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. 2018 Feb 27;51(1):53-62.
doi: 10.1267/ahc.17035. Epub 2018 Feb 21.

Expression of Draxin in Lung Carcinomas

Younosuke Sato  1 , Akira Matsuo  1 , Shinji Kudoh  1 , Liu Fang  1   2 , Koki Hasegawa  3 , Yohei Shinmyo  4 , Takaaki Ito  1
Affiliations

Expression of Draxin in Lung Carcinomas

Younosuke Sato et al. Acta Histochem Cytochem. .

Abstract

Guidance molecules, such as Netrin-1, and their receptors have important roles in controlling axon pathfinding, modulate biological activities of various cancer cells, and may be a useful target for cancer therapy. Dorsal repulsive axon guidance protein (Draxin) is a novel guidance molecule that binds not only common guidance molecule receptors with Netrin-1, but also directly binds the EGF domain of Netrin-1 through a 22-amino-acid peptide (22aa). By immunostaining, Draxin was positively expressed in small cell carcinoma, adenocarcinoma (ADC), and squamous cell carcinoma of the lung. In addition, western blot analysis revealed that Draxin was expressed in all histological types of lung cancer cell lines examined. Knockdown of Draxin in an ADC cell line H358 resulted in altered expression of molecules associated with proliferation and apoptosis. The Ki-67 labeling index of Draxin-knockdown ADC cells was increased compared to that of control ADC cells. In H358 cells, treatment of 22aa induced phosphorylation of histone H3, but did not change apoptosis-associated enzymes. These data suggest that Draxin might be involved in cell proliferation and apoptosis in lung adenocarcinoma cells VSports手机版. .

Keywords: Draxin; Netrin-1; cell proliferation; dependence receptor; lung cancer V体育安卓版. .

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V体育安卓版 - Figures

Fig. 1.
Fig. 1.
Immunohistochemical analysis of the expression of Draxin, Netrin-1, and their receptors, DCC and Neogenin, in normal lung tissue. The surface of alveolar and bronchiolar epithelial cells was positively stained fot Netrin-1. Draxin and dependence receptors were weakly or poorly stained in alveolar and bronchiolar epithelial cells. Bar = 20 μm.
Fig. 2.
Fig. 2.
Immunohistochemical analysis of the expression of Draxin, Netrin-1, and their receptors, DCC and Neogenin, in lung cancer tissue. Draxin was positively stained in all types of lung cancer, especially in ADC and SCC. Draxin was clearly expressed in lung cancer compared with that of Netrin-1 and receptors. Bar = 20 μm.
Fig. 3.
Fig. 3.
Western blot analysis of Draxin, Netrin-1, and their receptors, DCC and Neogenin, in lung cancer cell lines. Western blot analysis was performed using 13 human lung cancer cell lines including seven SCLC, three ADC, and three SCC cell lines. β-actin served as an internal control.
Fig. 4.
Fig. 4.
Evaluation of the relationship between Draxin and tumorigenesis. (A) Western blot analysis of Draxin knockdown in human lung adenocarcinoma cells (H358). Three biological replicates were analyzed. Representative examples of no treatment (NT), transfected control siRNA (Si-cont.), and Draxin siRNA (Si-Draxin) are shown. Expression of phosphorylated-histone H3 (ser10) and CyclinD1 as mitotic markers, antibodies against Caspase3 (CASP3), Cleaved Caspase3 (cCASP3), Caspase8 (CASP8), Caspase9 (CASP9), B-cell lymphoma 2 (Bcl-2), and phosphorylated-Bcl-2 (pBcl-2) as apoptosis-related markers, and antibodies against AKT and phosphorylated-AKT (pAKT) as markers of cell signaling activity. Draxin knockdown was successfully performed in Si- Draxin cells. All markers showed similar expression patterns between NT and Si-cont. Draxin siRNA induced the suppression of CASP3, CASP8 and CASP9. In addition, Draxin siRNA elevated of pHH3 levels. β-actin served as an internal control. (B) Ki-67 (MIB-1) index analysis of Draxin knockdown in human lung adenocarcinoma cell (H358). Upper panel, transfected control siRNA (control) where a moderate number of adenocarcinoma cells were positive for Ki-67. Lower panel, Draxin siRNA (Si-Draxin) where most adenocarcinoma cells were positive for Ki-67. Bar = 100 μm. (C) The average Ki-67 index in transfected control siRNA cells was 41.9%. The average Ki-67 index in Draxin siRNA cells was 63.6%. * p < 0.01.
Fig. 5.
Fig. 5.
Western blot analysis of adenocarcinoma cells (H358) treated with Draxin 22-amino-acid peptide. Four biological replicates were analyzed. Representatives of no treatment (NT), and treatment treated with Draxin 22-amino acid peptide in an amount of 1 μmol, 10 μmol and 100 μmol are shown. Reactivity of antibody markers are consistent with western blot analysis of Draxin knockdown in H358.
Fig. 6.
Fig. 6.
NNK-induced lung tumors in wild and Draxin gene-deficient mice. (A) Adenoma in a wild mouse. (B) Adenocarcinoma in a wild mouse is well to moderately-differentiated papillary type. Tumor size was about 2 mm. (C) Adenoma in a Draxin gene-deficient mouse. (D) Adenocarcinoma in a Draxin gene-deficient mouse is well to moderately-differentiated papillary type. Tumor size was about 2 mm. Bar = 20 μm.
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