Identification and Functional Characterization of Anti-metastasis and Anti-angiogenic Activities of Triethylene Glycol Derivatives

Figures

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  Figure 1. Effect of water extract (WEX) of Ashwagandha leaves and TEG on cell migration and invasion capacity of HUVECs. (A) VEGF-treated cells showed mild increase in migration; VEGF + Avastin (positive control)-treated cells showed inhibition. Both WEX and TEG showed dose-dependent inhibition of migration. (B) VEGF-treated cells showed enhanced invasion; VEGF + Avastin (positive control)-treated cells showed inhibition. Both WEX and TEG showed dose-dependent inhibition of invasion. Migrated and invaded cells were counted in three randomly selected fields. Data are shown as mean ± standard deviation (SD) of three independent fields/well. Maximum inhibition of cell migration was observed in cells treated with 5% WEX and 2.5% TEG. WEX (5%) and TEG (0.5 to 2.5%) caused strong inhibition of cell invasion. (C) Effect of WEX and TEG on tube-formation in HUVEC. VEGF-treated cells showed mild increase in tube formation capacity of HUVEC. Avastin and FP3 (positive controls), WEX and TEG showed dose-dependent inhibition of tube formation. Statistical analysis from three independent experiments is depicted as *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 2. Cytotoxicity curves. (A) Cytotoxicity of TEG (TD-1) and its 10 derivatives (TD-2 to TD-11) (Supplementary Figure S1) on H1299, U2OS, and A549 cells; TD-10 and TD-11 demonstrated the most remarkable cytotoxicity in each cell line. (B) Dose dependent cytotoxicity of TD-10 and TD-11 in A549 cells is shown. Statistical analysis is depicted as *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 3. Effect of TEG and its derivatives TD-10 and TD-11 on cell invasion capacity and tube-forming capacity of HUVEC. (A) VEGF-treated cells showed mild increase in invasion. TEG, TD-10, and TD-11 treated cells showed inhibition of VEGF-induced invasion; TD-10 and TD-11 showed stronger inhibition than TEG. Avastin was used as a positive control. (B) VEGF was used to induce tube formation. TEG, TD-10 and TD-11 treated cells showed inhibition of tube formation; TD-10 and TD-11 showed stronger inhibition than TEG. Avastin was used as a positive control. Statistical analysis is depicted as significant *p < 0.05, **p < 0.01, ***p < 0.001 and NS for insignificant change.

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  Figure 4. Effect of low doses of TD-10 and TD-11 on cell viability of A549 cells. (A) Crystal violet-stained photographs of cells treated with 0.005 and 0.05% of TD-10 and TD-11 are shown. (B) Cell cycle profile of control and treated cells showing mild growth arrest at G1 phase in 0.005 % TD-10 and TD-11 treated (48 h) cells, and lack of apoptosis. (C) Consolidated data from three independent experiments showing dose dependent accumulation of cells in G1 phase of the cell cycle in TD-10 and TD-11 treated cells. (D) Growth curve of cells treated (48 h) with low doses of TD-10 and TD-11 showing lack of toxicity for 0.005% TD-10 and TD-11 during 48 h treatment. (E) Colony forming efficiency of control, TD-10 and TD-11 treated (10 days) cells showing dose dependent decrease. (F) Migration capacity of cells treated with non-toxic conditions of TD-10 and TD-11 treatment. Both TD-10 and TD-11 caused inhibition in cell migration. Images of Wound-Scratch assay are shown in Supplementary Figure S2a. Statistical analysis from three independent experiments is depicted as *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant.

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  Figure 5. Effect of TD-10 and TD-11 (0.005%; 48 h) on proteins involved in cell migration, apoptosis and proliferation. (A,B) Immunostaining and immunoblotting of A549 cells treated with TEG derivatives. Downregulation of proteins involved in cell migration and EMT (mortalin, MMP2, vimentin, VEGF, β-catenin, SMAD2/3) was recorded. Downregulation of phosphorylated RB and E2F-1, Cyclin D1, and CDK4 signified moderate inhibition of cell proliferation and migration. Changes in p53, BCL2, Caspase 9 expression were mild and signified lack of apoptosis. (C,D) Mortalin and VEGF ELISA revealed downregulation in both TD-10 and TD-11 treated cells. Statistical analysis is depicted as *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 6. In vivo tumor progression assays using U343 cells in subcutaneous xenograft model. (A) TD-10 and TD-11 mice showed significant delay in tumor growth. (B) Body weight of control and treated mice did not show any difference. (C) Immunohistochemistry showing decrease in VEGF and CD31 expression in treated mice as compared to control.