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Isoindolone derivative QSN-10c induces leukemic cell apoptosis and suppresses angiogenesis via PI3K/AKT signaling pathway inhibition.

Lv WW, Qin SN, Chen CQ, Zhang JJ, Ren TS, Xu YN, Zhao QC - Acta Pharmacol. Sin. (2014)

Bottom Line: Furthermore, QSN-10c dose-dependently decreased the Δψm in K562 cells, increased the release of cytochrome c and the level of Bax, and decreased the level of Bcl-2, suggesting that QSN-10c-induced apoptosis of K562 cells was mediated via the mitochondrial apoptotic pathway.QSN-10c treatment did not alter the Δψm in HUVECs, but dose-dependently inhibited the expression of VEGF, inhibited the tube formation and cell migration in vitro, and significantly suppressed the number of ISVs in zebrafish embryos in vivo.QSN-10c is a novel antitumor compound that exerts both antitumor and anti-angiogenic effects via inhibiting the PI3K/AKT/GSK3β signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China [2] Shenyang Pharmaceutical University, Shenyang 110016, China.

ABSTRACT

Aim: 2-(4,6-Dimethoxy-1,3-dioxoisoindolin-2-yl) ethyl 2-chloroacetate (QSN-10c) is one of isoindolone derivatives with antiproliferative activity against human umbilical vein endothelial cells (HUVECs). The aim of this study was to investigate its antitumor activity in vitro and anti-angiogenic effects in vitro and in vivo.

Methods: K562 leukemic cells and HUVECs were used for in vitro studies. Cell viability was examined using MTT assay. Cell apoptosis and mitochondrial transmembrane potential (Δψm) were detected with flow cytometry. Tube formation and migration of HUVECs were studied using two-dimensional Matrigel assay and wound-healing migration assay, respectively. VEGF levels were analyzed with RT-PCR and Western blotting. A zebrafish embryo model was used for in vivo anti-angiogenic studies. The molecular mechanisms for apoptosis in K562 cells and antiangiogenesis were measured with Western blotting.

Results: In antitumor activity studies, QSN-10c suppressed the viability of K562 cells and induced apoptosis in dose- and time-dependent manners. Furthermore, QSN-10c dose-dependently decreased the Δψm in K562 cells, increased the release of cytochrome c and the level of Bax, and decreased the level of Bcl-2, suggesting that QSN-10c-induced apoptosis of K562 cells was mediated via the mitochondrial apoptotic pathway. In anti-angiogenic activity studies, QSN-10c suppressed the viability of HUVECs and induced apoptosis in dose dependent manners. QSN-10c treatment did not alter the Δψm in HUVECs, but dose-dependently inhibited the expression of VEGF, inhibited the tube formation and cell migration in vitro, and significantly suppressed the number of ISVs in zebrafish embryos in vivo. Furthermore, QSN-10c dose-dependently suppressed the phosphorylation of AKT and GSK3β in both HUVECs and K562 cells.

Conclusion: QSN-10c is a novel antitumor compound that exerts both antitumor and anti-angiogenic effects via inhibiting the PI3K/AKT/GSK3β signaling pathway.

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Effects of QSN-10c on VEGF expression in HUVECs. (A) Electrophoresis bands expressed with VEGF and internal standard GAPDH mRNA in various groups treated with different concentrations of thalidomide and QSN-10c. The upper bands were corresponded to VEGF, and the lower to GAPDH. (B) Relative VEGF mRNA levels were quantified by integral optical density analysis of the bands from (A) and then normalization to GAPDH in HUVECs. Cells received thalidomide as positive control. Data are expressed as percentages of the vehicle control (100%) in mean±SD from three independent experiments. bP<0.05, cP<0.01 compared with control. (C) HUVECs were treated with QSN-10c for 24 h. Cell extract was prepared and subjected to Western blot using anti-VEGF antibody. β-Actin served as a loading control. QSN-10c decreased VEGF protein expression in HUVECs in a dose dependent manner as detected by Western blot.
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fig7: Effects of QSN-10c on VEGF expression in HUVECs. (A) Electrophoresis bands expressed with VEGF and internal standard GAPDH mRNA in various groups treated with different concentrations of thalidomide and QSN-10c. The upper bands were corresponded to VEGF, and the lower to GAPDH. (B) Relative VEGF mRNA levels were quantified by integral optical density analysis of the bands from (A) and then normalization to GAPDH in HUVECs. Cells received thalidomide as positive control. Data are expressed as percentages of the vehicle control (100%) in mean±SD from three independent experiments. bP<0.05, cP<0.01 compared with control. (C) HUVECs were treated with QSN-10c for 24 h. Cell extract was prepared and subjected to Western blot using anti-VEGF antibody. β-Actin served as a loading control. QSN-10c decreased VEGF protein expression in HUVECs in a dose dependent manner as detected by Western blot.

Mentions: To investigate whether QSN-10c modulates the VEGF-mediated autocrine effects on endothelial cells, RT-PCR and Western blot were performed to examine VEGF expression in HUVECs. The results indicated that 24 h of treatment with 50–100 μmol/L QSN-10c clearly decreased VEGF mRNA levels (Figure 7A, 7B, P<0.05). We further determined the effect of QSN-10c on VEGF expression by Western blot. QSN-10c decreased VEGF protein in a dose-dependent manner compared with the control (Figure 7C). These results suggest that QSN-10c-mediated VEGF inhibition potentially contributes to reduced angiogenesis by HUVECs.


