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. 2016 Jun 14;7(24):37000-37012.
doi: 10.18632/oncotarget.9454.

Glutaredoxin 3 promotes nasopharyngeal carcinoma growth and metastasis via EGFR/Akt pathway and independent of ROS

Feng He  1 , Lili Wei  2 , Wenqi Luo  2 , Zhipeng Liao  1 , Bo Li  1 , Xiaoying Zhou  1 , Xue Xiao  1 , Jingping You  1 , Yufeng Chen  1 , Shixing Zheng  1 , Ping Li  2 , Mariko Murata  3 , Guangwu Huang  1 , Zhe Zhang  1
Affiliations

VSports最新版本 - Glutaredoxin 3 promotes nasopharyngeal carcinoma growth and metastasis via EGFR/Akt pathway and independent of ROS

Feng He et al. Oncotarget. .

Abstract

Glutaredoxin 3 (GLRX3) is antioxidant enzyme, maintaining a low level of ROS, thus contributing to the survival and metastasis of several types of cancer. However, the expression and functions of GLRX3 have not been addressed in nasopharyngeal carcinoma (NPC). In this study, we found that GLRX3 was overexpressed in NPC. Knockdown of GLRX3 in NPC cell lines inhibited proliferation in vitro, tumorignesis in vivo, and colony formation. In addition, GLRX3 knockdown decreased the migration and invasion capacity of NPC cells by reversing the epithelial-mesenchymal transition (EMT). Furthermore, stabilization of GLRX3 was positively related to with epidermal growth factor receptor (EGFR) expression and negatively with ROS generation. Phosphorylation of Akt, a key downstream effector, was induced by EGFR signaling but did not rely on increasing ROS level in NPC cells. GLRX3 might be an oncoprotein in NPC, playing important roles in increasing redox reaction and activating EGFR/ Akt signals, so it may be a therapeutic target for NPC. VSports手机版.

Keywords: Akt; EGFR; glutaredoxin 3; nasopharyngeal carcinoma V体育安卓版. .

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VSports在线直播 - Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. mRNA level of GLRX3 in nasopharyngeal carcinoma (NPC) and normal nasopharyngeal epithelia (NNE)
(A) Real-time PCR of the mRNA level of GLRX3 in 6 NPC cell lines and a non-cancerous nasopharyngeal epithelial cell line NP69. (B) Relative GLRX3 mRNA expression in NPC primary biopsies (n = 20) and NNE samples (n = 20). Boxes indicate 25 to 75 percentile, horizontal line indicates the mean, and bars indicate 10 and 90 percentile (*p < 0.05).
Figure 2
Figure 2. Immunohistochemical staining of GLRX3 protein expression in NPC (n = 59) and NNE tissue (n = 30)
Magnification ×400.
Figure 3
Figure 3. Knockdown of GLRX3 suppresses the growth of NPC cell line in vitro
(A) Western blot confirmation of the silencing effect of shGLRX3 construct in HONE1 and CNE2 cell lines. (B) MTT assay of growth curves of shGLRX3-HONE1 and shGLRX3-CNE2 and their shCtrl cell line. Data are mean ± SD of five independent experiments. (C) Representative colony images and quantification of colonies in CNE2 cells with and without GLRX3 knockdown. Data are mean ± SD of three independent experiments (**p < 0.01).
Figure 4
Figure 4. Knockdown of GLRX3 reduced tumorigenicity of NPC cells in nude mice
(A) Tumors from nude mice 2 weeks after inoculation. (B) Immunohistochemical staining of GLRX3 protein expression in tumors from nude mice. Magnification ×400. (C) Growth curves of tumors derived from HONE1 and CNE2 cells with and without shGLRX3 in nude mice. The volume of tumors was measured every 2 days after inoculation. Data are mean ± SD from five experiments.
Figure 5
Figure 5. Knockdown of GLRX3 inhibits migration and invasion of NPC cells
(A) Wound-healing assay. Images were taken at 0 and 12 h after introducing a scratch in shGLRX3-HONE1/CNE2 and shCtrl-HONE1/CNE2 cells. (B) Gap closure is measured as mean ± SD of three independent experiments. (C) Transwell invasion assay of invasive capacity of HONE1 and CNE2 cells. The violet color dots represent cells penetrating through matrix gels. (E) Real-time RT-PCR assay of E-cadherin, β-catenin, Vimentin and MMP9 mRNA levels (D) and western blot assay of protein levels (E). Data are mean ± SD from three experiments. (**p < 0.01)
Figure 6
Figure 6. Knockdown of GLRX3 in NPC cells decreases pAKT independent of reactive oxygen species (ROS) generation
(A) Western blot analysis of protein levels of pAkt and Akt in shGLRX3-HONE1/CNE2 cell lines. (B) ROS were detected by DCFH-DA staining with green fluorescent signal (magnification ×200). (C) The intensity of ROS was calculated as relative light unit. Data are mean±SD from three experiments (D–E) ROS detection in shGLRX3-HONE1 and shGLRX3-CNE2 cells treated with and without ROS inhibitor 5 mM NAC for 48 h, followed by intensity analysis. (F) Western blot analysis of pAkt protein level. Data are mean ± SD from three experiments. (**p < 0.01)
Figure 7
Figure 7. Epidermal growth factor receptor (EGFR) is essential for the effects of GLRX3 on inhibiting pAkt
(A–C) Real-time RT-PCR assay of mRNA levels of EGFR in shGLRX3-HONE1/CNE2 and shCtrl-HONE1/CNE2 cells (A), and western blot (B) and immunofluorescence (C) assay of protein levels. Data are mean ± SD from three experiments. (D) pAkt detection in shGLRX3-HONE1 and shGLRX3-CNE2 cells treated with and without EGFR stimulator 100 ng/ml EGF for 48 h, followed by western blot analysis. (**p < 0.01)
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