Introduction. Sulforaphane (SFN), a dietary isothiocyanate isolated from Brassicaceae, has been shown to prevent different cancers in laboratory animals [1]. In particular, SFN protects against colon carcinogenesis in vivo and causes a G0/G1 growth arrest and apoptosis in human colon cancer cells, by inducing p21Cip1/Waf1[2]. SFN also acts as an inhibitor of histone deacetylases (HDACs) in human embryonic kidney 293 cells and HCT116 colon cancer cells [3]. The objective of this study is to evaluate the ability of the HDAC inhibitor SFN to induce cytotoxic and cytostatic effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. The cells were plated in 6-well dishes at equal density, grown for 24 hours, and then treated with 5 M SFN. 3H-thymidine (1 μCi/mL) was added for the last 6 hours of the incubation. The cells were washed in ice-cold PBS and 5 % trichloroacetic acid was added for 30 minutes at 4 °C, and then incubated with 0.5 N NaOH for 1 hour at 50 °C. Cell lysates were assayed for protein content by Bio Rad assay kit and measured for incorporated radioactivity. Counts were normalized for total cellular protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and the acetylation status of the histone classes were assayed by Western Blot and HPLC/MS. Results. SFN negatively affects HT29 growth in a dose- and time-dependent manner with a 24h IC50 equal to 22.3 M  2.4. At concentrations as low as 5 M, a significant inhibition of cell proliferation is observed, accompanied by a 47% decrease of the mitotic index, with respect to the control. Histones extracted from SFN treated HT29 cells show a 63% increase in the acetylation status; in particular SFN markedly prolonges the half- life of the acetyl groups on histone H4.

Cell cycle arrest induced by sulforaphane in human colon carcinoma cells HT29 is associated with the hyperacetylation of histone H4.

PAROLIN, CAROLA ELEONORA;CALONGHI, NATALIA;PAGNOTTA, ELEONORA;NALDI, MARINA;ANGELONI, CRISTINA;HRELIA, SILVANA;BIAGI, PIERLUIGI;MASOTTI, LANFRANCO
2006

Abstract

Introduction. Sulforaphane (SFN), a dietary isothiocyanate isolated from Brassicaceae, has been shown to prevent different cancers in laboratory animals [1]. In particular, SFN protects against colon carcinogenesis in vivo and causes a G0/G1 growth arrest and apoptosis in human colon cancer cells, by inducing p21Cip1/Waf1[2]. SFN also acts as an inhibitor of histone deacetylases (HDACs) in human embryonic kidney 293 cells and HCT116 colon cancer cells [3]. The objective of this study is to evaluate the ability of the HDAC inhibitor SFN to induce cytotoxic and cytostatic effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. The cells were plated in 6-well dishes at equal density, grown for 24 hours, and then treated with 5 M SFN. 3H-thymidine (1 μCi/mL) was added for the last 6 hours of the incubation. The cells were washed in ice-cold PBS and 5 % trichloroacetic acid was added for 30 minutes at 4 °C, and then incubated with 0.5 N NaOH for 1 hour at 50 °C. Cell lysates were assayed for protein content by Bio Rad assay kit and measured for incorporated radioactivity. Counts were normalized for total cellular protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and the acetylation status of the histone classes were assayed by Western Blot and HPLC/MS. Results. SFN negatively affects HT29 growth in a dose- and time-dependent manner with a 24h IC50 equal to 22.3 M  2.4. At concentrations as low as 5 M, a significant inhibition of cell proliferation is observed, accompanied by a 47% decrease of the mitotic index, with respect to the control. Histones extracted from SFN treated HT29 cells show a 63% increase in the acetylation status; in particular SFN markedly prolonges the half- life of the acetyl groups on histone H4.
SIB-SINU: ASPETTI BIOCHIMICI E NUTRIZIONALI DELL'INTERAZIONE GENI-NUTRIENTI.
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C. Parolin; N. Calonghi; E. Pagnotta; M. Naldi; C. Angeloni; S. Hrelia; P.L. Biagi; L. Masotti.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/29620
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