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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

Nitric Oxide detection in xenografted glioma cells.(A) Embryos injected with CM-Dil labeled were cells imaged at 1dpi (A) and 4 dpi (A’). (B) Embryos injected with CellTrackerTM Green labeled cells were incubated at 4 dpi with DAR (20μM). The white arrow indicates the outgrowth. (B’) Green labeled glioma cells, (B”) red DAR signal. The specific co-localization is reported in the merge (B”‘). (C) CM-Dil labeled glioma cells transplanted in the yolk and incubated with DAF (5 μM). Specific co-localization is reported in the merge (C”‘). White arrows indicate the tumor area.
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pone.0120435.g001: Nitric Oxide detection in xenografted glioma cells.(A) Embryos injected with CM-Dil labeled were cells imaged at 1dpi (A) and 4 dpi (A’). (B) Embryos injected with CellTrackerTM Green labeled cells were incubated at 4 dpi with DAR (20μM). The white arrow indicates the outgrowth. (B’) Green labeled glioma cells, (B”) red DAR signal. The specific co-localization is reported in the merge (B”‘). (C) CM-Dil labeled glioma cells transplanted in the yolk and incubated with DAF (5 μM). Specific co-localization is reported in the merge (C”‘). White arrows indicate the tumor area.

Mentions: Around 100–200 GV1A1 glioma cells labeled with CM-Dil or CellTracker Green were injected into the yolk sac of 2 dpf embryos, which were then observed daily from 1 to 4 dpi to follow tumor formation (S1 Fig). An immune rejection is not possible at this stage as the thymus is not yet functional; according to previous reports, adaptive immunity is not reached before 4 weeks after hatching [27] and only a very small number of T cells could only be observed from 9 dpf [28]. At 1 dpi, the fluorescent cells were detected in the yolk sac and at 4 dpi tumors had significantly grown and formed masses (Fig. 1A).


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)

Nitric Oxide detection in xenografted glioma cells.(A) Embryos injected with CM-Dil labeled were cells imaged at 1dpi (A) and 4 dpi (A’). (B) Embryos injected with CellTrackerTM Green labeled cells were incubated at 4 dpi with DAR (20μM). The white arrow indicates the outgrowth. (B’) Green labeled glioma cells, (B”) red DAR signal. The specific co-localization is reported in the merge (B”‘). (C) CM-Dil labeled glioma cells transplanted in the yolk and incubated with DAF (5 μM). Specific co-localization is reported in the merge (C”‘). White arrows indicate the tumor area.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120435.g001: Nitric Oxide detection in xenografted glioma cells.(A) Embryos injected with CM-Dil labeled were cells imaged at 1dpi (A) and 4 dpi (A’). (B) Embryos injected with CellTrackerTM Green labeled cells were incubated at 4 dpi with DAR (20μM). The white arrow indicates the outgrowth. (B’) Green labeled glioma cells, (B”) red DAR signal. The specific co-localization is reported in the merge (B”‘). (C) CM-Dil labeled glioma cells transplanted in the yolk and incubated with DAF (5 μM). Specific co-localization is reported in the merge (C”‘). White arrows indicate the tumor area.
Mentions: Around 100–200 GV1A1 glioma cells labeled with CM-Dil or CellTracker Green were injected into the yolk sac of 2 dpf embryos, which were then observed daily from 1 to 4 dpi to follow tumor formation (S1 Fig). An immune rejection is not possible at this stage as the thymus is not yet functional; according to previous reports, adaptive immunity is not reached before 4 weeks after hatching [27] and only a very small number of T cells could only be observed from 9 dpf [28]. At 1 dpi, the fluorescent cells were detected in the yolk sac and at 4 dpi tumors had significantly grown and formed masses (Fig. 1A).

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