Triptolide Inhibits Bcr-Abl Transcription and Induces Apoptosis in STI571-resistant Chronic Myelogenous Leukemia Cells Harboring T315I Mutation

Abstract

Purpose: Resistance to STI571 is an emerging problem for patients with chronic myelogenous leukemia (CML). Mutation in the kinase domain of Bcr-Abl is the predominant mechanism of the acquired resistance to STI571. In the present study, we investigated the effect of triptolide on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. Experimental Design: CML cell lines (KBM5 versus KBM5-T315I, BaF3-Bcr-Abl versus BaF3-Bcr-Abl-T315I) and primary cells from CML patients with clinical resistance to STI571 were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. Nude mouse xenograft model was also used to evaluate the antitumor activity. Results: Triptolide potently down-regulated the mRNA and protein levels of Bcr-Abl independently of the caspase or proteosome activation in CML cells. It induced mitochondrial-dependent apoptosis in Bcr-Abl-T315I CML cells and primary cells from CML patients with clinical resistance to STI571. Additionally, triptolide inhibited the growth of STI571-sensitive KBM5 and STI571-resistant KBM5-T315I CML cells in nude mouse xenografts. Triptolide also down-regulated the expression of survivin, Mcl-1, and Akt in CML cells, which suggests that it may have multiple targets. Conclusions: These findings suggest that triptolide is a promising agent to overcome STI571-resistant CML cells, and warrant a clinical trial of triptolide derivatives for CML with Bcr-Abl-T315I mutation.

Targeting Activating Transcription Factor 3 by Galectin-9 Induces Apoptosis and Overcomes Various Types of Treatment Resistance in Chronic Myelogenous Leukemia

“…Cell lines and reagents, and gene trasfection CML-derived BV173, KT-1, KCL22, K562 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), KBM5 (13) and MYL (14) cell lines, Jurkat T cell, an immortalized T lymphocyte cell line, and HL60, Bcr-Abl-negative myelogenous leukemia cell line were maintained in RPMI 1640 with 10% FCS, 2 mmol/L L-glutamate, and penicillin/streptomycin. IM-resistant KBM5/IMR with Abl T315I mutation was generated by continuous culture in medium containing 1.0 μmol/L IM (15).…”

The Antitumor Activity of Homoharringtonine against Human Mast Cells Harboring the KIT D816V Mutation

“…Mcl-1 localizes to the mitochondria and other intracellular membranes and has a relatively short half-life (31). Overexpression of Mcl-1 protects tumor cells from apoptosis induced by diverse agents, including flavopiridol, triptolide, and sorafenib (25,27,43). Mcl-1 is overexpressed in neoplastic cells in hematologic malignancies, including CML and SM (1,44).…”

“…STI571 was a product of Novartis Pharmaceuticals (27). Rabbit antibodies against human myeloid cell leukemia-1 (Mcl-1; S-19), KIT (CD117), and phospho-KIT (Y568/570), Bax, Bcl-2, and caspase-3 were from Santa Cruz Biotechnology; mouse monoclonal antibody against poly(ADP-ribose) polymerase (PARP) was from BD Pharmingen; rabbit anti-Akt, phospho-Akt (S473), extracellular signal-regulated kinase 1/2 (ERK1/2), and phospho-ERK1/2 (T202/Y204) were from Cell Signaling Technology; mouse anti-phospho-STAT3 (Y705, clone 9E12), STAT3, phospho-STAT5A/B (Y694/Y699; clone 8-5-2), rabbit anti-STAT5A, and platelet-derived growth factor receptor α (PDGFRα) were from Upstate Technology; rabbit anti-Bim was from Stressgen; mouse anti-actin was from Sigma; and mouse CD45-FITC and CD117-phycoerythrin were from eBioscience.…”

“…An MTS assay (CellTiter 96 Aqueous One Solution reagent, Promega Corp.) was used to evaluate cell viability, as described previously (27). The drug concentration resulting in 50% inhibition of cell growth (IC 50 ) was determined.…”

“…Apoptosis was evaluated by Annexin V-fluoroisothiocyanate apoptosis detection kit according to the instruction of the manufacturer (Sigma-Aldrich) and analyzed with use of FACSCalibur flow cytometer (13,27,31).…”