Nutr Res Pract.  2011 Aug;5(4):288-293.

Radish (Raphanus sativus L. leaf) ethanol extract inhibits protein and mRNA expression of ErbB2 and ErbB3 in MDA-MB-231 human breast cancer cells

• Kim WK
• Kim JH
• Jeong DH
• Chun YH
• Kim SH
• Cho KJ
• Chang MJ

Abstract

In this study, we investigated the effects of the ethanol extract of aerial parts of Raphanus sativus L. (ERL) on breast cancer cell proliferation and gene expression associated with cell proliferation and apoptosis in MDA-MB-231 human breast cancer cells. The MDA-MB-231 cells were cultured in the presence or absence of various concentrations (100, 200, or 300 microg/mL) of ERL. ERL significantly decreased cell proliferation after 48 h of incubation (P < 0.05). The protein and mRNA expression of ErbB2 were decreased significantly in a dose-dependent manner (P < 0.05). The protein expression of ErbB3 was decreased significantly at an ERL concentration of 300 microg/mL (P < 0.05), and mRNA expression of ErbB3 was decreased significantly in a dose-dependent manner (P < 0.05). The protein expression of Akt was decreased significantly at the ERL concentration of 200 microg/mL (P < 0.05), and the protein expression of pAkt was decreased significantly in a dose-dependent manner (P < 0.05). The mRNA expression of Akt was decreased significantly at the ERL concentration of 200 microg/mL ERL (P < 0.05). The protein and mRNA expression of Bax were increased significantly at ERL concentrations of 200 microg/mL or higher (P < 0.05). The protein expression of Bcl2 was increased significantly at ERL concentrations of 100 microg/mL or higher (P < 0.05), and mRNA expression of Bcl2 was increased significantly at an ERL concentration of 300 microg/mL (P < 0.05). In conclusion, we suggest that Raphanus sativus, L. inhibits cell proliferation via the ErbB-Akt pathway in MDA-MB-231 cells.

Keyword

Raphanus sativus L. ethanol extract; epidermal growth factor receptor; apoptosis; cell proliferation; MDA-MB-231 cell

MeSH Terms

Apoptosis
Breast
Breast Neoplasms
Cell Proliferation
Ethanol
Gene Expression
Humans
Raphanus
Receptor, Epidermal Growth Factor
RNA, Messenger
Ethanol
RNA, Messenger
Receptor, Epidermal Growth Factor

Figure

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  Fig. 1 ERL inhibits cell proliferation in MDA-MB-231 cells. MDA-MB-231 cells were plated at a density of 2.5×104 cells/ml in a 24 well plate with DMEM/F12 supplemented with 10% FBS. Monolayers were then serum-starved with DMEM/F12 supplemented with 5 µg/ml transferrin, 5 ng/ml selenium, and 1 mg/ml bovine serum albumin for 24 h. After serum starvation, the monolayers were incubated in serum-free medium with 0, 100, 200, or 300 µg/mL ERL for 0, 24, or 48 h. Each bar represents the mean ± S.E. of three independent experiments. Different letters indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

• 

  Fig. 2 ERL reduces protein and mRNA expression of ErbB2 in MDA-MB-231 cells. For ErbB2 protein expression, MDA-MB-231 cells were plated in a 100 mm dish at a density of 1×105 cells/dish with DMEM/F12 supplemented with 10% FBS for 48 h. The cells were then incubated in serum free medium for 24 h, after which they were incubated in the presence of ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Equal amounts of cell lysates (30 µg) were then resolved by SDS-PAGE, transferred to a membrane, and probed with ErbB2 (A). For ErbB2 mRNA expression, the cells were cultured in serum-free medium with ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Total RNA was isolated and RT-PCR was performed (B). a) Photographs of the bands, which are representative of three independent experiments. b) Quantitative analysis of the bands. Each bar represents the mean ± S.E. of three independent experiments. Different letters indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

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  Fig. 3 ERL reduces protein and mRNA expression of ErbB3 in MDA-MB-231 cells. For ErbB3 protein expression, MDA-MB-231 cells were seeded in a 100 mm dish at a density of 1×105 cells/dish with DMEM/F12 supplemented with 10% FBS for 48 h. The cells were then incubated in serum free medium for 24 h, after which they were incubated in the presence of ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Equal amounts of cell lysates (30 µg) were then resolved by SDS-PAGE, transferred to a membrane, and probed with ErbB3 (A). For ErbB3 mRNA expression, the cells were cultured in serum-free medium with ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Total RNA was isolated and RT-PCR was performed (B). a) Photographs of the bands, which are representative of three independent experiments. b) Quantitative analysis of the bands. Each bar represents the mean ± SE of three independent experiments. Different letters indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

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  Fig. 4 ERL reduces protein and mRNA expression of Akt MDA-MB-231 cells. For Akt and pAkt protein expression, MDA-MB-231 cells were seeded in a 100 mm dish at a density of 1×105 cells/dish with DMEM/F12 supplemented with 10% FBS for 48 h. The cells were then incubated in serum free medium for 24 h, after which they were incubated in the presence of ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Equal amounts of cell lysates (30 µg) were then resolved by SDS-PAGE, transferred to a membrane, and probed with Akt (A) and pAkt (B). For Akt mRNA expression, the cells were cultured in serum-free medium with ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Total RNA was isolated and RT-PCR was performed (C). a) Photographs of the bands, which are representative of three independent experiments. b) Quantitative analysis of the bands. Each bar represents the mean ± SE of three independent experiments. Different letters indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

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  Fig. 5 ERL increases protein and mRNA expression of Bax in MDA-MB-231 cells. For Bax protein expression, MDA-MB-231 cells were seeded in a 100 mm dish at a density of 1×105 cells/dish with DMEM/F12 supplemented with 10% FBS for 48 h. The cells were then incubated in serum free medium for 24 h, after which they were incubated in the presence of ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Equal amounts of cell lysates (30 µg) were then resolved by SDS-PAGE, transferred to a membrane, and probed with Bax (A). For Bax mRNA expression, the cells were cultured in serum-free medium with ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Total RNA was isolated and RT-PCR was performed (B). a) Photographs of the bands, which are representative of three independent experiments. b) Quantitative analysis of the bands. Each bar represents the mean ± S.E. of three independent experiments. Different letters indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

• 

  Fig. 6 ERL increases protein and mRNA expression of Bcl2 in MDA-MB-231 cells. For Bcl2 protein expression, MDA-MB-231 cells were seeded in a 100 mm dish at a density of 1×105 cells/dish with DMEM/F12 supplemented with 10% FBS for 48 h. The cells were then incubated in serum free medium for 24 h, after which they were incubated in the presence of ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Equal amounts of cell lysates (30 µg) were then resolved by SDS-PAGE, transferred to a membrane, and probed with Bcl2 (A). For mRNA expression, the cells were cultured in serum-free medium with ERL at concentrations of 0, 100, 200, or 300 µg/mL for 48 h. Total RNA was isolated and RT-PCR was performed (B). a) Photographs of the bands, which are representative of three independent experiments. b) Quantitative analysis of the bands. Each bar represents the mean ± S.E. of three independent experiments. Different letters indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

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