INHIBITORY EFFECTS OF QUERCETIN ON ANGIOGENESIS IN T-ALL CELL LINES AND ZEBRAFISH IN VIVO
(Abstract release date: 05/19/16)
EHA Library. Shen L. 06/09/16; 134488; PB1588
Dr. Lijing Shen
Contributions
Contributions
Abstract
Abstract: PB1588
Type: Publication Only
Background
Microvessel dendity was significantly increased in organs from patients with T cell acute lymphocyte Leukemia (T-ALL). Notch1 activating mutations was identified in more than 60% of T-ALL case, and has been demonstrated as one of the most important pathways in angiogenesis. Quercetin, a kind of polyphenolic bioflavonoid, has showed strong anti-tumor effects and antiangiogenesis on various cancers.
Aims
we constructed Notch1 overexpression transgenic zebrafish, then, treated zebrafish embryos and T-ALL cell lines (A3 and Molt-4) with quercetin to explore its role in anti-angiogenesis of T-ALL.
Methods
Quercetin (>95% pure) was dissolved in <0.1% dimethylsulfoxide. Using T lymphocytes specific promoter Rag2, we introduced human intracellular domain Notch1 (ICN1) gene into Tg(fli1:EGFP) zebrafish, tagged with RFP gene. In previously work, we vertified that these fish are closely resemble the main aspects of human ALL. Zebrafish embryos were collected and treated with various concentrations of quercetin (0, 80, 160, 240µM) dissolved in 4mL egg water and incubated in six-well plates (20 embryos per well) at 28.5°C from 14 hours post fertilization (hpf) to 86 hpf. Morphological changes and vessel density were observed and imaged with fluorescence microscope. The subintestinal vessels (SIVs) and intersegmental artery (ISV) of the zebrafish were chosen for the measurement of the length by image analyse software Image-Pro Plus 6.0. Cell line growth inhibition assay was performed at different concentrations of quercetin（0, 15, 30, 60, 90, and 120μM）with Cell Counting Kit-8. A3 and Molt-4 cells apoptosis was measured with AnnexinV-FITC/PI Apoptosis Detection Kit after treated with 30μM or 60μM of quercetin at 24, 48 and 72h. Quantitative real-time PCR and western blot analyses were used to detect the changes of angiogenic factors after dosing. All statistical analyses were carried out in Graphpad Prism 5.
Results
It showed that quercetin can significantly block the SIVs and ISV formation in Notch1 transgenic zebrafish model. Two of the T-ALL cell lines (A3 and Molt-4) have high expression of Notch1. The IC50 at 48h for quercetin in A3 and Molt-4 were (35.17±1.46)μM and (33.24±2.35)μM, respectively. Their proliferation was significantly inhibited by quercetin in a dose- and time-dependent manner. Furthermore, the mRNA and protein levels of VEGF, DLL4, Notch1 and Hes1 were gradual inhibited with the concentration and exposure time increasing. After treatment with various concentrations (30µM and 60µM) of quercetin for 48h, the maximal apoptosis rates were (70.24±3.58) % in A3 cells, (61.05±2.37) % in Molt-4 cells, respectively. In contrast, less than 5% of untreated cells underwent apoptosis under the same conditions.
Conclusion
These finding suggested that quercetin could inhibit angiogenesis by targeting VEGF regulated DLL4/Notch signaling pathway and supposed to be a potential drug candidate for T-ALL therapy.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Angiogenesis, Notch, Zebra fish
Type: Publication Only
Background
Microvessel dendity was significantly increased in organs from patients with T cell acute lymphocyte Leukemia (T-ALL). Notch1 activating mutations was identified in more than 60% of T-ALL case, and has been demonstrated as one of the most important pathways in angiogenesis. Quercetin, a kind of polyphenolic bioflavonoid, has showed strong anti-tumor effects and antiangiogenesis on various cancers.
Aims
we constructed Notch1 overexpression transgenic zebrafish, then, treated zebrafish embryos and T-ALL cell lines (A3 and Molt-4) with quercetin to explore its role in anti-angiogenesis of T-ALL.
