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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

QRT-PCR analysis of nos gene expression in control and xenografted embryos.(A) In xenografted embryos, rat Nos2 is highly expressed in comparison to Nos1 and Nos3. (B) Zebrafish nos1 gene is strongly expressed in transplanted and control embryos. A significant difference is only observed for nos2a. Error bars represent SE, * p≤ 0, 05, **<0,01 (one way ANOVA).
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pone.0120435.g003: QRT-PCR analysis of nos gene expression in control and xenografted embryos.(A) In xenografted embryos, rat Nos2 is highly expressed in comparison to Nos1 and Nos3. (B) Zebrafish nos1 gene is strongly expressed in transplanted and control embryos. A significant difference is only observed for nos2a. Error bars represent SE, * p≤ 0, 05, **<0,01 (one way ANOVA).

Mentions: NO is catalyzed from L-arginine by nitric oxide synthase (NOS) enzymes, which have been identified in vertebrates, invertebrates and bacteria. Three isozymes exist in mammals: NOS1, NOS2 and NOS3 [30], and in zebrafish, NOS1, NOS2a and NOS2b [31–33]. In order to determine which Nos are expressed by the glioma cells in vitro and which are induced by the xenografts in zebrafish we performed qRT-PCR analyses. Nos2 was clearly overexpressed in the xenografted glioma cells when compared to Nos1 and Nos3 (Fig. 3A). In contrast, Nos2 expression in cultured glioma cells depended on the cell density and was very variable (not shown). In zebrafish, nos1 was the highest expressed NO synthase gene in both, control and xenografted embryos; there was no significant difference between control and xenografted groups. The same was the case for nos2b. However, for nos2a, the nos isoform that corresponds to mammalian Nos2, a significant increase was observed in the xenografted embryos (Fig. 3B).


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)

QRT-PCR analysis of nos gene expression in control and xenografted embryos.(A) In xenografted embryos, rat Nos2 is highly expressed in comparison to Nos1 and Nos3. (B) Zebrafish nos1 gene is strongly expressed in transplanted and control embryos. A significant difference is only observed for nos2a. Error bars represent SE, * p≤ 0, 05, **<0,01 (one way ANOVA).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120435.g003: QRT-PCR analysis of nos gene expression in control and xenografted embryos.(A) In xenografted embryos, rat Nos2 is highly expressed in comparison to Nos1 and Nos3. (B) Zebrafish nos1 gene is strongly expressed in transplanted and control embryos. A significant difference is only observed for nos2a. Error bars represent SE, * p≤ 0, 05, **<0,01 (one way ANOVA).
Mentions: NO is catalyzed from L-arginine by nitric oxide synthase (NOS) enzymes, which have been identified in vertebrates, invertebrates and bacteria. Three isozymes exist in mammals: NOS1, NOS2 and NOS3 [30], and in zebrafish, NOS1, NOS2a and NOS2b [31–33]. In order to determine which Nos are expressed by the glioma cells in vitro and which are induced by the xenografts in zebrafish we performed qRT-PCR analyses. Nos2 was clearly overexpressed in the xenografted glioma cells when compared to Nos1 and Nos3 (Fig. 3A). In contrast, Nos2 expression in cultured glioma cells depended on the cell density and was very variable (not shown). In zebrafish, nos1 was the highest expressed NO synthase gene in both, control and xenografted embryos; there was no significant difference between control and xenografted groups. The same was the case for nos2b. However, for nos2a, the nos isoform that corresponds to mammalian Nos2, a significant increase was observed in the xenografted embryos (Fig. 3B).

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