A Synthetic <i>dl</i>-Nordihydroguaiaretic acid (Nordy), Inhibits Angiogenesis, Invasion and Proliferation of Glioma Stem Cells within a Zebrafish Xenotransplantation Model

<div><p>The zebrafish (<i>Danio rerio</i>) and their transparent embryos represent a promising model system in cancer research. Compared with other vertebrate model systems, we had previously shown that the zebrafish model provides many advantages over mouse or chicken models to study tumor invasion, angiogenesis, and tumorigenesis. In this study, we systematically investigated the biological features of glioma stem cells (GSCs) in a zebrafish model, such as tumor angiogenesis, invasion, and proliferation. We demonstrated that several verified anti-angiogenic agents inhibited angiogenesis that was induced by xenografted-GSCs. We next evaluated the effects of a synthetic dl-nordihydroguaiaretic acid compound (<i>dl</i>-NDGA or “Nordy”), which revealed anti-tumor activity against human GSCs <i>in vitro</i> by establishing parameters through studying its ability to suppress angiogenesis, tumor invasion, and proliferation. Furthermore, our results indicated that Nordy might inhibit GSCs invasion and proliferation through regulation of the arachidonate 5-lipoxygenase (Alox-5) pathway. Moreover, the combination of Nordy and a VEGF inhibitor exhibited an enhanced ability to suppress angiogenesis that was induced by GSCs. By contrast, even following treatment with 50 µM Nordy, there was no discernible effect on zebrafish embryonic development. Together, these results suggested efficacy and safety of using Nordy <i>in vivo</i>, and further demonstrated that this model should be suitable for studying GSCs and anti-GSC drug evaluation.</p></div

The response to Nordy treatment on the survival rate, and development of zebrafish embryos.

<p>A. Quantitative analysis of the survival rate of naïve zebrafish embryos following treatment with various concentrations of Nordy. B. Showing the representative bright field images of native zebrafish embryos that were incubated with 50 µM Nordy. C. The representative images of vascular development of Tg (<i>fli1</i>:EGFP)<i>^y1</i> embryos following treatment with 50 µM Nordy.</p

Analysis of angiogenesis induced by U87 GSCs with/without Nordy treatment.

<p>A. The representative merged images of angiogenesis as induced by U87 GSCs with/without Nordy treatment. The images at higher magnification show the new vessels that were induced by U87 GSCs. B. Quantitative analysis of the length of newly formed vessels that were induced by U87 GSCs with/without Nordy treatment at 2 dpi. C. Quantitative analysis of the percentage of angiogenic embryos with/without Nordy treatment at 2 dpi. D. Showing the secreted VEGF¹⁶⁵ volume of U87 GSCs with/without Nordy treatment <i>in vitro</i>. E. Showing the VEGF¹⁶⁵ mRNA level in U87 GSCs with/without Nordy treatment as assayed by qRT-PCR at 2 dpi. F. Showing the representative merged images of angiogenesis that were induced by U87 GSCs treated with/without Nordy and/or vatalanib. The images at higher magnification showed new vessels induced by tumor cells. G. Showing quantitative analysis of the length of newly formed vessels induced by U87 GSCs with/without Nordy and/or Vatalanib treatment. The yellow arrow indicates newly formed angiogenic vessels on embryonic yolk <i>sac</i> ball. Red: injected RFP-labeled U87 GSCs cells; green: GFP fluorescence of vasculature in Tg (<i>fli1</i>:EGFP)<i>^y1</i> embryos.</p

The effects of verified VEGF receptor tyrosine kinase inhibitor on angiogenesis that was induced by GSCs within zebrafish embryos.

<p>A. The representative images of the inhibition of angiogenesis with axitinib, suntinib and vatalanib treatment. B. Quantitative analysis of the length of newly formed vessels that were induced by U87 GSCs with/without axitinib, suntinib and vatalanib treatment at 2 dpi. (P<0.0001)</p

Quantitation of invading U87 CD133⁺ GSCs within zebrafish embryos injected with/without 50 µM Nordy treatment at 2 dpi.

<p>The data were obtained from three replicate experiments of 50 injected embryos for each experiment.</p

CD133 positive U87 GSCs were suppressed by Nordy treatment within zebrafish embryos.

