13 research outputs found
Differential inhibition of the TGF-b signaling pathway in HCC cells using the small molecule inhibitor LY2157299 and the D10 monoclonal antibody against TGF-β receptor Type II
We investigated blocking the TGF-b signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761) and a neutralizing humanized antibody (D10) against TGF-bRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-bRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-b receptor cycling in cancer cells
Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II.
We investigated blocking the TGF-β signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761) and a neutralizing humanized antibody (D10) against TGF-βRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-β receptor cycling in cancer cells
Comparison of the effect between LY2157299 and the fully humanized monoclonal antibody D10 on HCC cell morphology, actin cytoskeleton and E-cadherin expression.
<p>HepG2 cells were seeded onto glass coverslips at 3 x104 cells/ml in 24-well plates overnight and treated with control (A, G) or LY2157299 (E, K) or D10 (C, I) alone in serum-free medium. Then, cells were stimulated with TGF-β1 in the absence (B, H) or presence of LY2157299 (F, L) or D10 (D, J). LY2157299, D10 or TGF-β1 were added daily and after 72 hours cells were live-imaged under a phase contrast microscope. For immunofluorescence, cells were fixed with 4% paraformaldehyde, stained with TRITC-phalloidin and anti-E-cadherin antibody and examined under a NIKON eclipse E1000 fluorescence microscope. (M) Cell area was quantified using a combination of two digital image analysis softwares (ImageJ and Adobe Photoshop) and results were plotted as cell area (µm<sup>2</sup>) per random field.**P<0.01.</p
Comparison of the effect between LY2157299 and the fully humanized monoclonal antibody D10 in HCC cell migration.
<p>LY2157299 and D10 were tested in two different HCC cell lines, (A) HepG2 and (B) HLE, that were allowed to migrate on collagen I or matrigel. LY2157299, but not D10, was able to inhibit cell migration of both cell lines on both extracellular matrix components. An anti-isotype IgG was used as internal negative control. Results of representative experiments are shown, and values are expressed as mean ± standard deviation. *P<0.05 **P<0.001 versus TGF-β1.</p
Comparison of the effect of two TGF-βR inhibitor analogs, LY2109761 and LY2157299, on HCC cell migration on different extracellular matrix components.
<p>(A) HLE and (B) HLF cells were allowed to migrate for 16 hours on fibronectin (Fn), vitronectin (Vn), laminin-5 (Ln-5) and fibrinogen (Fg) in the presence or absence (vehicle) of increasing concentrations of LY2109761 or LY2157299. All conditions were performed in duplicate, and each experiment was repeated at least three times. Results of representative experiments are shown, and values are expressed as mean ± standard deviation.</p
RNA expression levels of TGF-β receptor I, TGF-β receptor II and TGF-β1 in different HCC cell lines evaluated by real-time PCR.
<p>Values are expressed as relative gene expression versus GAPDH and represent the mean ±SD of three independent experiments.</p
Immunofluorescence staining of TGF-β receptor II in frozen HCC tissues.
<p>Tissues from 30 patients with HCC were stained using D10, a fully humanized monoclonal antibody directed against TGF-β receptor II (red) and alpha smooth muscle actin with the monoclonal antibody clone 1A4 (green). In 11 out of 30 HCC tissues (36.6%) positivity for TGF-β receptor II was obtained; representative cases of TGF-β receptor II-positive (patient #22 and #29) or -negative (patients #8 and #19) tissues are shown.</p
Effect of LY2109761 and LY2157299 on SMAD2 phosphorylation upon TGF-β1.
<p>(A) Two different HCC cell lines HLE and HLF were pretreated for 16 hours with 100 nM of LY2109761 or LY2157299 and then stimulated with 2 ng of TGF-β1 for 30 min. Phosphorylation of SMAD2 was detected in immunoblots using a rabbit polyclonal antibody directed against Phospho-Smad2 (Ser465/467). (B) Effect of LY2109761 and LY2157299 on E-cadherin expression in HLE and HLF. Increasing expression of E-cadherin is observed in HLE and HLF cells after treatment with both inhibitors for 48 hours.</p
Immunofluorescence staining of p-Smad2 in frozen HCC tissues.
<p>Tissues from 30 patients with HCC were stained using an anti-p-Smad2 polyclonal antibody (red) and alpha smooth muscle actin with the monoclonal antibody clone 1A4 (green). In 20 out of 30 HCC tissues (69.0%) positivity for p-Smad2 was obtained; representative cases of p-Smad2-positive (patient #1 and #3) or -negative (patients #7 and #9) tissues are shown.</p
Effect of LY2157299 and D10 on CTGF and VEGF gene expression in HCC cell lines.
<p>(A) HepG2 and (B) HLE cells were preincubated with LY2157299 (10 µM) or D10 (25 ng/mL) and then stimulated with TGF-β1 (5 ng/mL) in the presence or absence of LY2157299 or D10. After 48 hours, RNA was extracted and real-time PCR was performed. Values are expressed as relative gene expression versus GAPDH and represent the mean ±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001.</p