Methods
Quercetin (>95% pure) was dissolved in <0.1% dimethylsulfoxide. Using T lymphocytes specific promoter Rag2, we introduced human intracellular domain Notch1 (ICN1) gene into Tg(fli1:EGFP) zebrafish, tagged with RFP gene. In previously work, we vertified that these fish are closely resemble the main aspects of human ALL. Zebrafish embryos were collected and treated with various concentrations of quercetin (0, 80, 160, 240µM) dissolved in 4mL egg water and incubated in six-well plates (20 embryos per well) at 28.5°C from 14 hours post fertilization (hpf) to 86 hpf. Morphological changes and vessel density were observed and imaged with fluorescence microscope. The subintestinal vessels (SIVs) and intersegmental artery (ISV) of the zebrafish were chosen for the measurement of the length by image analyse software Image-Pro Plus 6.0. Cell line growth inhibition assay was performed at different concentrations of quercetin（0, 15, 30, 60, 90, and 120μM）with Cell Counting Kit-8. A3 and Molt-4 cells apoptosis was measured with AnnexinV-FITC/PI Apoptosis Detection Kit after treated with 30μM or 60μM of quercetin at 24, 48 and 72h. Quantitative real-time PCR and western blot analyses were used to detect the changes of angiogenic factors after dosing. All statistical analyses were carried out in Graphpad Prism 5.
Results
It showed that quercetin can significantly block the SIVs and ISV formation in Notch1 transgenic zebrafish model. Two of the T-ALL cell lines (A3 and Molt-4) have high expression of Notch1. The IC50 at 48h for quercetin in A3 and Molt-4 were (35.17±1.46)μM and (33.24±2.35)μM, respectively. Their proliferation was significantly inhibited by quercetin in a dose- and time-dependent manner. Furthermore, the mRNA and protein levels of VEGF, DLL4, Notch1 and Hes1 were gradual inhibited with the concentration and exposure time increasing. After treatment with various concentrations (30µM and 60µM) of quercetin for 48h, the maximal apoptosis rates were (70.24±3.58) % in A3 cells, (61.05±2.37) % in Molt-4 cells, respectively. In contrast, less than 5% of untreated cells underwent apoptosis under the same conditions.
Conclusion
These finding suggested that quercetin could inhibit angiogenesis by targeting VEGF regulated DLL4/Notch signaling pathway and supposed to be a potential drug candidate for T-ALL therapy.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Angiogenesis, Notch, Zebra fish
Abstract: PB1588
Type: Publication Only
Background
Microvessel dendity was significantly increased in organs from patients with T cell acute lymphocyte Leukemia (T-ALL). Notch1 activating mutations was identified in more than 60% of T-ALL case, and has been demonstrated as one of the most important pathways in angiogenesis. Quercetin, a kind of polyphenolic bioflavonoid, has showed strong anti-tumor effects and antiangiogenesis on various cancers.
Aims
we constructed Notch1 overexpression transgenic zebrafish, then, treated zebrafish embryos and T-ALL cell lines (A3 and Molt-4) with quercetin to explore its role in anti-angiogenesis of T-ALL.
Methods
Quercetin (>95% pure) was dissolved in <0.1% dimethylsulfoxide. Using T lymphocytes specific promoter Rag2, we introduced human intracellular domain Notch1 (ICN1) gene into Tg(fli1:EGFP) zebrafish, tagged with RFP gene. In previously work, we vertified that these fish are closely resemble the main aspects of human ALL. Zebrafish embryos were collected and treated with various concentrations of quercetin (0, 80, 160, 240µM) dissolved in 4mL egg water and incubated in six-well plates (20 embryos per well) at 28.5°C from 14 hours post fertilization (hpf) to 86 hpf. Morphological changes and vessel density were observed and imaged with fluorescence microscope. The subintestinal vessels (SIVs) and intersegmental artery (ISV) of the zebrafish were chosen for the measurement of the length by image analyse software Image-Pro Plus 6.0. Cell line growth inhibition assay was performed at different concentrations of quercetin（0, 15, 30, 60, 90, and 120μM）with Cell Counting Kit-8. A3 and Molt-4 cells apoptosis was measured with AnnexinV-FITC/PI Apoptosis Detection Kit after treated with 30μM or 60μM of quercetin at 24, 48 and 72h. Quantitative real-time PCR and western blot analyses were used to detect the changes of angiogenic factors after dosing. All statistical analyses were carried out in Graphpad Prism 5.
