Nutr Res Pract.  2013 Apr;7(2):89-95.

Inorganic sulfur reduces cell proliferation by inhibiting of ErbB2 and ErbB3 protein and mRNA expression in MDA-MB-231 human breast cancer cells

• Ha AW
• Hong KH
• Kim HS
• Kim WK

Abstract

Dietary inorganic sulfur is the minor component in our diet, but some studies suggested that inorganic sulfur is maybe effective to treat cancer related illness. Therefore, this study aims to examine the effects of inorganic sulfur on cell proliferation and gene expression in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells were cultured the absence or presence of various concentrations (12.5, 25, or 50 micromol/L) of inorganic sulfur. Inorganic sulfur significantly decreased proliferation after 72 h of incubation (P < 0.05). The protein expression of ErbB2 and its active form, pErbB2, were significantly reduced at inorganic sulfur concentrations of 50 micromol/L and greater than 25 micromol/L, respectively (P < 0.05). The mRNA expression of ErbB2 was significantly reduced at an inorganic sulfur concentration of 50 micromol/L (P < 0.05). The protein expression of ErbB3 and its active form, pErbB3, and the mRNA expression of ErbB3 were significantly reduced at inorganic sulfur concentrations greater than 25 micromol/L (P < 0.05). The protein and mRNA expression of Akt were significantly reduced at an inorganic sulfur concentration of 50 micromol/L (P < 0.05), but pAkt was not affected by inorganic sulfur treatment. The protein and mRNA expression of Bax were significantly increased with the addition of inorganic sulfur concentration of 50 micromol/L (P < 0.05). In conclusion, cell proliferation was suppressed by inorganic sulfur treatment through the ErbB-Akt pathway in MDA-MB-231 cells.

Keyword

Inorganic sulfur; epidermal growth factor receptor; apoptosis; cell proliferation; MDA-MB-231 cell

MeSH Terms

Apoptosis
Breast
Breast Neoplasms
Cell Proliferation
Diet
Gene Expression
Humans
Receptor, Epidermal Growth Factor
RNA, Messenger
Sulfur
RNA, Messenger
Receptor, Epidermal Growth Factor
Sulfur

Figure

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  Fig. 1 Inorganic sulfur inhibited the 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, monolayers were incubated in serum-free medium with 0, 12.5, 25, or 50 µmol/L sulfur for 0, 24, or 72 h. 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. 2 Inorganic sulfur reduced the protein and mRNA expression of ErbB2 in MDA-MB-231 cells. For ErbB2 and pErbB2 protein expression, MDA-MB-231 cells were plated in a 100 mm dish at a density of 1 × 106 cells/dish with DMEM/F12 supplemented with 10% FDS for 48 h. The cells were then incubated in serum free medium for 24 h, after which they were then incubated in the presence of inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 h. Equal amounts of cell lysates (30 µg) were then resolved by SDS-PAGE, transferred to a membrane and probed with ErbB2 (A) and pErbB2 (B). For ErbB2 mRNA expression, cells were cultured in serum-free medium with inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 h. Total RNA was isolated and RT-PCR was performed (C). a) Photographs of the bands, which were 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. 3 Inorganic sulfur reduced the protein and mRNA expression of ErbB3 in MDA-MB-231 cells. For ErbB3 and pErbB3 protein expression, MDA-MB-231 cells were seeded in a 100 mm dish at a density of 1 × 106 cells/dish with DMEM/F12 supplemented with 10% FDS for 48 h. The cells were then incubated in serum free medium for 24 h, after which they were then incubated in the presence of inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 h. Equal amounts of cell lysates (30 µg) were then resolved by SDS-PAGE, transferred to a membrane and probed with ErbB3 (A) and pErbB3 (B). For ErbB3 mRNA expression, cells were cultured in serum-free medium with inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 h. Total RNA was isolated and RT-PCR was performed (C). a) Photographs of the bands, which were 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 Inorganic sulfur reduced the 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 × 106 cells/dish with DMEM/F12 supplemented with 10% FDS for 48 h. Cells were then incubated in serum free medium for 24 h, after which they were then incubated in the presence of inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 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, cells were cultured in serum-free medium with inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 h. Total RNA was isolated and RT-PCR was performed (C). a) Photographs of the bands, which were 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 Inorganic sulfur increased the 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 × 106 cells/dish with DMEM/F12 supplemented with 10% FDS for 48 h. Cells were then incubated in serum free medium for 24 h, after which they were then incubated in the presence of inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 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, cells were cultured in serum-free medium with inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 h. Total RNA was isolated and RT-PCR was performed (B). a) Photographs of the bands, which were 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. 6 Inorganic sulfur did not influence the 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 × 106 cells/dish with DMEM/F12 supplemented with 10% FDS for 48 h. Cells were then incubated in serum free medium for 24 h, after which they were then incubated in the presence of inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 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, cells were cultured in serum-free medium with inorganic sulfur at concentrations of 0, 12.5, 25, or 50 µmol/L for 72 h. Total RNA was isolated and RT-PCR was performed (B). a) Photographs of the bands, which were 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. 7 Inorganic sulfur reduced the Bcl2/Bax ratio of protein expression in MDA-MB-231 cells. Each bar represents the mean ± SE from 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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