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 p21Waf1[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 antiproliferative and differentiating effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. Cell proliferation was assayed by measuring 3H-thymidine incorporation and by normalizing counts to mg protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and by Western Blot by labelling a nitrocellulose membrane with an anti-acetylated lysine antibody. Histone classes were separated and identified by HPLC/MS, and the post-translational modifications were determined. The effects of SFN on cell differentiation and invasivity were detected after 120h administration by immunofluorescence microscopy, by the kinetic determination of alkaline phosphatase activity, and by means of a cell invasion kit. 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 from SFN treated cells show a 63% increase in the acetylation status; in particular SFN selectively prolongs the half- life of the acetyl groups on histone H4. These data are associated to a diminished cell invasivity, that decreases of 25% in SFN treated HT29. Antiproliferative and differentiating effects of SFN can therefore be attributed to the inhibition of HDAC. References. [1] Chung F.L., et al. (2000) Carcinogenesis 21, 2287–2291. [2] Shen G., et al. (2006) Cancer Chemother Pharmacol 57, 317–327. [3] Myzak M.C. et al. (2004) Cancer Research 64, 5767–5774
C. Parolin, N. Calonghi, E. Pagnotta, M. Naldi, C. Angeloni, S. Hrelia, et al. (2006). The cell cycle arrest induced by sulforaphane in human colon carcinoma cells HT29 is associated with the hyperacetylation of histone H4..
The 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 p21Waf1[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 antiproliferative and differentiating effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. Cell proliferation was assayed by measuring 3H-thymidine incorporation and by normalizing counts to mg protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and by Western Blot by labelling a nitrocellulose membrane with an anti-acetylated lysine antibody. Histone classes were separated and identified by HPLC/MS, and the post-translational modifications were determined. The effects of SFN on cell differentiation and invasivity were detected after 120h administration by immunofluorescence microscopy, by the kinetic determination of alkaline phosphatase activity, and by means of a cell invasion kit. 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 from SFN treated cells show a 63% increase in the acetylation status; in particular SFN selectively prolongs the half- life of the acetyl groups on histone H4. These data are associated to a diminished cell invasivity, that decreases of 25% in SFN treated HT29. Antiproliferative and differentiating effects of SFN can therefore be attributed to the inhibition of HDAC. References. [1] Chung F.L., et al. (2000) Carcinogenesis 21, 2287–2291. [2] Shen G., et al. (2006) Cancer Chemother Pharmacol 57, 317–327. [3] Myzak M.C. et al. (2004) Cancer Research 64, 5767–5774I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
