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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

Alkaline phosphatase assay of xenografted DAF-FM-DA treated embryos.Embryos were injected with red CM-Dil labeled tumor cells, incubated at 4 dpi in DAF (5μM) and fixed at 4dpi before detection of alkaline phosphatase activity. (A) Bright field image of the embryos before treatments. (B) Neovascularisation was observed in 12 of 14 (85.5%) embryos presenting NO production in tumor environment. (D) Fluorescence detection of glioma cells, (C) DAF signal, (E) merge. The white arrows indicate the part of the tumor mass that co-localizes with DAF fluorescence signal. (F) A control embryo with absence of vessels in this region of the yolk.
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pone.0120435.g004: Alkaline phosphatase assay of xenografted DAF-FM-DA treated embryos.Embryos were injected with red CM-Dil labeled tumor cells, incubated at 4 dpi in DAF (5μM) and fixed at 4dpi before detection of alkaline phosphatase activity. (A) Bright field image of the embryos before treatments. (B) Neovascularisation was observed in 12 of 14 (85.5%) embryos presenting NO production in tumor environment. (D) Fluorescence detection of glioma cells, (C) DAF signal, (E) merge. The white arrows indicate the part of the tumor mass that co-localizes with DAF fluorescence signal. (F) A control embryo with absence of vessels in this region of the yolk.

Mentions: NO is a key mediator in angiogenesis and tumor progression, but is also known to promote or inhibit vessel formation depending on concentration and duration of exposure [16]. We analyzed blood vessel formation using alkaline phosphatase labeling [7] in xenografted embryos in which we detected tumor NO production. Neovascularisation in close proximity of the tumors was observed in 85% (12/14) of the embryos presenting NO production in the tumor area (Fig. 4B). In these embryos, the network profile of phosphatase alkaline detection overlapped with the NO production profile obtained with DAF labeling (Fig. 4C and 4E) and the glioma mass (Fig. 4D and 4E). Overlap of CM-Dil and DAF signal is only partial and indicated by the arrow in Fig. 4E. In non-injected embryos, there are no vessels in this region, as reported earlier by Nicoli et al. 2007 [34], also shown in the control embryo (Fig. 4F).


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)

Alkaline phosphatase assay of xenografted DAF-FM-DA treated embryos.Embryos were injected with red CM-Dil labeled tumor cells, incubated at 4 dpi in DAF (5μM) and fixed at 4dpi before detection of alkaline phosphatase activity. (A) Bright field image of the embryos before treatments. (B) Neovascularisation was observed in 12 of 14 (85.5%) embryos presenting NO production in tumor environment. (D) Fluorescence detection of glioma cells, (C) DAF signal, (E) merge. The white arrows indicate the part of the tumor mass that co-localizes with DAF fluorescence signal. (F) A control embryo with absence of vessels in this region of the yolk.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120435.g004: Alkaline phosphatase assay of xenografted DAF-FM-DA treated embryos.Embryos were injected with red CM-Dil labeled tumor cells, incubated at 4 dpi in DAF (5μM) and fixed at 4dpi before detection of alkaline phosphatase activity. (A) Bright field image of the embryos before treatments. (B) Neovascularisation was observed in 12 of 14 (85.5%) embryos presenting NO production in tumor environment. (D) Fluorescence detection of glioma cells, (C) DAF signal, (E) merge. The white arrows indicate the part of the tumor mass that co-localizes with DAF fluorescence signal. (F) A control embryo with absence of vessels in this region of the yolk.
Mentions: NO is a key mediator in angiogenesis and tumor progression, but is also known to promote or inhibit vessel formation depending on concentration and duration of exposure [16]. We analyzed blood vessel formation using alkaline phosphatase labeling [7] in xenografted embryos in which we detected tumor NO production. Neovascularisation in close proximity of the tumors was observed in 85% (12/14) of the embryos presenting NO production in the tumor area (Fig. 4B). In these embryos, the network profile of phosphatase alkaline detection overlapped with the NO production profile obtained with DAF labeling (Fig. 4C and 4E) and the glioma mass (Fig. 4D and 4E). Overlap of CM-Dil and DAF signal is only partial and indicated by the arrow in Fig. 4E. In non-injected embryos, there are no vessels in this region, as reported earlier by Nicoli et al. 2007 [34], also shown in the control embryo (Fig. 4F).

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