title: Novel mechanism of aberrant ZIP4 expression with zinc supplementation in oral tumorigenesis.
abstract: Zrt-Irt-like protein 4 (ZIP4) is critical molecule for proper mammalian development and releasing zinc from vesicular compartments. Recent studies suggested that ZIP4 plays an important role of tumor progression in pancreatic, prostate, and hepatocellular cancers, however, little is known about the detail mechanism of ZIP4 in their cancers. In the present study, we examined the possibility of ZIP4 as a new molecular target for oral squamous cell carcinoma (OSCC). We evaluated ZIP4 expression in OSCC-derived cell lines and primary OSCC samples by quantitative RT-PCR, immunoblotting, and immunohistochemistry (IHC). We also analyzed the clinical correlation between ZIP4 status and clinical behaviors in patients with OSCC. In addition, ZIP4 knockdown cells (shZIP4 cells) and ZnCl2treatment were used for functional experiments, including cellular proliferation assay, zinc uptake assay, and cell-cycle analysis. ZIP4 mRNA and protein were up-regulated significantly in OSCCs compared with normal counterparts in vitro and in vivo. IHC showed that ZIP4 expression in the primary OSCC was positively correlated with primary tumoral size. The shZIP4 cells showed decrease accumulation of intercellular zinc and decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors and down-regulation of cyclins and cyclin-dependent kinases. Since cellular growth of OSCC cells after treatment with zinc was significantly greater than control cells, we speculated that intercellular ZnCl2accumulation is an important factor for cellular growth. Consistent with our hypothesis, not only decreased zinc uptake by ZIP4 knockdown but also chelating agent, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), showed inhibitory effects of cellular proliferation. Therefore, our data provide evidence for an essential role of ZIP4 and intracellular zinc for tumoral growth in OSCC, suggesting that zinc uptake might be a potential therapeutic targeting event for OSCCs.
authors.affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Ishida
authors.affiliation: Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan. Electronic address: kasamatsua@faculty.chiba-u.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kasamatsu
authors.affiliation: Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Endo-Sakamoto
authors.affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Dai
authors.initials: D
authors.lastname: Nakashima
authors.affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Nao
authors.initials: N
authors.lastname: Koide
authors.affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Takahara
authors.affiliation: Division of Oral Surgery, Kashima Rosai Hospital, Ibaraki, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: Division of Oral Surgery, Chiba Rosai Hospital, Chiba, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Iyoda
authors.affiliation: Department of Medical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Shiiba
authors.affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Tanzawa
authors.affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan. Electronic address: uzawak@faculty.chiba-u.jp.
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Uzawa
mesh_terms: D063646:Carcinogenesis; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002614:Chelating Agents / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D019587:Dietary Supplements; D018450:Disease Progression; D004305:Dose-Response Relationship, Drug; D005029:Ethylenediamines / Q000737:chemistry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D009062:Mouth Neoplasms / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*; D017967:Zinc Compounds / Q000737:chemistry
chemical_list: D027682:Cation Transport Proteins; D002614:Chelating Agents; D002712:Chlorides; D005029:Ethylenediamines; D034741:RNA, Small Interfering; C486791:SLC39A4 protein, human; D017967:Zinc Compounds; C016837:zinc chloride; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
keywords: ZIP4 expression; ZRT- and IRT-like protein 4; Zinc