A novel coumarin derivative DBH2 inhibits proliferation and induces apoptosis of chronic myeloid leukemia cells

Abstract

With the development of tyrosine kinase inhibitor (TKI) resistance, finding the novel effective chemotherapeutic agent is of seminal importance for chronic myelogenous leukemia (CML) treatment. This study aims to find the effective anti-leukemic candidates and investigate the possible underlying mechanism. We synthesized the novel coumarin derivatives and evaluated their anti-leukemic activity. Cell viability assay revealed that compound DBH2 exhibited the potent inhibitory activity on the proliferation of CML K562 cells and TKI resistant K562 cells. Morphological observation and flow cytometry confirmed that DBH2 could selectively induce cell apoptosis and cell cycle arrest at G2/M phase of the K562 cells, which was further confirmed on the bone marrow cells from CML transgenic model mice and CD34⁺ bone marrow leukemic cells from CML patients. Treatments of DBH2 in combination with imatinib could prolong the survival rate of SCL-tTA-BCR/ABL transgenic model mice significantly. Quantitative RT-PCR revealed that DBH2 inhibited the expression of STAT3 and STAT5 in K562 cells, and caspase-3 knockout alleviated the DBH2 induced apoptosis. Furthermore, DBH2 could induce the expression of PARP1 and ROCK1 in K562 cells, which may play the important role in caspase-dependent apoptosis. Our results concluded that coumarin derivative DBH2 serves as a promising candidate for the CML treatment, especially in the combination with imatinib for the TKI resistant CML, and STAT/caspase-3 pathway was involved in the molecular mechanism of anti-leukemic activity of DBH2.

Keywords: Apoptosis; Caspase; Chronic myeloid leukemia; Coumarin; STAT.

Figure 1

Compound DBH2 induced apoptosis of K562 cells and G2/M cell cycle arrest. (A) The representative flow cytometric results showed the apoptosis of cells by staining with FITC-labeled Annexin V and PI. (B) The percentages of apoptotic cells-based flow cytometric staining, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 vs. vehicle control, n = 5. (C) Morphological observation was performed to show typical apoptotic characteristics in K562 cells after 100 μg/mL DBH2 treatment post 24 h under transmission electron microscopy (TEM). Arrows in red color indicated the apoptotic-body in nuclear, and magnification was 20,000 times. (D) The representative flow cytometry data of K562 cells after 100 μg/mL compound DBH2 treatment or 1% DMSO as vehicle control with Annexin-V and PI staining for cell cycle distribution. (E) Statistical analysis of cell cycle arrest in K562 cells. ∗∗P < 0.01 vs. vehicle control, n = 3.

Figure 2

Compound DBH2 induced apoptosis of bone marrow cells from BCR-ABL gene transgenic mice. (A) The scheme of experimental procedure for bone marrow cells from SCL-tTA-BCR/ABL transgenic model mice. (B) The representative flow cytometry data of murine bone marrow cells after compound DBH2 treatment or 1% DMSO as vehicle control with Annexin-V and PI staining. (C) The quantitative analysis of apoptosis-based flow cytometry analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 vs. vehicle, ^####P < 0.0001 vs. WT, n = 4.

Figure 3

The effect of DBH2 in combination with imatinib (IM) on bone marrow cells from BCR-ABL gene transgenic mice in vitro and leukemic development in vivo. (A) Murine model was established and bone marrow transplantation was performed. After 2 months, bone marrow cells were collected and administrated with DBH2 (50, 100 and 200 μg/mL) and IM (1 μM) in vitro. (B) Flow cytometry analysis of murine bone marrow cells treated by DBH2 with or without IM. (C) The quantitative analysis of apoptosis-based flow cytometry analysis. ^###P < 0.001 vs. vehicle control, ∗P < 0.05, ∗∗P < 0.01 vs. DBH2 alone treatment, n = 3. (D) Animal survival observation after intraperitoneal administration of 100 mg/kg DBH2, 50 mg/kg IM or DBH2 combined with IM (100 mg/kg; 50 mg/kg) for 32 days. ∗P < 0.05 vs. vehicle control, ^#P < 0.05 vs. IM alone, n = 8.

Figure 4

DBH2 promoted apoptosis targeting leukemic stem cells and STAT pathway. (A) Representative flow cytometric results of compound DBH2 (50, 100, and 200 μg/mL) and 1% DMSO treated bone marrow cells from CML patients. (B) The quantitative analysis of DBH2 induced bone marrow (BM) cells and CD34⁺ LSCs apoptosis from CML patients. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. vehicle control, n = 3. (C) Expression of STAT3 or STAT5 mRNA in K562 cells after compound DBH2 (50, 100, and 200 μg/mL) treatment. (D) The quantitative analysis of relative expression of STAT3 or STAT5 mRNA after DBH2 (50, 100, and 200 μg/mL) and 1% DMSO treatment. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. control, n = 3.

Figure 5

Caspase-3 mediated the apoptosis of leukemic cells induced by compound DBH2. (A) Representative flow cytometric results of Gr-1⁺/Mac-1⁺bone marrow myeloid cells from wild type (WT) and caspase-3 gene knock-out mice. (B) The quantitative analysis of Gr-1⁺/Mac-1⁺bone marrow myeloid cells between WT and caspase-3 knock-out groups. ∗∗∗∗P < 0.0001 vs. WT, n = 6. (C) Representative flow cytometric results of bone marrow myeloid cells from WT mice or caspase-3 knock-out mice treated by compound DBH2 (50, 100, and 200 μg/mL) with 1% DMSO as vehicle control. (D) The quantitative analysis of apoptosis in bone marrow myeloid cells from WT mice or caspase-3 knock-out mice after DBH2 (50, 100, and 200 μg/mL) treatment. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. vehicle control, ^#P < 0.05 vs. WT group, n = 6.

Figure 6

Compound DBH2 up-regulated the expression of PARP1 and ROCK1 in K562 cells. (A) The representative Western blot staining for PARP1 protein in the vehicle treatment control group and compound DBH2 (50, 100, and 200 μg/mL) treatment groups. (B) The quantitative analysis of PARP1 protein expression in the control and compound DBH2 treatment groups in K562 cells. ∗∗P < 0.05, ∗∗∗∗P < 0.0001 vs. vehicle control, n = 3. (C) The representative Western blot staining for ROCK1 protein in the vehicle treatment control group and compound DBH2 (50, 100, and 200 μg/mL) treatment groups. (D) The quantitative analysis of ROCK1 protein expression in the control and compound DBH2 treatment groups in K562 cells. ∗∗∗∗P < 0.0001 vs. vehicle control, n = 3.