Synergistic effects of tea polyphenols and ascorbic acid on human lung adenocarcinoma SPC-A-1 cells

Abstract

Tea polyphenols have been shown to have anticancer activity in many studies. In the present study, we investigated effects of theaflavin-3-3'-digallate (TF(3)), one of the major theaflavin monomers in black tea, in combination with ascorbic acid (AA), a reducing agent, and (-)-epigallocatechin-3-gallate (EGCG), the main polyphenol presented in green tea, in combination with AA on cellular viability and cell cycles of the human lung adenocarcinoma SPC-A-1 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that the 50% inhibition concentrations (IC(50)) of TF(3), EGCG, and AA on SPC-A-1 cells were 4.78, 4.90, and 30.62 micromol/L, respectively. The inhibitory rates of TF(3) combined with AA (TF(3)+AA) and EGCG combined with AA (EGCG+AA) at a molar ratio of 1:6 on SPC-A-1 cells were 54.4% and 45.5%, respectively. Flow cytometry analysis showed that TF(3)+AA and EGCG+AA obviously increased the cell population in the G(0)/G(1) phase of the SPC-A-1 cell cycle from 53.9% to 62.8% and 60.0%, respectively. TF(3)-treated cells exhibited 65.3% of the G(0)/G(1) phase at the concentration of its IC(50). Therefore, TF(3)+AA and EGCG+AA had synergistic inhibition effects on the proliferation of SPC-A-1 cells, and significantly held SPC-A-1 cells in G(0)/G(1) phase. The results suggest that the combination of TF(3) with AA or EGCG with AA enhances their anticancer activity.

Fig. 1

Chemical structures of EGCG and theaflavins (a) EGCG; (b) Theaflavins. TF₁ (R₁=R₂=OH), TF₂A (R₁=gallate, R₂=OH), TF₂B (R₁=OH, R₂=gallate), TF₃ (R₁=R₂=gallate)

Fig. 1

Chemical structures of EGCG and theaflavins (a) EGCG; (b) Theaflavins. TF₁ (R₁=R₂=OH), TF₂A (R₁=gallate, R₂=OH), TF₂B (R₁=OH, R₂=gallate), TF₃ (R₁=R₂=gallate)

Fig. 2

Combination index (CI) plots of interactions between TF₃, EGCG, and AA SPC-A-1 cells were treated with tea polyphenol monomers and AA at a fixed molar ratio: (a) TF₃:AA =1:6 and (b) EGCG:AA=1:6. Using the mutually exclusive or mutually non-exclusive isobologram equation, the affected fraction (f _a)-CI plots for SPC-A-1 cells were constructed by computer analysis of the data generated from the median effect analysis. CI<1 occurred over a wide range of inhibition levels, indicating synergism

Fig. 2

Combination index (CI) plots of interactions between TF₃, EGCG, and AA SPC-A-1 cells were treated with tea polyphenol monomers and AA at a fixed molar ratio: (a) TF₃:AA =1:6 and (b) EGCG:AA=1:6. Using the mutually exclusive or mutually non-exclusive isobologram equation, the affected fraction (f _a)-CI plots for SPC-A-1 cells were constructed by computer analysis of the data generated from the median effect analysis. CI<1 occurred over a wide range of inhibition levels, indicating synergism

Fig. 3

Cell cycle analysis of SPC-A-1 cells treated with the indicated concentrations of drugs for 48 h by flow cytometry analysis After cells were fixed and stained with propidium iodide, and the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using the FAC Star flow cytometry. (a) Control; (b) Ascorbic acid (30.62 μmol/L); (c) TF₃ (4.78 μmol/L); (d) EGCG (4.90 μmol/L); (e) TF₃ (4.78 μmol/L)+AA (30.62 μmol/L); (f) EGCG (4.90 μmol/L)+AA (30.62 μmol/L)

Fig. 3

Cell cycle analysis of SPC-A-1 cells treated with the indicated concentrations of drugs for 48 h by flow cytometry analysis After cells were fixed and stained with propidium iodide, and the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using the FAC Star flow cytometry. (a) Control; (b) Ascorbic acid (30.62 μmol/L); (c) TF₃ (4.78 μmol/L); (d) EGCG (4.90 μmol/L); (e) TF₃ (4.78 μmol/L)+AA (30.62 μmol/L); (f) EGCG (4.90 μmol/L)+AA (30.62 μmol/L)

Fig. 3

Cell cycle analysis of SPC-A-1 cells treated with the indicated concentrations of drugs for 48 h by flow cytometry analysis After cells were fixed and stained with propidium iodide, and the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using the FAC Star flow cytometry. (a) Control; (b) Ascorbic acid (30.62 μmol/L); (c) TF₃ (4.78 μmol/L); (d) EGCG (4.90 μmol/L); (e) TF₃ (4.78 μmol/L)+AA (30.62 μmol/L); (f) EGCG (4.90 μmol/L)+AA (30.62 μmol/L)

Fig. 3

Cell cycle analysis of SPC-A-1 cells treated with the indicated concentrations of drugs for 48 h by flow cytometry analysis After cells were fixed and stained with propidium iodide, and the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using the FAC Star flow cytometry. (a) Control; (b) Ascorbic acid (30.62 μmol/L); (c) TF₃ (4.78 μmol/L); (d) EGCG (4.90 μmol/L); (e) TF₃ (4.78 μmol/L)+AA (30.62 μmol/L); (f) EGCG (4.90 μmol/L)+AA (30.62 μmol/L)

Fig. 3

Cell cycle analysis of SPC-A-1 cells treated with the indicated concentrations of drugs for 48 h by flow cytometry analysis After cells were fixed and stained with propidium iodide, and the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using the FAC Star flow cytometry. (a) Control; (b) Ascorbic acid (30.62 μmol/L); (c) TF₃ (4.78 μmol/L); (d) EGCG (4.90 μmol/L); (e) TF₃ (4.78 μmol/L)+AA (30.62 μmol/L); (f) EGCG (4.90 μmol/L)+AA (30.62 μmol/L)

Fig. 3

Cell cycle analysis of SPC-A-1 cells treated with the indicated concentrations of drugs for 48 h by flow cytometry analysis After cells were fixed and stained with propidium iodide, and the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using the FAC Star flow cytometry. (a) Control; (b) Ascorbic acid (30.62 μmol/L); (c) TF₃ (4.78 μmol/L); (d) EGCG (4.90 μmol/L); (e) TF₃ (4.78 μmol/L)+AA (30.62 μmol/L); (f) EGCG (4.90 μmol/L)+AA (30.62 μmol/L)