A medicinal and edible formula YH0618 ameliorates the toxicity induced by Doxorubicin via regulating the expression of Bax/Bcl-2 and FOXO4

Figures

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  Figure 1. UPLC determination of YH0618. In the UPLC fingerprint at an absorbance of 260 nm, the peaks corresponding to daidzin, daidzein, genistin and genistein were identified.

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  Figure 2. YH0618 selectively inhibited DOX cytotoxicity in normal liver and kidney cells but not in breast cancer cells. (A) MDA-MB-231, MCF-7, L02 and HEK-293 cells were treated with various concentrations (0 μM to 10 μM) of DOX for 48 h to determine the IC50 value of DOX on these cells by MTT assay. Data were represented as a percentage of the vehicle-treated control. (B) MDA-MB-231, MCF-7, L02 and HEK-293 cells were incubated with YH0618 (6.25 μg/ml to 100 μg/ml) for 48 h and subjected to an MTT assay to determine the proliferation rate. (C) The MDA-MB-231, MCF-7, L02 and HEK-293 cells were incubated with DOX (IC50 value) and YH0618 (6.25 μg/ml to 100 μg/ml) for 48 h, and then subjected to an MTT assay. Data were represented as a percentage of the vehicle-treated control. *P<0.05, **P<0.01 vs. DOX control

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  Figure 3. YH0618 potentiated the effect of DOX on apoptosis in breast cancer MCF-7 cells and MDA-MB-231 cells, but suppressed the effect of DOX on apoptosis in normal liver L02 cells and kidney HEK293 cells. All cells were incubated with DOX (IC50) and/or YH0618 (25, 50 and 100 μg/ml) for 16 h, and then analyzed by flow cytometry using Annexin V/PI staining to discriminate the live cells (Q4), early apoptotic cells (Q3), necrosis or late apoptotic cells (Q2), and dead cells (Q1). (A) MCF-7 cells were incubated with DOX (2.2 μM) and/or YH0618. (B) MDA-MA-231 cells were incubated with DOX(1.6 μM) and/or YH0618. L02 cells were incubated with DOX (6 μM) and/or YH0618. (D) HEK-293 cells were incubated with DOX (6 μM) and/or YH0618.

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  Figure 4. YH0618 selectively inhibited DOX-induced apoptosis in normal liver and kidney cells but increased the apoptosis in breast cancer cells. Data were presented as a percentage of the vehicle-treated control. ###P<0.001 vs. Control; ***P<0.001, ** P<0.01 presented DOX+ YH0618 vs. DOX

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  Figure 5. Apoptosis related protein expressions were determined by western blot analysis in DOX and/or YH0618-treated L02 cells. (A, B) The ratio of Bax/Bcl-2 was increased significantly in DOX group versus control group, which was recovered in DOX (6 μM)+ YH0618 (100 μg/ml) group in L02 cells. (A, C) The increased level of FOXO4 was shown in DOX group versus control group, which was attenuated in DOX + YH0618 group in L02 cells (F=26.77). *P<0.05, **P<0.01, ***P<0.001vs. control group; #P<0.05, ##P<0.01, ###P<0.001 presented vs. DOX group.

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  Figure 6. Apoptosis related protein expressions were determined by Western blot analysis in DOX and/or YH0618-treated HEK-293 cells. (A, B) The ratio of Bax/Bcl-2 was increased significantly in DOX group versus control group, which was recovered in DOX (6 μM)+ YH0618 (100 μg/ml) group in HEK293 cells. (A, C) The increased level of FOXO4 was shown in DOX group versus control group, which was attenuated in DOX + YH0618 group in HEK-293 cells (F=38.66). *P<0.05, **P<0.01, ***P<0.001vs. control group; ##P<0.01, ###P<0.001 presented vs. DOX group.

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  Figure 7. The effect of YH0618 on body weight and tumor in mice. (A) The body weights between control and YH0618-treated group had little differences. All values represented as means ± SD, n = 9, * P < 0.05, ** P<0.01 vs. tumor control. (B) At the end of three weeks, the tumors were collected and weighed. DOX and YH0618 alone treatment reduced the tumor size compared with the control groups (**P<0.01). Co-treatment significantly reduced the tumor size compared with other treatment groups (F=65.27,**P<0.01). ** P<0.01 vs. tumor control.

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  Figure 8. YH0618 significantly reduced DOX-induced hair loss. (A) The visualized appearance of alopecia in 7-week-old C57BL/6 mice at day 0, 3 and 21 after administration of DOX (5 mg/kg) and/or YH0618 (4.5 g/kg). (B) Histopathological appearance of the skin collected, at the end of the experiment, from the C57BL/6 mice. DOX reduced the hair follicle, and damaged sebaceous gland and piloerector muscle, but YH0618 improved the conditions.

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  Figure 9. Histological changes in the MMTV-PyMT (+/-) female mice liver, kidney, heart and lung that received DOX (5 mg/kg) and/or YH0618 (4.5 g/kg). The histopathological appearance of liver (A), kidney (B), heart (C) and lung (D) collected, at the end of the 21 day after administration of DOX (5 mg/kg) and/or YH0618 (4.5 g/kg).