Original Article
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 14, 2014; 20(22): 6844-6859
Published online Jun 14, 2014. doi: 10.3748/wjg.v20.i22.6844
Role of the Na+/K+/2Cl- cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma
Atsushi Shiozaki, Yoshito Nako, Daisuke Ichikawa, Hirotaka Konishi, Shuhei Komatsu, Takeshi Kubota, Hitoshi Fujiwara, Kazuma Okamoto, Mitsuo Kishimoto, Yoshinori Marunaka, Eigo Otsuji
Atsushi Shiozaki, Yoshito Nako, Daisuke Ichikawa, Hirotaka Konishi, Shuhei Komatsu, Takeshi Kubota, Hitoshi Fujiwara, Kazuma Okamoto, Eigo Otsuji, Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
Mitsuo Kishimoto, Department of Pathology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
Yoshinori Marunaka, Departments of Molecular Cell Physiology and Bio-Ionomics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
Yoshinori Marunaka, Japan Institute for Food Education and Health, St. Agnes’ University, Kyoto 602-8013, Japan
Author contributions: Shiozaki A and Nako Y contributed equally to this work; Shiozaki A and Nako Y designed the study, wrote the manuscript and performed the majority of the experiments; Ichikawa D, Konishi H, Komatsu S, Kubota T, Fujiwara H, Okamoto K and Kishimoto M collected and analyzed the clinicopathological data obtained from ESCC patients; Marunaka Y and Otsuji E were involved in editing the manuscript.
Supported by Grants-in-Aid for Young Scientists (B), NO. 22791295, NO. 23791557, and NO. 24791440; and a Grant-in-Aid for Scientific Research (C), NO.22591464 and NO. 24591957, from the Japan Society for the Promotion of Science
Correspondence to: Atsushi Shiozaki, Assistant Professor, Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan. shiozaki@koto.kpu-m.ac.jp
Telephone: +81-75-2515527 Fax: +81-75-2515522
Received: October 28, 2013
Revised: January 17, 2014
Accepted: February 17, 2014
Published online: June 14, 2014
Abstract

AIM: To investigate the role of Na+/K+/2Cl- cotransporter 1 (NKCC1) in the regulation of genes involved in cell cycle progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC).

METHODS: An immunohistochemical analysis was performed on 68 primary tumor samples obtained from ESCC patients that underwent esophagectomy. NKCC1 expression in human ESCC cell lines was analyzed by Western blotting. Knockdown experiments were conducted using NKCC1 small interfering RNA, and the effects on cell cycle progression were analyzed. The gene expression profiles of cells were analyzed by microarray analysis.

RESULTS: Immunohistochemical staining showed that NKCC1 was primarily found in the cytoplasm of carcinoma cells and that its expression was related to the histological degree of differentiation of SCC. NKCC1 was highly expressed in KYSE170 cells. Depletion of NKCC1 in these cells inhibited cell proliferation via G2/M phase arrest. Microarray analysis identified 2527 genes with altered expression levels in NKCC1depleted KYSE170. Pathway analysis showed that the top-ranked canonical pathway was the G2/M DNA damage checkpoint regulation pathway, which involves MAD2L1, DTL, BLM, CDC20, BRCA1, and E2F5.

CONCLUSION: These results suggest that the expression of NKCC1 in ESCC may affect the G2/M checkpoint and may be related to the degree of histological differentiation of SCCs. We have provided a deeper understanding of the role of NKCC1 as a mediator and/or a biomarker in ESCC.

Keywords: Na+/K+/2Cl- cotransporter 1, Esophageal cancer, Cell cycle

Core tip: The objectives of the present study were to investigate the role of Na+/K+/2Cl- cotransporter 1 (NKCC1) in the regulation of genes involved in cell cycle progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC). An immunohistochemical analysis revealed that the expression of NKCC1 in ESCC samples was related to the histological type. Microarray results suggested that NKCC1 exhibits marked effects on the expression of genes related to G2/M cell cycle progression. A deeper understanding of the role of NKCC1 may lead to its use as an important biomarker and/or a novel therapeutic target for ESCC treatment.