Inhibition of 32Dp210 cells harboring T315I mutation by a novel derivative of emodin correlates with down-regulation of BCR-ABL and its downstream signaling pathways.

Abstract

The clinical outcome of chronic myeloid leukemia (CML) patients has been changed dramatically due to the development of imatinib (IM). However, the emergence of IM resistance, commonly associated with point mutations within the BCR-ABL kinase domain, remains a major clinical problem. Here, we investigated the effects of E35, a novel derivative of emodin, on the IM-resistant 32Dp210-T315I cells. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide and colony formation assay. Induction of apoptosis was confirmed by DNA fragmentation assay and annexin V/PI staining assay. Real-time quantitative PCR was used to access the BCR-ABL gene expression. Changes of related signaling molecules were detected through Western blot. E35 was found to potently inhibit proliferation of 32Dp210-T315I cells with an average IC50 of 2.4 µM at 48 h. Colony formation was almost fully suppressed in 1.0 μM E35 group. DNA fragmentation and annexin V/PI staining assay exhibited the typical DNA fragmentation and the increased proportion of early apoptotic cells, respectively. The induction of apoptosis was associated with increase of Bax to Bcl-2 expression ratio and activation of caspase cascades involving decrease of pro-caspase 9 and pro-caspase 3 and increase of PARP cleavage. The protein expression of P210(BCR-ABL) and p-P210(BCR-ABL) was down-regulated in the presence of E35, although the mRNA levels remained almost unchanged. Moreover, the activation of the P210(BCR-ABL) downstream signaling pathways including CrkL, Akt/mTOR and MEK/ERK was fully suppressed by E35. Our study indicated that E35 might be a potential antileukemia agent against IM resistance in CML.