INTRODUCTION The endogenous lipoperoxidation product 9-hydroxystearic acid (9HSA) is an inhibitor of HDAC1 and its administration to HT29, a colon adenocarcinoma cell line, induces a proliferative arrest mediated by a direct activation of the p21 <sup> WAF 1</sup> gene, bypassing p53 [1]. In a recent work the interaction of 9-HSA with the catalytic site of the 3D model of HDAC1 has been tested with a docking procedure. Noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one: in fact the energies of interaction are -8.45 kcal/mol and -1.97 kcal/mol for the (R) and (S) isomer, respectively, and the estimated free energies of binding are -6.31 kcal/mol and +4.98 kcal/mol for (R) and (S), respectively [2]. In this work (R) and (S)-9HSA were isolated and their biological activity tested in HT29. MATERIALS AND METHODS (R)-9HSA and (S)-9HSA were obtained in enantiomerically pure forms, through a multistep procedure starting from Dimorphotheca sinuata seeds. Cell viability was evaluated by MTT assay. HT29 cells were treated with 50 μM of (R)-9HSA or (S)-9HSA after 24 hours of seeding and their effects on cell growth were evaluated by flow citometry. The effect of the two enantiomers on the HDAC1 activity was tested as histone acetylation level by pulse labelling experiments with <sup>3H-acetate. Histone isoforms were separated and identified by HPLC/MS to determine post-translational modifications. RESULTS At the concentration of 50 μM R 9HSA shows a stronger antiproliferative effect in HT29 cells in comparison with the S isomer, as indicated by a more remarkable arrest in G0/G1 associated to the higher hyperacetylation of nucleosamal histones. These results show for the first time that (R)-9HSA induces antiproliferative effects at concentrations lower than those of the racemic mixture and they suggest that the interaction with HDAC1 is strictly stereospecific. [1] Calonghi N. et al. (2004) BBRC 314:138-142.
N. Calonghi, E. Pagnotta, C. Parolin, M. Naldi, C. Mangano, C. Boga, et al. (2006). ANTIPROLIFERATIVE ACTIVITY OF ENANTIOMERICALLY PURE (R) AND (S)-9-HYDROXYSTEARIC ACID IN HT29 COLON CANCER CELLS..
ANTIPROLIFERATIVE ACTIVITY OF ENANTIOMERICALLY PURE (R) AND (S)-9-HYDROXYSTEARIC ACID IN HT29 COLON CANCER CELLS.
CALONGHI, NATALIA;PAGNOTTA, ELEONORA;PAROLIN, CAROLA ELEONORA;NALDI, MARINA;MANGANO, CHIARA;BOGA, CARLA;MASOTTI, LANFRANCO
2006
Abstract
INTRODUCTION The endogenous lipoperoxidation product 9-hydroxystearic acid (9HSA) is an inhibitor of HDAC1 and its administration to HT29, a colon adenocarcinoma cell line, induces a proliferative arrest mediated by a direct activation of the p21 WAF 1 gene, bypassing p53 [1]. In a recent work the interaction of 9-HSA with the catalytic site of the 3D model of HDAC1 has been tested with a docking procedure. Noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one: in fact the energies of interaction are -8.45 kcal/mol and -1.97 kcal/mol for the (R) and (S) isomer, respectively, and the estimated free energies of binding are -6.31 kcal/mol and +4.98 kcal/mol for (R) and (S), respectively [2]. In this work (R) and (S)-9HSA were isolated and their biological activity tested in HT29. MATERIALS AND METHODS (R)-9HSA and (S)-9HSA were obtained in enantiomerically pure forms, through a multistep procedure starting from Dimorphotheca sinuata seeds. Cell viability was evaluated by MTT assay. HT29 cells were treated with 50 μM of (R)-9HSA or (S)-9HSA after 24 hours of seeding and their effects on cell growth were evaluated by flow citometry. The effect of the two enantiomers on the HDAC1 activity was tested as histone acetylation level by pulse labelling experiments with 3H-acetate. Histone isoforms were separated and identified by HPLC/MS to determine post-translational modifications. RESULTS At the concentration of 50 μM R 9HSA shows a stronger antiproliferative effect in HT29 cells in comparison with the S isomer, as indicated by a more remarkable arrest in G0/G1 associated to the higher hyperacetylation of nucleosamal histones. These results show for the first time that (R)-9HSA induces antiproliferative effects at concentrations lower than those of the racemic mixture and they suggest that the interaction with HDAC1 is strictly stereospecific. [1] Calonghi N. et al. (2004) BBRC 314:138-142.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
