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, 2018, 3545376
eCollection

Biochanin A Induces S Phase Arrest and Apoptosis in Lung Cancer Cells

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Biochanin A Induces S Phase Arrest and Apoptosis in Lung Cancer Cells

Yan Li et al. Biomed Res Int.

Abstract

Lung cancer is among the most common malignancies with a poor 5-year survival rate reaching only 16%. Thus, new effective treatment modalities and drugs are urgently needed for the treatment of this malignancy. In this study, we conducted the first investigation of the effects of Biochanin A on lung cancer and revealed the mechanisms underlying its potential anticancer effects. Biochanin A decreased cell viability in a time-dependent and dose-dependent manner and suppressed colony formation in A549 and 95D cells. In addition, Biochanin A induced S phase arrest and apoptosis and decreased mitochondrial membrane potential (ΔΨm) in A549 and 95D cells in a dose-dependent manner. Our results of subcutaneous xenograft models showed that the growth of Biochanin A group was significantly inhibited compared with that of control groups. Finally, P21, Caspase-3, and Bcl-2 were activated in Biochanin A-treated cells and Biochanin A-treated xenografts which also demonstrated that Biochanin A induced cell cycle arrest and apoptosis in lung cancer cells by regulating expression of cell cycle-related proteins and apoptosis-related proteins. In conclusion, this study suggests that Biochanin A inhibits the proliferation of lung cancer cells and induces cell cycle arrest and apoptosis mainly by regulating cell cycle-related protein expression and activating the Bcl-2 and Caspase-3 pathways, thus suggesting that Biochanin A may be a promising drug to treat lung cancer.

Figures

Figure 1
Figure 1
Chemical structure of Biochanin A. The molecular formula of Biochanin A is C16H12O5 and its molecular weight is 284.26.
Figure 2
Figure 2
Biochanin A inhibits the proliferation of A549 and 95D cells. (a) Cells were treated with varying concentrations of Biochanin A, and the cell proliferation and IC 50 were determined by MTT assay on days 1, 2, and 3. Each value represents the mean ± SD (n = 3). (b) Biochanin A suppressed colony formation of A549 and 95D cells. Cells were treated with Biochanin A (0, 5, 10, and 20μmol/L) and were allowed to form colonies in fresh medium for 14 days. These results were from 1 representative experiment of 3 independent trials. The photomicrographic difference (left panel) and influence of colonies (mean ± SD, n = 3) (right panel) in colony formation are shown. p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 3
Figure 3
Biochanin A induces S phase arrest in A549. Cells were treated with 0, 50, 100, and 200 μmol/L Biochanin A for 48 h and the DNA content was analyzed by flow cytometry. The percentages of cells in the G1, S, and G2/M phases of the cell cycle are shown. These results were from 1 representative experiment of 3 independent trials.
Figure 4
Figure 4
Biochanin A induces S phase arrest in 95D. Cells were treated with 0, 60, 120, and 240 μmol/L Biochanin A for 48 h and the DNA content was analyzed by flow cytometry. The percentages of cells in the G1, S, and G2/M phases of the cell cycle are shown. These results were from 1 representative experiment of 3 independent trials.
Figure 5
Figure 5
Biochanin A induces apoptosis in A549 cells. Cells were incubated with Biochanin A (0, 50, 100, and 200μmol/L) for 48 h, followed by staining with annexin V/PI. The Q3 quadrant (annexin V−/PI−), Q4 quadrant (annexin V+/PI−), and Q2 quadrant (annexin V+/PI+) indicate the percentage of normal cells, early apoptosis, and late apoptosis, respectively. These results were from 1 representative experiment of 3 independent trials. Values represent the mean ± SD (n = 3). P < 0.05; ∗∗P< 0.01; ∗∗∗P < 0.001.
Figure 6
Figure 6
Biochanin A induces apoptosis in 95D cells. Cells were incubated with Biochanin A (0, 60, 120, and 240μmol/L) for 48 h, followed by staining with annexin V/PI. These results were from 1 representative experiment of 3 independent trials. Values represent the mean ± SD (n = 3). P < 0.05; ∗∗P< 0.01; ∗∗∗P < 0.001.
Figure 7
Figure 7
Biochanin A decreases mitochondrial membrane potential (ΔΨm) in A549 cells. Flow cytometric analysis of ΔΨm. A549 cells were treated with Biochanin A (0, 50, 100, and 200μmol/L) followed by rhodamine 123 staining. Cells with high ΔΨm are marked “survival” and those with low ΔΨm are marked “apoptosis”. Percentages (%) of cells with high ΔΨm (survival) and low ΔΨm (apoptosis) are shown. These results were from 1 representative experiment of 3 independent trials. Values represent the mean ± SD (n = 3). P < 0.05; ∗∗P< 0.01; ∗∗∗P < 0.001.
Figure 8
Figure 8
Biochanin A decreases mitochondrial membrane potential (ΔΨm) in 95D cells. Flow cytometric analysis of ΔΨm. 95D cells were treated with Biochanin A (0, 60, 120, and 240μmol/L) followed by rhodamine 123 staining. Percentage (%) of cells with high ΔΨm (survival) and low ΔΨm (apoptosis) are shown. These results were from 1 representative experiment of 3 independent trials. Values represent the mean ± SD (n = 3). P < 0.05; ∗∗P< 0.01; ∗∗∗P < 0.001.
Figure 9
Figure 9
Biochanin A inhibits the proliferation of lung cancer cell lines A549 in vivo. Representative examples of tumors formed in nude mice injected with the indicated cells.
Figure 10
Figure 10
Biochanin A inhibits the proliferation of lung cancer cell lines A549 in vivo. (a) The bar graph of average tumor weights in the subcutaneous xenograft model. (b) Tumor growth curves are summarized in the line chart. P < 0.05; ∗∗P< 0.01; ∗∗∗P < 0.001.
Figure 11
Figure 11
Biochanin A inhibits the proliferation of lung cancer cell lines 95D in vivo. Representative examples of tumors formed in nude mice injected with the indicated cells.
Figure 12
Figure 12
Biochanin A inhibits the proliferation of lung cancer cell lines 95D in vivo. (a) The bar graph of average tumor weights in the subcutaneous xenograft models. (b) Tumor growth curves are summarized in the line chart. P < 0.05; ∗∗P< 0.01; ∗∗∗P < 0.001.
Figure 13
Figure 13
Effect of Biochanin A on the signal pathway of cell cycle-related proteins and apoptosis-related proteins in lung cancer cells in vitro. (a) The protein expression of cell cycle-related proteins in A549 cells in vitro. (b) The protein expression of apoptosis-related proteins in A549 cells in vitro. (c) The protein expression of cell cycle-related proteins in 95D cells in vitro. (d) The protein expression of apoptosis-related proteins in 95D cells in vitro.
Figure 14
Figure 14
Effect of Biochanin A on the signal pathway of cell cycle-related proteins and apoptosis-related proteins in lung cancer cells in vivo. (a) The protein expression of cell cycle-related proteins in A549 cells in vivo. (b) The protein expression of apoptosis-related proteins in A549 cells in vivo. (c) The protein expression of cell cycle-related proteins in 95D cells in vivo. (d) The protein expression of apoptosis-related proteins in 95D cells in vivo.

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