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

QRT-PCR analysis of Vegfa expression in control and xenografted embryos.(A) Glioma cell Vegfa expression significantly decreases in presence of the NO scavenger CPTIO. Error bars represent SE, **<0, 01, Studtent’s t-test. The same effect was observed for zebrafish vegfaa (C), compared to non xenografted embryos and CPTIO treatment. Error bars represent SE, * p ≤ 0, 05 (one way ANOVA). (B) Embryos were stained with DAF-FM-DA at 4 dpi and separated in DAF signal positive and negative groups. Vegfa mRNA level is higher in the DAF positive group. One representative experiment from three independent experiments is shown. (D) Tg (fli: EGFP) CPTIO treated embryos that did not receive xenograft were lysed and endothelial cells sorted by FACS. The bars represent the mean percentage of sorted endothelial cells in presence and absence of CPTIO. Error bars represent SE, * p < 0, 05, Student’s t-test. (E) Zebrafish embryos were incubated with increasing concentrations of GTN. Expression of zebrafish vegfaa is dose dependent at the indicated concentrations. One representative experiment from three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120435.g005: QRT-PCR analysis of Vegfa expression in control and xenografted embryos.(A) Glioma cell Vegfa expression significantly decreases in presence of the NO scavenger CPTIO. Error bars represent SE, **<0, 01, Studtent’s t-test. The same effect was observed for zebrafish vegfaa (C), compared to non xenografted embryos and CPTIO treatment. Error bars represent SE, * p ≤ 0, 05 (one way ANOVA). (B) Embryos were stained with DAF-FM-DA at 4 dpi and separated in DAF signal positive and negative groups. Vegfa mRNA level is higher in the DAF positive group. One representative experiment from three independent experiments is shown. (D) Tg (fli: EGFP) CPTIO treated embryos that did not receive xenograft were lysed and endothelial cells sorted by FACS. The bars represent the mean percentage of sorted endothelial cells in presence and absence of CPTIO. Error bars represent SE, * p < 0, 05, Student’s t-test. (E) Zebrafish embryos were incubated with increasing concentrations of GTN. Expression of zebrafish vegfaa is dose dependent at the indicated concentrations. One representative experiment from three independent experiments is shown.

Mentions: In order to find a correlation between NO production and angiogenesis, we measured the mRNA levels of rat Vegfa and zebrafish vegfa in the presence and absence of CPTIO, a NO scavenger. QRT-PCR revealed a decrease of glioma Vegfa expression (Fig. 5A). The link between NO production and Vegfa expression was verified by comparing DAF-positive and DAF-negative tumors. DAF negative tumors expressed less Vegfa than the NO producing tumors (Fig. 5B). Two vegfa genes exist in zebrafish: vegfaa and vegfab [35]. Zebrafish vegfaa levels also decreased in presence of the CPTIO (Fig. 5C). As the expression of vegfab remained unchanged in xenografted embryos, results are not shown.


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 Vegfa expression in control and xenografted embryos.(A) Glioma cell Vegfa expression significantly decreases in presence of the NO scavenger CPTIO. Error bars represent SE, **<0, 01, Studtent’s t-test. The same effect was observed for zebrafish vegfaa (C), compared to non xenografted embryos and CPTIO treatment. Error bars represent SE, * p ≤ 0, 05 (one way ANOVA). (B) Embryos were stained with DAF-FM-DA at 4 dpi and separated in DAF signal positive and negative groups. Vegfa mRNA level is higher in the DAF positive group. One representative experiment from three independent experiments is shown. (D) Tg (fli: EGFP) CPTIO treated embryos that did not receive xenograft were lysed and endothelial cells sorted by FACS. The bars represent the mean percentage of sorted endothelial cells in presence and absence of CPTIO. Error bars represent SE, * p < 0, 05, Student’s t-test. (E) Zebrafish embryos were incubated with increasing concentrations of GTN. Expression of zebrafish vegfaa is dose dependent at the indicated concentrations. One representative experiment from three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120435.g005: QRT-PCR analysis of Vegfa expression in control and xenografted embryos.(A) Glioma cell Vegfa expression significantly decreases in presence of the NO scavenger CPTIO. Error bars represent SE, **<0, 01, Studtent’s t-test. The same effect was observed for zebrafish vegfaa (C), compared to non xenografted embryos and CPTIO treatment. Error bars represent SE, * p ≤ 0, 05 (one way ANOVA). (B) Embryos were stained with DAF-FM-DA at 4 dpi and separated in DAF signal positive and negative groups. Vegfa mRNA level is higher in the DAF positive group. One representative experiment from three independent experiments is shown. (D) Tg (fli: EGFP) CPTIO treated embryos that did not receive xenograft were lysed and endothelial cells sorted by FACS. The bars represent the mean percentage of sorted endothelial cells in presence and absence of CPTIO. Error bars represent SE, * p < 0, 05, Student’s t-test. (E) Zebrafish embryos were incubated with increasing concentrations of GTN. Expression of zebrafish vegfaa is dose dependent at the indicated concentrations. One representative experiment from three independent experiments is shown.
Mentions: In order to find a correlation between NO production and angiogenesis, we measured the mRNA levels of rat Vegfa and zebrafish vegfa in the presence and absence of CPTIO, a NO scavenger. QRT-PCR revealed a decrease of glioma Vegfa expression (Fig. 5A). The link between NO production and Vegfa expression was verified by comparing DAF-positive and DAF-negative tumors. DAF negative tumors expressed less Vegfa than the NO producing tumors (Fig. 5B). Two vegfa genes exist in zebrafish: vegfaa and vegfab [35]. Zebrafish vegfaa levels also decreased in presence of the CPTIO (Fig. 5C). As the expression of vegfab remained unchanged in xenografted embryos, results are not shown.

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