title: Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells.
abstract: OBJECTIVE
  To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin (IL)-24 in hepatocellular carcinoma (HCC) cells.
  
  
  METHODS
  γ-secretase inhibitors (GSIs) were used to down-regulate Notch1. HepG2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h. Cell viability was measured by MTT assay. The cellular and nuclear morphology was observed under a fluorescence microscope. To further verify the apoptotic phenotype, cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining. The expression of Notch1, SNAIL1, SNAIL2, E-cadherin, IL-24, XIAP and VEGF was detected by Western blot. The invasion and migration capacities of HCC cells were detected by wound healing assays. Notch1 and Snail were down-regulated by RNA interference, and the target proteins were analyzed by Western blot. To investigate the mechanism of apoptosis, we analyzed HepG2 cells treated with siNotch1 or siCON plus IL-24 or not for 48 h by caspase-3/7 activity luminescent assay.
  
  
  RESULTS
  GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in HepG2 cells. The addition of 50 ng/mL IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%, respectively. Treatment with IL-24 alone did not induce any cytotoxic effect. In SMMC7721 cells with the addition of IL-24 to GSI-I (2.5 μmol/L), the reduction of cell viability was only about 25%. Following GSI-I/IL-24 combined treatment for 6 h, the apoptotic rate of HepG2 cells was 47.2%, while no significant effect was observed in cells treated with the compounds employed separately. Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in HepG2 cells. Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I. Furthermore, the increased GSI-I and IL-24 in HepG2 cell was associated with downregulation of MMP-2, XIAP and VEGF. In the absence of treatment, HepG2 cells could migrate into the scratched space in 24 h. With IL-24 or GSI-I treatment, the wound was still open after 24 h. And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I. Treatment of Notch-1 silenced HepG2 cells with 50 ng/mL IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination. Caspase-3/7 activity was increased in the presence of siNotch1 plus IL-24 treatment.
  
  
  CONCLUSIONS
  Down-regulation of Notch1 by GSI-I or siRNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of HepG2 cells.
authors.affiliation: Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Han
authors.affiliation: Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China.
authors.forename: Shi-Hai
authors.initials: SH
authors.lastname: Liu
authors.affiliation: Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China.
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Guo
authors.affiliation: Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Wang
authors.affiliation: Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China.
authors.forename: Yu-Kun
authors.initials: YK
authors.lastname: Cao
authors.affiliation: Bing Han, Jian-Ping Wang, Yu-Kun Cao, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D002465:Cell Movement / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D007378:Interleukins / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D009361:Neoplasm Invasiveness; D009842:Oligopeptides / Q000494:pharmacology*; D034622:RNA Interference; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D000099180:Interleukin-24
chemical_list: D053829:Amyloid Precursor Protein Secretases; D004791:Enzyme Inhibitors; D007378:Interleukins; D009842:Oligopeptides; D051881:Receptor, Notch1; D000099180:Interleukin-24; C497761:NOTCH1 protein, human; C523462:benzyloxycarbonyl-leucyl-leucyl-norleucinal
keywords: Hepatocellular carcinoma; Interleukin-24; Invasion; Migration; Notch signaling pathway; γ-secretase inhibitor