Isoindolone derivative QSN-10c induces leukemic cell apoptosis and suppresses angiogenesis via PI3K/AKT signaling pathway inhibition.

Lv WW, Qin SN, Chen CQ, Zhang JJ, Ren TS, Xu YN, Zhao QC - Acta Pharmacol. Sin. (2014)

Effects of QSN-10c on VEGF expression in HUVECs. (A) Electrophoresis bands expressed with VEGF and internal standard GAPDH mRNA in various groups treated with different concentrations of thalidomide and QSN-10c. The upper bands were corresponded to VEGF, and the lower to GAPDH. (B) Relative VEGF mRNA levels were quantified by integral optical density analysis of the bands from (A) and then normalization to GAPDH in HUVECs. Cells received thalidomide as positive control. Data are expressed as percentages of the vehicle control (100%) in mean±SD from three independent experiments. bP<0.05, cP<0.01 compared with control. (C) HUVECs were treated with QSN-10c for 24 h. Cell extract was prepared and subjected to Western blot using anti-VEGF antibody. β-Actin served as a loading control. QSN-10c decreased VEGF protein expression in HUVECs in a dose dependent manner as detected by Western blot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4814032&req=5

fig7: Effects of QSN-10c on VEGF expression in HUVECs. (A) Electrophoresis bands expressed with VEGF and internal standard GAPDH mRNA in various groups treated with different concentrations of thalidomide and QSN-10c. The upper bands were corresponded to VEGF, and the lower to GAPDH. (B) Relative VEGF mRNA levels were quantified by integral optical density analysis of the bands from (A) and then normalization to GAPDH in HUVECs. Cells received thalidomide as positive control. Data are expressed as percentages of the vehicle control (100%) in mean±SD from three independent experiments. bP<0.05, cP<0.01 compared with control. (C) HUVECs were treated with QSN-10c for 24 h. Cell extract was prepared and subjected to Western blot using anti-VEGF antibody. β-Actin served as a loading control. QSN-10c decreased VEGF protein expression in HUVECs in a dose dependent manner as detected by Western blot.
Mentions: To investigate whether QSN-10c modulates the VEGF-mediated autocrine effects on endothelial cells, RT-PCR and Western blot were performed to examine VEGF expression in HUVECs. The results indicated that 24 h of treatment with 50–100 μmol/L QSN-10c clearly decreased VEGF mRNA levels (Figure 7A, 7B, P<0.05). We further determined the effect of QSN-10c on VEGF expression by Western blot. QSN-10c decreased VEGF protein in a dose-dependent manner compared with the control (Figure 7C). These results suggest that QSN-10c-mediated VEGF inhibition potentially contributes to reduced angiogenesis by HUVECs.

Bottom Line: Furthermore, QSN-10c dose-dependently decreased the Δψm in K562 cells, increased the release of cytochrome c and the level of Bax, and decreased the level of Bcl-2, suggesting that QSN-10c-induced apoptosis of K562 cells was mediated via the mitochondrial apoptotic pathway.QSN-10c treatment did not alter the Δψm in HUVECs, but dose-dependently inhibited the expression of VEGF, inhibited the tube formation and cell migration in vitro, and significantly suppressed the number of ISVs in zebrafish embryos in vivo.QSN-10c is a novel antitumor compound that exerts both antitumor and anti-angiogenic effects via inhibiting the PI3K/AKT/GSK3β signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China [2] Shenyang Pharmaceutical University, Shenyang 110016, China.

ABSTRACT

Aim: 2-(4,6-Dimethoxy-1,3-dioxoisoindolin-2-yl) ethyl 2-chloroacetate (QSN-10c) is one of isoindolone derivatives with antiproliferative activity against human umbilical vein endothelial cells (HUVECs). The aim of this study was to investigate its antitumor activity in vitro and anti-angiogenic effects in vitro and in vivo.

Methods: K562 leukemic cells and HUVECs were used for in vitro studies. Cell viability was examined using MTT assay. Cell apoptosis and mitochondrial transmembrane potential (Δψm) were detected with flow cytometry. Tube formation and migration of HUVECs were studied using two-dimensional Matrigel assay and wound-healing migration assay, respectively. VEGF levels were analyzed with RT-PCR and Western blotting. A zebrafish embryo model was used for in vivo anti-angiogenic studies. The molecular mechanisms for apoptosis in K562 cells and antiangiogenesis were measured with Western blotting.

Results: In antitumor activity studies, QSN-10c suppressed the viability of K562 cells and induced apoptosis in dose- and time-dependent manners. Furthermore, QSN-10c dose-dependently decreased the Δψm in K562 cells, increased the release of cytochrome c and the level of Bax, and decreased the level of Bcl-2, suggesting that QSN-10c-induced apoptosis of K562 cells was mediated via the mitochondrial apoptotic pathway. In anti-angiogenic activity studies, QSN-10c suppressed the viability of HUVECs and induced apoptosis in dose dependent manners. QSN-10c treatment did not alter the Δψm in HUVECs, but dose-dependently inhibited the expression of VEGF, inhibited the tube formation and cell migration in vitro, and significantly suppressed the number of ISVs in zebrafish embryos in vivo. Furthermore, QSN-10c dose-dependently suppressed the phosphorylation of AKT and GSK3β in both HUVECs and K562 cells.

Conclusion: QSN-10c is a novel antitumor compound that exerts both antitumor and anti-angiogenic effects via inhibiting the PI3K/AKT/GSK3β signaling pathway.

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