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Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis.

Ming H, Lan Y, He F, Xiao X, Zhou X, Zhang Z, Li P, Huang G - Chin J Cancer (2015)

Bottom Line: Inactivation of CYB5R2 is associated with lymph node metastasis in NPC.A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis.Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, P.R. China. yskm128@163.com.

ABSTRACT

Background: Cytochrome b5 reductase 2 (CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma (NPC). Inactivation of CYB5R2 is associated with lymph node metastasis in NPC. This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.

Methods: Polymerase chain reaction (PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1. NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2, and data was validated by real-time PCR. A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis. An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor (VEGF).

Results: In CYB5R2-transfected NPC cells, PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), interferon beta 1 (IFNB1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) and down-regulated levels of integrin beta 5 (ITGB5), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), transforming growth factor beta receptor 1 (TGFBR1), and VEGF. The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited. Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.

Conclusion: CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis, thereby suppressing tumor formation.

No MeSH data available.


Related in: MedlinePlus

Ectopic expression of cytochrome b5 reductase 2 (CYB5R2) in nasopharyngeal carcinoma (NPC) cell lines CNE2 and HONE1. Relative expression of CYB5R2 in NPC cell lines was confirmed by real-time polymerase chain reaction (PCR) 48 h after transfection of CYB5R2 or empty vector plasmids. Data are presented as mean ± standard deviation (SD) of three experiments. *P < 0.05.
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Fig1: Ectopic expression of cytochrome b5 reductase 2 (CYB5R2) in nasopharyngeal carcinoma (NPC) cell lines CNE2 and HONE1. Relative expression of CYB5R2 in NPC cell lines was confirmed by real-time polymerase chain reaction (PCR) 48 h after transfection of CYB5R2 or empty vector plasmids. Data are presented as mean ± standard deviation (SD) of three experiments. *P < 0.05.

Mentions: To determine the mechanisms involved in the tumor suppressive effect of CYB5R2 on NPC cells, we first confirmed that CYB5R2 mRNA level was increased in NPC cells (CNE2 and HONE1) after transfection with a CYB5R2 expression plasmid (Figure 1). We then used PCR assays to evaluate changes in the mRNA levels of 84 genes related to cell proliferation, apoptosis, cell cycle, angiogenesis, invasion, and metastasis in HONE1 cells that were transfected with CYB5R2 or an empty vector. The mRNA levels of 12 genes were significantly altered by CYB5R2 overexpression (up- or down-regulated by at least 1.5-fold compared with empty vector control) (Table 2). Several genes involved in apoptosis, signal transduction and transcription, cell cycle control, and DNA damage repair [Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), and cyclin-dependent kinase inhibitor 2A (CDKN2A)] were up-regulated. Some genes involved in angiogenesis (vascular endothelial growth factor A (VEGFA) [16]), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), and transforming growth factor beta receptor 1 (TGFBR1) were down-regulated, whereas one, interferon beta 1 (IFNB1), was up-regulated. The mRNA levels of several genes involved in adhesion [integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), and integrin beta 5 (ITGB5)] were up-regulated or down-regulated with CYB5R2 expression. These changes in the gene expression in CYB5R2-transfected NPC cells (CNE2 and HONE1) was verified by real-time PCR (Figure 2).Figure 1


Cytochrome b5 reductase 2 suppresses tumor formation in nasopharyngeal carcinoma by attenuating angiogenesis.

Ming H, Lan Y, He F, Xiao X, Zhou X, Zhang Z, Li P, Huang G - Chin J Cancer (2015)

Ectopic expression of cytochrome b5 reductase 2 (CYB5R2) in nasopharyngeal carcinoma (NPC) cell lines CNE2 and HONE1. Relative expression of CYB5R2 in NPC cell lines was confirmed by real-time polymerase chain reaction (PCR) 48 h after transfection of CYB5R2 or empty vector plasmids. Data are presented as mean ± standard deviation (SD) of three experiments. *P < 0.05.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4593386&req=5