Results
It showed that quercetin can significantly block the SIVs and ISV formation in Notch1 transgenic zebrafish model. Two of the T-ALL cell lines (A3 and Molt-4) have high expression of Notch1. The IC50 at 48h for quercetin in A3 and Molt-4 were (35.17±1.46)μM and (33.24±2.35)μM, respectively. Their proliferation was significantly inhibited by quercetin in a dose- and time-dependent manner. Furthermore, the mRNA and protein levels of VEGF, DLL4, Notch1 and Hes1 were gradual inhibited with the concentration and exposure time increasing. After treatment with various concentrations (30µM and 60µM) of quercetin for 48h, the maximal apoptosis rates were (70.24±3.58) % in A3 cells, (61.05±2.37) % in Molt-4 cells, respectively. In contrast, less than 5% of untreated cells underwent apoptosis under the same conditions.
Conclusion
These finding suggested that quercetin could inhibit angiogenesis by targeting VEGF regulated DLL4/Notch signaling pathway and supposed to be a potential drug candidate for T-ALL therapy.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Angiogenesis, Notch, Zebra fish
Type: Publication Only
Background
Microvessel dendity was significantly increased in organs from patients with T cell acute lymphocyte Leukemia (T-ALL). Notch1 activating mutations was identified in more than 60% of T-ALL case, and has been demonstrated as one of the most important pathways in angiogenesis. Quercetin, a kind of polyphenolic bioflavonoid, has showed strong anti-tumor effects and antiangiogenesis on various cancers.
Aims
we constructed Notch1 overexpression transgenic zebrafish, then, treated zebrafish embryos and T-ALL cell lines (A3 and Molt-4) with quercetin to explore its role in anti-angiogenesis of T-ALL.
Methods
Quercetin (>95% pure) was dissolved in <0.1% dimethylsulfoxide. Using T lymphocytes specific promoter Rag2, we introduced human intracellular domain Notch1 (ICN1) gene into Tg(fli1:EGFP) zebrafish, tagged with RFP gene. In previously work, we vertified that these fish are closely resemble the main aspects of human ALL. Zebrafish embryos were collected and treated with various concentrations of quercetin (0, 80, 160, 240µM) dissolved in 4mL egg water and incubated in six-well plates (20 embryos per well) at 28.5°C from 14 hours post fertilization (hpf) to 86 hpf. Morphological changes and vessel density were observed and imaged with fluorescence microscope. The subintestinal vessels (SIVs) and intersegmental artery (ISV) of the zebrafish were chosen for the measurement of the length by image analyse software Image-Pro Plus 6.0. Cell line growth inhibition assay was performed at different concentrations of quercetin（0, 15, 30, 60, 90, and 120μM）with Cell Counting Kit-8. A3 and Molt-4 cells apoptosis was measured with AnnexinV-FITC/PI Apoptosis Detection Kit after treated with 30μM or 60μM of quercetin at 24, 48 and 72h. Quantitative real-time PCR and western blot analyses were used to detect the changes of angiogenic factors after dosing. All statistical analyses were carried out in Graphpad Prism 5.
Results
It showed that quercetin can significantly block the SIVs and ISV formation in Notch1 transgenic zebrafish model. Two of the T-ALL cell lines (A3 and Molt-4) have high expression of Notch1. The IC50 at 48h for quercetin in A3 and Molt-4 were (35.17±1.46)μM and (33.24±2.35)μM, respectively. Their proliferation was significantly inhibited by quercetin in a dose- and time-dependent manner. Furthermore, the mRNA and protein levels of VEGF, DLL4, Notch1 and Hes1 were gradual inhibited with the concentration and exposure time increasing. After treatment with various concentrations (30µM and 60µM) of quercetin for 48h, the maximal apoptosis rates were (70.24±3.58) % in A3 cells, (61.05±2.37) % in Molt-4 cells, respectively. In contrast, less than 5% of untreated cells underwent apoptosis under the same conditions.
Conclusion
These finding suggested that quercetin could inhibit angiogenesis by targeting VEGF regulated DLL4/Notch signaling pathway and supposed to be a potential drug candidate for T-ALL therapy.
Session topic: E-poster
Keyword(s): Acute lymphoblastic leukemia, Angiogenesis, Notch, Zebra fish
{{ help_message }}
{{filter}}