<p>A. The representative high-invasion phenotypes of injected GSCs with/without Nordy treatment. B. The inhibitory effect of Nordy on the invasion of CD133 positive U87 GSCs within zebrafish embryos. The percentages of invasive cells in adoptively transferred embryos (low, medium, or high-invasion) were measured at 2 dpi. The data were obtained from three replicate experiments with the number of embryos: n = 120 for the non-Nordy treated group, n = 113 for the Nordy treated group. C. The relationship between angiogenesis induced by GSCs and degree of invasion. N = 34 represented the low-invasion group, n = 33 represented the medium-invasion group, and n = 39 represented the high-invasion group. Red: adoptively transferred RFP-labeled U87 GSCs cells; green: represents the GFP fluorescence of angiogenesis in Tg (<i>fli1</i>:EGFP)<i>^y1</i> embryos.</p

Effects of Nordy on the proliferation of GSCs within zebrafish embryos.

<p>A. Representative merged images of GSC proliferation with/without Nordy treatment within zebrafish embryos. B. Quantitative analysis of the emitted fluorescence of RFP-labeled GSCs with/without Nordy treatment at 2 dpi and 4 dpi. At higher magnification, the images showed the injected the RFP labeled GSCs accumulate within the embryos (yellow broken box). Red: injected U87-RFP cells; green: GFP fluorescence of angiogenesis in Tg (<i>fli1</i>:EGFP)<i>^y1</i> embryos.</p

Invasive U87 sphere cells express CD133.

<p>A. U87 sphere cells with various invasion capability within zebrafish embryos. The extent of invasion was classified in three degrees: Low: less than 5 migrated cells; Medium: 5–20 migrated cells; High: more than 20 migrated cells. Representative images at higher magnification show the invasive RFP-labeled U87 sphere cell masses (red) in the tail region of the embryos <i>via</i> EGFP-labeled host vessels (green). B. Detection of CD133 expression on non-invasive and invasive U87 sphere cells at 2 dpi by immunofluorecent staining. All of U87 sphere cells within injected embryos were stained with monoclonal anti-CD133 antibody (1∶300) and examined by confocal microscopy. Green: Tg (<i>fli1</i>:EGFP)^y1 microvessels; red: RFP-labeled U87 sphere cells; blue: CD133 positive U87 cells. C. Quantitative analysis of CD133-expressing cells in non-invasive cell group (n = 713) and high-invasive cell group (n = 175) at 2 dpi. (<i>p</i><0.001).</p

The establishment of U87 glioma sphere cell invasion model in zebrafish embryos.

<p>A. Dual color confocal image shows that U87 sphere cells (RFP labeled, red) were microinjected into the middle of yolk <i>sac</i> within Tg (<i>fli1</i>:EGFP)^y1 transgenic zebrafish embryos (EGFP labeled, green). B. Different numbers of U87-RFP glioma sphere cells were microinjected into Tg (<i>fli1</i>:EGFP)^y1 embryos (n = 300 in each group), and the percentage of embryos with invasive tumor cells was quantitated. C. The survival rate of Tg (<i>fli1</i>:EGFP)^y1 zebrafish embryos microinjected with different numbers of U87-RFP glioma sphere cells (n = 300 in each group). D. Representative dual color confocal images of RFP-labeled U87 sphere cells within Tg (<i>fli1</i>:EGFP)^y1 zebrafish embryos at the different invasive stages. Red: RFP-labeled U87 sphere cells; Green: Tg (<i>fli1</i>:EGFP)^y1 microvessels.</p

MMP-9 mediates invasion and spread of CD133⁺ U87 GSCs in zebrafish embryos.

<p>A. The MMP-2 and MMP-9 RNA in CD133⁻ U87 cells and CD133⁺ U87 GSCs were examined by qRT-PCR. B. The MMP-2 and MMP-9 proteins in CD133⁻ U87 cells and CD133⁺ U87 GSCs examined by Western blot. C. The inhibitory effect of MMP-9 inhibitor (AG-L-66085) on the invasion of CD133⁺ U87 GSCs within zebrafish embryos. The embryos xenografted with CD133⁺ U87 GSCs were treated with 2 µM AG-L-66085 or DMSO control. The percentages of invasive cells in injected embryos (low, medium, or high-invasion) were measured at 2 dpi. The data were obtained from three replicate experiments with the number of embryos: n = 123 for DMSO control group, n = 119 for MMP-9 inhibitor group, and n = 144 for negative control group (<i>p</i><0.001).</p