Limits...
The impact of tumor nitric oxide production on VEGFA expression and tumor growth in a zebrafish rat glioma xenograft model.

Yousfi N, Pruvot B, Lopez T, Magadoux L, Franche N, Pichon L, Salvadori F, Solary E, Garrido C, Laurens V, Chluba J - PLoS ONE (2015)

Bottom Line: Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA.In the xenografted embryos we also found increased zebrafish vegfa expression.We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 866, 'Equipe Labellisée Ligue Contre le Cancer', Dijon, France; University of Burgundy, UFR SVTE, Dijon, France.

ABSTRACT
To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

No MeSH data available.


Related in: MedlinePlus

Histology of xenografted tumors and detection of NO in cells.Embryos engrafted with red fluorescent glioma cells and labelled with DAF-FM-DA. Embryos were fixed at 4dpi and sectioned at a 5μm thickness. NO production, detected with DAF signal (A’, A”) was observed in cytoplasm and cell nuclei of tumor cells (yellow arrow). (B) Magnification of yellow framed areas. DAPI labeling was used to detect nuclei (B”‘), enabling detection of the large glioma nuclei and of a dividing tumor cell (white arrow); purple arrows indicate nuclei from zebrafish cells. (C), (D) In vivo images of CM-Dil labelled glioma cells taken with a biphoton confocal microscope in 4 dpi DAF treated embryos. White arrows indicate tumor cells. (C) CM-Dil labelled red fluorescent tumor cells, (C”) DAF signal, (C”‘) merge. (D) Red fluorescent glioma cells in DAF and CPTIO treated embryos, (D’) CPTIO decreases DAF fluorescence intensity, showing the specificity of the DAF fluorescence. (D”) merge. White arrows indicate the tumor cells. Scale bars are 10 μM
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359111&req=5

pone.0120435.g002: Histology of xenografted tumors and detection of NO in cells.Embryos engrafted with red fluorescent glioma cells and labelled with DAF-FM-DA. Embryos were fixed at 4dpi and sectioned at a 5μm thickness. NO production, detected with DAF signal (A’, A”) was observed in cytoplasm and cell nuclei of tumor cells (yellow arrow). (B) Magnification of yellow framed areas. DAPI labeling was used to detect nuclei (B”‘), enabling detection of the large glioma nuclei and of a dividing tumor cell (white arrow); purple arrows indicate nuclei from zebrafish cells. (C), (D) In vivo images of CM-Dil labelled glioma cells taken with a biphoton confocal microscope in 4 dpi DAF treated embryos. White arrows indicate tumor cells. (C) CM-Dil labelled red fluorescent tumor cells, (C”) DAF signal, (C”‘) merge. (D) Red fluorescent glioma cells in DAF and CPTIO treated embryos, (D’) CPTIO decreases DAF fluorescence intensity, showing the specificity of the DAF fluorescence. (D”) merge. White arrows indicate the tumor cells. Scale bars are 10 μM

Mentions: To identify the nitric oxide producing cells sections of xenografted DAF labeled embryos (Fig. 2A and 2B) were performed. The DAF signal is conserved in histological sections, so it could be observed in cytoplasm and in the nucleus of tumor cells as described previously by Saini et al. [29]. Tumor cells were detected by red fluorescent cell tracker spot signals in perinuclear vesicles and large DAPI labeled nuclei. The size difference of the nuclei allowed us to distinguish rat glioma cells from zebrafish cells (Fig. 2B). In addition, DAPI labeling revealed a dividing tumor cell showing that the cells proliferate at 4 dpi (Fig. 2B). Live imaging of 4 dpi xenografted embryos with a biphoton confocal microscope confirmed NO in rat glioma cells (Fig. 2C and 2D). This DAF signal is specific as it disappeared after treatment with the NO scavenger CPTIO (Fig. 2D).


The impact of tumor nitric oxide production on VEGFA expression and tumor growth in a zebrafish rat glioma xenograft model.

Yousfi N, Pruvot B, Lopez T, Magadoux L, Franche N, Pichon L, Salvadori F, Solary E, Garrido C, Laurens V, Chluba J - PLoS ONE (2015)

Histology of xenografted tumors and detection of NO in cells.Embryos engrafted with red fluorescent glioma cells and labelled with DAF-FM-DA. Embryos were fixed at 4dpi and sectioned at a 5μm thickness. NO production, detected with DAF signal (A’, A”) was observed in cytoplasm and cell nuclei of tumor cells (yellow arrow). (B) Magnification of yellow framed areas. DAPI labeling was used to detect nuclei (B”‘), enabling detection of the large glioma nuclei and of a dividing tumor cell (white arrow); purple arrows indicate nuclei from zebrafish cells. (C), (D) In vivo images of CM-Dil labelled glioma cells taken with a biphoton confocal microscope in 4 dpi DAF treated embryos. White arrows indicate tumor cells. (C) CM-Dil labelled red fluorescent tumor cells, (C”) DAF signal, (C”‘) merge. (D) Red fluorescent glioma cells in DAF and CPTIO treated embryos, (D’) CPTIO decreases DAF fluorescence intensity, showing the specificity of the DAF fluorescence. (D”) merge. White arrows indicate the tumor cells. Scale bars are 10 μM
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359111&req=5

pone.0120435.g002: Histology of xenografted tumors and detection of NO in cells.Embryos engrafted with red fluorescent glioma cells and labelled with DAF-FM-DA. Embryos were fixed at 4dpi and sectioned at a 5μm thickness. NO production, detected with DAF signal (A’, A”) was observed in cytoplasm and cell nuclei of tumor cells (yellow arrow). (B) Magnification of yellow framed areas. DAPI labeling was used to detect nuclei (B”‘), enabling detection of the large glioma nuclei and of a dividing tumor cell (white arrow); purple arrows indicate nuclei from zebrafish cells. (C), (D) In vivo images of CM-Dil labelled glioma cells taken with a biphoton confocal microscope in 4 dpi DAF treated embryos. White arrows indicate tumor cells. (C) CM-Dil labelled red fluorescent tumor cells, (C”) DAF signal, (C”‘) merge. (D) Red fluorescent glioma cells in DAF and CPTIO treated embryos, (D’) CPTIO decreases DAF fluorescence intensity, showing the specificity of the DAF fluorescence. (D”) merge. White arrows indicate the tumor cells. Scale bars are 10 μM
Mentions: To identify the nitric oxide producing cells sections of xenografted DAF labeled embryos (Fig. 2A and 2B) were performed. The DAF signal is conserved in histological sections, so it could be observed in cytoplasm and in the nucleus of tumor cells as described previously by Saini et al. [29]. Tumor cells were detected by red fluorescent cell tracker spot signals in perinuclear vesicles and large DAPI labeled nuclei. The size difference of the nuclei allowed us to distinguish rat glioma cells from zebrafish cells (Fig. 2B). In addition, DAPI labeling revealed a dividing tumor cell showing that the cells proliferate at 4 dpi (Fig. 2B). Live imaging of 4 dpi xenografted embryos with a biphoton confocal microscope confirmed NO in rat glioma cells (Fig. 2C and 2D). This DAF signal is specific as it disappeared after treatment with the NO scavenger CPTIO (Fig. 2D).

Bottom Line: Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA.In the xenografted embryos we also found increased zebrafish vegfa expression.We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 866, 'Equipe Labellisée Ligue Contre le Cancer', Dijon, France; University of Burgundy, UFR SVTE, Dijon, France.

ABSTRACT
To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

No MeSH data available.


Related in: MedlinePlus