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. 2021 Feb 1;22(2):603-610.
doi: 10.31557/APJCP.2021.22.2.603.

Daidzein Induces Intrinsic Pathway of Apoptosis along with ER α/β Ratio Alteration and ROS Production

Vinod Kumar  1 , Shyam S Chauhan  1
Affiliations

Daidzein Induces Intrinsic Pathway of Apoptosis along with ER α/β Ratio Alteration and ROS Production

Vinod Kumar et al. Asian Pac J Cancer Prev. .

Abstract

Background: Low risk of breast cancer is observed among females consuming a moderate quantity of soy throughout their life. The present study was conducted to evaluate the anticancer potential of Daidzein, one of the major Isoflavones in soy using Human breast cancer cells MCF-7.

Methods: MCF-7 were subjected to various doses of Daidzein treatment to determine the IC50 value. Onset of apoptosis was ascertained by AnnexinV assay and caspase 3/7 activity post treatment. Expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 was also assessed to further confirm apoptotic mode of cell death. ROS production post treatment with Daidzein was assessed to ascertain the apoptosis via intrinsic pathway. Expression of ER α and ER β was evaluated by western blot analysis.

Results: Human breast cancer cells MCF-7 were found to be sensitive to Daidzein treatment, with an IC50 value of 50µM. Increased percentage of treated cells stained with Annexin V confirmed apoptosis mediated cell death. Activity of Caspase 3/7 activity was found to be 1.4-fold higher in Daidzein treated cells than control cells, confirming apoptosis. Daidzein caused over expression of Bax and down-regulated expression of Bcl2. There has been an outburst of ROS in Daidzein treated cells indicating that Daidzein induces apoptosis via intrinsic pathway. A decrease in the expression of ER α and increase in levels of ER β has been observed which are conducive indicator of apoptosis.

Conclusions: In conclusion, the present study suggests that Daidzein induces apoptosis in MCF-7 cells by mitochondrial pathway along with lowering the ratio of ER α/β and an outburst of Reactive Oxygen Species(ROS).

Keywords: Apoptosis; Bax/Bcl-2 Ratio; ER α/β ratio; Molecular docking; ROS.

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Figures

Figure 1
Figure 1
MCF-7 Cells Viability Analysis after Daidzein Treatment. MCF-7 cells were treated with varying concentrations of daidzein for 24, 48 and 72 h. After treatment, 20 μl of 5 mg/ml MTT was added and incubated for 4 hours at 37o C. After this, 100 µl of DMSO was added to each well to dissolve the resulting formazan crystals. Absorbance was measured at 490 nm in and ELISA reader. Data are presented as means of standard deviation from three independent experiments. Each experiment was conducted in triplicate (P = 0.047).
Figure 2
Figure 2
Daidzein Induced Apoptosis in MCF-7 Cells. 1×105 MCT-7 cells were treated with 50µM Diadzein for 48 hrs. and analysed for apoptosis by Annexin V assay by flow cytometry. After 48 hrs. 5 µl each of Annexin V and FITC PI were added to cells’ pellet and vortexed gently. Then the cells were incubated for 30 minutes at room temperature in dark and 400µl of binding buffer was added to each tube and subjected to analysis by flow cytometer (Becton Dickinson; Ex, 488 nm and Em, 530 nm). Flow cytometry analysis of MCF-7 cells shows apoptotic cells stained for Annexin V and Propidium Iodide (PI) after treatment with 50µM daidzein (panel b) as compared to untreated cells (panel a). In both the panels, Quadrant1; Annexin−/PI+ (necrosis), Quadrant2; Annexin+/PI+(late apoptotic), Quadrant3; Annexin+/PI−(early apoptotic cells) and Quadrant4; Annexin−/PI− (intact cells). There is an increase in apoptosis in daidzein treated cells as compared to untreated ones from 16.8% to 29.5%. Also, there is substantial increase in number of early apoptotic cells in treated cells ;15.8% to 41.9% as against control cells
Figure 3
Figure 3
Activity of Apoptotic Marker Caspase 3 /7 has been Observed to be more in Diadzein Treated MCF-7 Cells as Compared to Control Cells. There is an increase of 1.4 times in the activity of caspase3/7 in daidzein treated cell lines as compared to untreated ones. The data represent plot of thrice repeated analysis with a p value of 0.0194
Figure 4
Figure 4
Apoptotic Markers were Analyzed by Western Blotting of Lysates of MCF-7 Cells after Treatment with 50 μm Daidzein against Control Cells. 60 μg of protein was loaded for each sample. Representative blots for Bax, Bcl-2 , ER α, ERβ and α-tubulin show the expression profile of concerned markers (4a). α-tubulin has been used as an internal loading control. Densitometric analysis of Bax vs Bcl-2 (4b) shows and increase in the ratio of Bax/Bcl-2 by a factor of about ten times (p=.0084) and that of ER αand ER β (4c) shows a decrease in the ratio of ER α /ER β by about 6 times (p=.001) . These observations collectively indicate inhibition of growth of cells and induction of apoptosis by intrinsic pathway of apoptosis
Figure 5
Figure 5
Generation of Reactive Oxygen Species (ROS) in 50μm and 100 μm Daidzein Treated MCF-7 Cells. There is an outburst of ROS in 50 and 100 μm Daidzein treated cells as against control cells. As can be seen, the ROS emission is higher in 50 μm Daidzein treated cells in both the time frames, this is justified as the standard inhibitory concentration of daidzein which is capable of inducing apoptosis. Luminescence is higher in +ve control (0.03% H2O2). The data is statistically significant in all the four subsets of observation with p value in each case being 0.001
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