title: Hugl-1 induces apoptosis in esophageal carcinoma cells both in vitro and in vivo.
abstract: OBJECTIVE
  To determine whether the human giant larvae homolog 1 gene (Hugl-1/Llg1/Lgl1) exerts tumor suppressor effects in esophageal cancer.
  
  
  METHODS
  We constructed a Hugl-1 expression plasmid, pEZ-M29-Hugl1, for gene transfection. We transfected the pEZ-M29-Hugl1 plasmid into Eca109 esophageal cancer cell lines with Lipofectamine 2000 to overexpress Hugl-1. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the effects of the plasmid on Hugl-1 expression. In vitro cell proliferation and apoptosis were examined separately by cell counting Kit-8 (CCK-8) assay, flow cytometry, and Western blotting before and after the transfection of the plasmid into Eca109 cells. Cell cycle distribution was assessed with flow cytometry. The effect of Hugl-1 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice. Expression of Hugl-1 in xenograft tumor was analyzed by immunohistochemistry. The transferase-mediated dUTP nick end-labeling (TUNEL) technique was performed to detect and quantitate apoptotic cell.
  
  
  RESULTS
  The transfection efficiency was confirmed with real-time RT-PCR and Western blotting. Our results show that compared with control groups the mRNA levels and protein levels of Hugl-1 in pEZ-M29-Hugl1-treated group were remarkably increased (P < 0.05). The CCK-8 assay demonstrated that the growth of cells overexpressing Hugl-1 was significantly lower than control cells. Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing Hugl-1 (64.09% ± 3.14% vs 50.32% ± 4.60%, 64.09% ± 3.14% vs 49.13% ± 2.24%). Annexin V-fluorescein isothiocyanate revealed that apoptosis was significantly increased in cells overexpressing Hugl-1 compared with control group (17.33% ± 4.76% vs 6.90% ± 1.61%, 17.33% ± 4.76% vs 6.27% ± 0.38%). Moreover, we found that Hugl-1 changes the level of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax and the activation of both caspase-3 and caspase-9. With a TUNEL assay, we found that Hugl-1 markedly increased the apoptosis rate of Eca109 cells in vivo (60.50% ± 9.11% vs 25.00% ± 12.25%). It was shown that Hugl-1 represents a significantly more effective tumor suppressor gene alone in a xenograft tumor mouse model. This data suggest that Hugl-1 inhibited tumor growth and induced cell apoptosis in vivo.
  
  
  CONCLUSIONS
  These results suggest that Hugl-1 induces growth suppression and apoptosis in a human esophageal squamous cell carcinoma cell line both in vitro and in vivo.
authors.affiliation: Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Song
authors.forename: Xiu-Lan
authors.initials: XL
authors.lastname: Peng
authors.forename: Meng-Yao
authors.initials: MY
authors.lastname: Ji
authors.forename: Ming-Hua
authors.initials: MH
authors.lastname: Ai
authors.forename: Ji-Xiang
authors.initials: JX
authors.lastname: Zhang
authors.forename: Wei-Guo
authors.initials: WG
authors.lastname: Dong
mesh_terms: D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D015854:Up-Regulation
chemical_list: D051017:Apoptosis Regulatory Proteins; D003598:Cytoskeletal Proteins; C097906:LLGL1 protein, human
keywords: Apoptosis; Esophageal squamous cell carcinoma; Human giant larvae homolog 1; Proliferation