Introduction. Imatinib mesilate, an inhibitior of bcr-abl tyrosine kinases (TK) which was primarily designed to treat chronic myeloid leukaemia, is also an inhibitor of the c-kit receptor and is currently the drug of choice for the therapy of metastatic gastrointestinal stromal tumors (GISTs), which frequently express constitutively activated forms of the c-kit-receptor. Despite the fact that the majority of patients receiving imatinib respond to treatment, early relapse and drug resistance occur in a large percentage of them. The present study is focused on the ability of a new histone deacetylase (HDAC) inhibitor, 9-hydroxystearic acid (9-HSA) to enhance the cytotoxicity of imatinib in imatinib-resistant cell lines. Materials and methods. HT29, a colon adenocarcinoma cell line expressing the c-kit receptor and p53 mutated, was grown in Petri dishes, exposed for 24 hr to a high dose of imatinib, and maintained in drug-free medium for up to 7 days. Resistance to further treatment with the drug was characterized by loss of apoptotic response by means of annexin V staining. 9-HSA antiproliferative effect on growth inhibition in the resistant cell line (IR-HT-29) was assayed by measuring 3H-thymidine incorporation. Histone classes were separated and identified by HPLC/MS, and the post-translational modifications were determined both in IR-HT-29 and 9-HSA treated cells. Results. Pretreating IR-HT-29 with 9-HSA led to growth-inhibitory and apoptotic effects of imatinib similar to that in imatinib-sensitive HT-29 cell line. More precisely, 9-HSA treatment is able per se to trigger apoptosis and subsequent imatinib treatment amplifies the process. In order to understand the mechanisms of this effect, we analyzed the degree of acetylation, methylation and phosphorylation of histones in IR-HT-29, and in 9-HSA treated IR-HT-29. The results clearly show that histone code in IR-HT-29 was completely changed with respect to control sensitive HT-29, and that 9-HSA treatment induced the increase of the diacetyl-dimethyl H4 content, and several modifications on H3-1 histone. In conclusion our data indicate that the use of HDAC inhibitor 9-HSA may constitute a powerful strategy to enhance cytotoxic effects of imatinib in resistant cell lines.

9-HSA INDUCES APOPTOSIS AND INCREASES SENSITIVITY TO IMATINIB IN IMATINIB RESISTANT COLON CANCER CELLS

PAGNOTTA, ELEONORA;CALONGHI, NATALIA;PAROLIN, CAROLA ELEONORA;MANGANO, CHIARA;BOGA, CARLA;NALDI, MARINA;SANTUCCI, MARIA ALESSANDRA;MASOTTI, LANFRANCO
2006

Abstract

Introduction. Imatinib mesilate, an inhibitior of bcr-abl tyrosine kinases (TK) which was primarily designed to treat chronic myeloid leukaemia, is also an inhibitor of the c-kit receptor and is currently the drug of choice for the therapy of metastatic gastrointestinal stromal tumors (GISTs), which frequently express constitutively activated forms of the c-kit-receptor. Despite the fact that the majority of patients receiving imatinib respond to treatment, early relapse and drug resistance occur in a large percentage of them. The present study is focused on the ability of a new histone deacetylase (HDAC) inhibitor, 9-hydroxystearic acid (9-HSA) to enhance the cytotoxicity of imatinib in imatinib-resistant cell lines. Materials and methods. HT29, a colon adenocarcinoma cell line expressing the c-kit receptor and p53 mutated, was grown in Petri dishes, exposed for 24 hr to a high dose of imatinib, and maintained in drug-free medium for up to 7 days. Resistance to further treatment with the drug was characterized by loss of apoptotic response by means of annexin V staining. 9-HSA antiproliferative effect on growth inhibition in the resistant cell line (IR-HT-29) was assayed by measuring 3H-thymidine incorporation. Histone classes were separated and identified by HPLC/MS, and the post-translational modifications were determined both in IR-HT-29 and 9-HSA treated cells. Results. Pretreating IR-HT-29 with 9-HSA led to growth-inhibitory and apoptotic effects of imatinib similar to that in imatinib-sensitive HT-29 cell line. More precisely, 9-HSA treatment is able per se to trigger apoptosis and subsequent imatinib treatment amplifies the process. In order to understand the mechanisms of this effect, we analyzed the degree of acetylation, methylation and phosphorylation of histones in IR-HT-29, and in 9-HSA treated IR-HT-29. The results clearly show that histone code in IR-HT-29 was completely changed with respect to control sensitive HT-29, and that 9-HSA treatment induced the increase of the diacetyl-dimethyl H4 content, and several modifications on H3-1 histone. In conclusion our data indicate that the use of HDAC inhibitor 9-HSA may constitute a powerful strategy to enhance cytotoxic effects of imatinib in resistant cell lines.
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Pagnotta E.; Calonghi N.; Parolin C.; Mangano C.; Boga C.; Naldi M.; Santucci M.A.; Masotti L.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/38920
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