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

Purpose: 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.

Methods: 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.

Results: 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.

Conclusion: Our study indicated that E35 might be a potential antileukemia agent against IM resistance in CML.

Fig. 1

Chemical structure of emodin and E35. a Emodin: C₁₅H₁₀O₅, MW: 446.35. b E35: C₃₄H₅₀BrNO₅^.H₂O, MW: 631.29

Fig. 2

Proliferation inhibition of 32Dp210-T315I cells treated with E35, IM or nilotinib. a Effects of IM, nilotinib or E35 on the proliferation of 32Dp210-T315I cells. Cells were treated with IM, nilotinib or E35 at the indicated concentrations for 48 h and then assessed by MTT assay (^* P < 0.01, versus IM group). b Proliferation of 32Dp210-T315I cells with or without E35 at indicated incubation time and concentrations. c Proliferation of 32Dp210-T315I cells in the presence or absence of E35 at 48 h. Cells were treated with E35 at the indicated concentrations for 48 h. d Inhibition of the colony formation of 32Dp210-T315I cells by E35. Colony formation assay was conducted as described in “materials and methods” (*P < 0.01, versus control group)

Fig. 3

Induction of apoptosis in 32Dp210-T315I cells by E35. a Cells were treated with E35 (0–8.0 μM) for 48 h. DNA fragmentation assay was performed as described in “materials and methods.” b Cells were treated with E35 (0–3.0 μM) for 12 h. Annexin V/PI staining assay was performed. The annexin V⁻PI⁻, annexin V⁺PI⁻, annexin V⁺PI⁺ and annexin V⁻PI⁺ cells correspond to viable, early apoptotic, late apoptotic and necrotic cells, respectively. Results were presented as mean ± SD (n = 3) (^△ P > 0.05, versus control group, *P < 0.05, versus control group)

Fig. 4

Expression of the BCR-ABL mRNA and P210^BCR-ABL protein detected by RQ-PCR and Western blot in 32Dp210-T315I cells after exposure of E35. a The 32Dp210-T315I cells were exposed to varying concentrations of E35 (0–8 μM) for 48 h. The RQ-PCR detection of expression of the BCR-ABL gene was performed in triplicate as described in “materials and methods.” The ratios of BCR-ABL to ABL were used for quantitative analysis (^△ P > 0.05, versus control group). b, c The 32Dp210-T315I cells were exposed to 8 μM of E35 for different incubation time (12, 24 and 48 h) or to E35 at concentrations of 2, 4 and 8 μM for 48 h. And the expression of P210^BCR-ABL and p-P210^BCR-ABL proteins was analyzed by Western blot and normalized to β-actin intensity (*P < 0.05, versus control group)

Fig. 5

Detection of proteins associated with the CrkL, Akt/mTOR, MEK/ERK and mitochondrial-mediated apoptotic pathway in 32Dp210-T315I cells after exposure of E35. a Dose-dependent and time-dependent effects of E35 on the total and phosphorylated proteins expression of Crkl, Akt, mTOR, 4E-BP1, p70S6K and ERK in 32Dp210-T315I cells. b The 32Dp210-T315I cells were exposed to PD98059 (20 μM), rapamycin (500 nM), LY294002 (50 μM) and E35 (2 and 8 μM) for 48 h, respectively. The changes of molecules of Akt/mTOR and MEK/ERK signaling pathways were analyzed by Western blot. c Dose-dependent activation effects of E35 on the mitochondrial-mediated apoptotic pathway involving Bax, Bcl-2, pro-caspase 3, pro-caspase 9 and PARP

Fig. 5

Detection of proteins associated with the CrkL, Akt/mTOR, MEK/ERK and mitochondrial-mediated apoptotic pathway in 32Dp210-T315I cells after exposure of E35. a Dose-dependent and time-dependent effects of E35 on the total and phosphorylated proteins expression of Crkl, Akt, mTOR, 4E-BP1, p70S6K and ERK in 32Dp210-T315I cells. b The 32Dp210-T315I cells were exposed to PD98059 (20 μM), rapamycin (500 nM), LY294002 (50 μM) and E35 (2 and 8 μM) for 48 h, respectively. The changes of molecules of Akt/mTOR and MEK/ERK signaling pathways were analyzed by Western blot. c Dose-dependent activation effects of E35 on the mitochondrial-mediated apoptotic pathway involving Bax, Bcl-2, pro-caspase 3, pro-caspase 9 and PARP