver (in animals and humans) produces large amounts of IGF-1, and provides roughly 75% of the IGF-1 levels in plasma. In addition, hepatocytes do not express IGF-IRs, but they do so in regenerating liver and in some hepatocellular carcinomas (HCC). We investigated the role of the IGFIR and its signaling pathways in a number of HCC cell lines. We also generated stable cell lines using lentivirus-mediated shRNA targeting IGF-1R. We propose to investigate the therapeutic potential of shRNA targeting IGF-1R in human HCC. Results: A Fold Change of Day 0 Introduction: 400 300 200 100 0 HepG2 Huh-7 PLC Mahlavu Hep 3B P6 Cell Line Fig. 5. Colony formation in soft agar of HCC cells. P6 is a mouse cell line over-expressing IGF-1R.