Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jul 14, 2013; 19(26): 4127-4136
Published online Jul 14, 2013. doi: 10.3748/wjg.v19.i26.4127
Hugl-1 induces apoptosis in esophageal carcinoma cells both in vitro and in vivo
Jia Song, Xiu-Lan Peng, Meng-Yao Ji, Ming-Hua Ai, Ji-Xiang Zhang, Wei-Guo Dong
Jia Song, Xiu-Lan Peng, Meng-Yao Ji, Ming-Hua Ai, Ji-Xiang Zhang, Wei-Guo Dong, Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
Author contributions: Song J and Peng XL performed the majority of experiments; Ji MY, Ai MH and Zhang JX provided vital reagents and analytical tools and were also involved in editing the manuscript; Dong WG designed the study and wrote the manuscript.
Supported by The Fundamental Research Funds for the Central Universities, No. 302274546
Correspondence to: Dr. Wei-Guo Dong, Department of Gastroenterology, Renmin Hospital of Wuhan University, Zhidong Zhang Road 99, Wuhan 430060, Hubei Province, China. dwg@whu.edu.cn
Telephone: +86-27-88041911 Fax: +86-27-88042292
Received: February 27, 2013
Revised: April 15, 2013
Accepted: May 8, 2013
Published online: July 14, 2013

AIM: 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.

CONCLUSION: 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.

Keywords: Esophageal squamous cell carcinoma, Human giant larvae homolog 1, Proliferation, Apoptosis

Core tip: In this paper, we constructed a plasmid to express Hugl-1 which has significant homology to the Drosophila tumor suppressor gene lethal giant larvae. The human esophageal squamous cell carcinoma cell line Eca109 was used as the object of study. We found a positive correlation between Hugl-1 expression and cell apoptosis in Eca109 cells both in vitro and in vivo. These data suggest that Hugl-1 is a tumor suppressor gene in esophageal cancer and may provide a novel target for the treatment of esophageal cancer patients.