Fig1: Ectopic expression of cytochrome b5 reductase 2 (CYB5R2) in nasopharyngeal carcinoma (NPC) cell lines CNE2 and HONE1. Relative expression of CYB5R2 in NPC cell lines was confirmed by real-time polymerase chain reaction (PCR) 48 h after transfection of CYB5R2 or empty vector plasmids. Data are presented as mean ± standard deviation (SD) of three experiments. *P < 0.05.
Mentions: To determine the mechanisms involved in the tumor suppressive effect of CYB5R2 on NPC cells, we first confirmed that CYB5R2 mRNA level was increased in NPC cells (CNE2 and HONE1) after transfection with a CYB5R2 expression plasmid (Figure 1). We then used PCR assays to evaluate changes in the mRNA levels of 84 genes related to cell proliferation, apoptosis, cell cycle, angiogenesis, invasion, and metastasis in HONE1 cells that were transfected with CYB5R2 or an empty vector. The mRNA levels of 12 genes were significantly altered by CYB5R2 overexpression (up- or down-regulated by at least 1.5-fold compared with empty vector control) (Table 2). Several genes involved in apoptosis, signal transduction and transcription, cell cycle control, and DNA damage repair [Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), and cyclin-dependent kinase inhibitor 2A (CDKN2A)] were up-regulated. Some genes involved in angiogenesis (vascular endothelial growth factor A (VEGFA) [16]), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), and transforming growth factor beta receptor 1 (TGFBR1) were down-regulated, whereas one, interferon beta 1 (IFNB1), was up-regulated. The mRNA levels of several genes involved in adhesion [integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), and integrin beta 5 (ITGB5)] were up-regulated or down-regulated with CYB5R2 expression. These changes in the gene expression in CYB5R2-transfected NPC cells (CNE2 and HONE1) was verified by real-time PCR (Figure 2).Figure 1

Bottom Line: Inactivation of CYB5R2 is associated with lymph node metastasis in NPC.A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis.Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, 530021, P.R. China. yskm128@163.com.

ABSTRACT

Background: Cytochrome b5 reductase 2 (CYB5R2) is a potential tumor suppressor that inhibits cell proliferation and motility in nasopharyngeal carcinoma (NPC). Inactivation of CYB5R2 is associated with lymph node metastasis in NPC. This study aimed to explore the mechanisms contributing to the anti-neoplastic effects of CYB5R2.

Methods: Polymerase chain reaction (PCR) assays were used to analyze the transcription of 84 genes known to be involved in representative cancer pathways in the NPC cell line HONE1. NPC cell lines CNE2 and HONE1 were transiently transfected with CYB5R2, and data was validated by real-time PCR. A chick chorioallantoic membrane (CAM) embryo model was implanted with CYB5R2-expressing CNE2 and HONE1 cells to evaluate the effect of CYB5R2 on angiogenesis. An immunohistochemical assay of the CAM model was used to analyze the protein expression of vascular endothelial growth factor (VEGF).

Results: In CYB5R2-transfected NPC cells, PCR assays revealed up-regulated mRNA levels of Fas cell surface death receptor (FAS), FBJ murine osteosarcoma viral oncogene homolog (FOS), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), integrin beta 3 (ITGB3), metastasis suppressor 1 (MTSS1), interferon beta 1 (IFNB1), and cyclin-dependent kinase inhibitor 2A (CDKN2A) and down-regulated levels of integrin beta 5 (ITGB5), insulin-like growth factor 1 (IGF1), TEK tyrosine kinase (TEK), transforming growth factor beta receptor 1 (TGFBR1), and VEGF. The angiogenesis in the CAM model implanted with CYB5R2-transfected NPC cells was inhibited. Down-regulation of VEGF by CYB5R2 in NPC cells was confirmed by immunohistochemical staining in the CAM model.

Conclusion: CYB5R2 up-regulates the expression of genes that negatively modulate angiogenesis in NPC cells and down-regulates the expression of VEGF to reduce angiogenesis, thereby suppressing tumor formation.

No MeSH data available.


Related in: MedlinePlus