The synergistic effect of 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside combined with Adriamycin on MCF-7 breast cancer cells

Figures

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  Figure 1. The proliferative inhibition assay of THSG, ADM, and THSG in combination with ADM on different cell lines. Notes: Cells were exposed to various concentrations of THSG and ADM alone or in combination at 400:1 molar ratio for 48 hours. (A) Cell viability curves were plotted based on the MTT assay in MCF-7 cell lines. The synergistic effects between drugs were shown as CI-Fa plot (B); isobologram (C) was calculated with the Calcusyn™ software. (B) CI values are represented by points above (indicate antagonism) or below the lines (indicate synergism). (C) Isobologram analysis represents the individual doses of drugs in the achievement of 90% growth inhibition (Fa = 0.9), 75% growth inhibition (Fa = 0.75), and 50% growth inhibition (Fa = 0.5). (D) Cell viability of THSG was performed based on the MTT assay in H1299 cell lines. (E) Cell viability of THSG was performed based on the MTT assay in LNC cell lines. *P<0.05, **P<0.01 vs the Ctr group with THSG; ##P<0.01 vs the Ctr group with ADM; &P<0.05, &&P<0.01 vs the Ctr group with THSG. Abbreviations: ADM, Adriamycin; CI, combination index; Ctr, control; T+A, THSG + Adriamycin; THSG, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside.

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  Figure 2. The proliferative inhibition assay of THSG, ADM, and THSG in combination with ADM on MCF-7 cell lines. Notes: Cells were exposed to various concentrations of THSG and ADM alone or in combination at 400:1 molar ratio for 24 hours. (A) Cell viability curves were plotted based on the MTT assay in MCF-7 cell lines. The antagonism effects between drugs were shown as CI-Fa plot (B), isobologram (C) calculated with the Calcusyn™ software. (B) CI values are represented by points above (indicate antagonism) or below the lines (indicate synergism). (C) Isobologram analysis represents the individual doses of drugs in the achievement of 90% growth inhibition (Fa = 0.9), 75% growth inhibition (Fa = 0.75), and 50% growth inhibition (Fa = 0.5). *P<0.05, **P<0.01 vs the Ctr group with THSG; ##P<0.01 vs the Ctr group with ADM; &&P<0.01 vs the Ctr group with THSG. Abbreviations: ADM, Adriamycin; Ctr, control; T+A, THSG + Adriamycin; THSG, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside.

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  Figure 3. Effect of THSG and ADM individual and in combination on the apoptosis in MCF-7 cells. Notes: (A) The apoptosis rate after 48-hour treatment with 200 µM of THSG and 0.5 µM of ADM individual or in combination with flow cytometry. (B) Representative graph of late apoptosis (Q2 dots). (C) Representative graph of early apoptosis (Q4 dots). (D) Representative graph of late + early apoptosis (Q2+Q4 dots). (E) Representative photographs of TUNEL staining cells (green) and DAPI-positive cells (blue) in various groups. (F) Histogram of quantification of TUNEL was shown with the percentage of TUNEL-positive nuclei (green) relative to DAPI-positive total nuclei (blue). (G) Morphology and growth status under an inverted phase microscope. All data represent mean ± SEM of three independent experiments. Bars = 50 µm. *P<0.05, **P<0.01 vs the Ctr group. Abbreviations: ADM, Adriamycin; Ctr, control; PI, propidium iodide; T+A, THSG + Adriamycin; THSG, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside.

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  Figure 4. Effect of THSG and ADM in combination on the apoptosis in MCF-7 cells. Notes: (A) The apoptosis rate after 24-hour treatment with 200 µM of THSG and 0.5 µM of ADM in combination with flow cytometry. (B) Representative graph of late + early apoptosis (Q2+Q4 dots). (C) Representative graph of late apoptosis (Q2 dots). (D) Representative graph of early apoptosis (Q4 dots). All data represent mean ± SEM of three independent experiments. Bars = 50 µm. *P<0.05, **P<0.01 vs the Ctr group. Abbreviations: ADM, Adriamycin; Ctr, control; PI, propidium iodide; T+A, THSG + Adriamycin; THSG, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside.

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  Figure 5. Effects of THSG and ADM on the inhibition of VEGF/PI3K/Akt signaling pathway in MCF-7 cells. Notes: The expression levels of various proteins with 200 µM of THSG and 0.5 µM of ADM individual or in combination for 48 hours. (A) Representative protein band of Bax and Bcl-2. (B) Representative optical density of the tests protein Bax, Bcl-2, and Bax/Bcl-2. (C) Representative protein band of caspase-3 and cleaved caspase-3. (D) Representative optical density of caspase-3, cleaved caspase-3, and cleaved caspase-3/caspase-3. (E) Representative protein band of VEGF, p-PI3K, and p-Akt. (F) Representative optical density of VEGF, p-PI3K, and p-Akt. Data are presented as mean ± SEM of three independent experiments. *P<0.05, **P<0.01 vs the Ctr group. Abbreviations: ADM, Adriamycin; CASP3, caspase-3; C-CASP3, cleaved caspase-3; Ctr, control; T+A, THSG + Adriamycin; THSG, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside.

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  Figure 6. The proposed signal pathway with THSG and ADM in MCF-7 cells. Abbreviations: ADM, Adriamycin; THSG, 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside.