Suppression of chronic lymphocytic leukemia progression by CXCR4 inhibitor WZ811

Abstract

CXCR4 is a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of C-X-C chemokine receptor type 4 (CXCR4) is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and generally correlates with a poor prognosis. A highly potent competitive antagonist of CXCR4, WZ811, recently has been identified with suppression of cancer cells aggressive in a variety of cancers. However, the effects of WZ811 on chronic lymphocytic leukemia cells have not yet been defined. The effect of WZ811 on chronic lymphocytic leukemia cells TF-1 and UT-7 cells in proliferation, colony formation, and cell migration in vitro were measured respectively. Decreased in cell viability, colony formation, migration, and survival with cell cycle arrest and higher sensitivity to docetaxel in vitro was observed upon WZ811 treatment. In mouse xenograft models developed with human leukemia cells, WZ811 exhibited tumor growth inhibition. Collectively, we have demonstrated that CXCR4 inhibition by WZ811 has the potential for the treatment of human hematological malignancies. This study demonstrated that WZ811 may be a novel approach in the treatment of chronic lymphocytic leukemia.

Keywords: CXCR4; Chronic lymphocytic leukemia; WZ811; docetaxel.

Figures

Figure 1

CXCR4 is up-expressed in chronic lymphocytic leukemia. A: The expression level of CXCR4 in chronic lymphocytic leukemia cells was examined using western blot assay. B: CXCR4 levels were further confirmed using RT-PCR. The data are presented as mean ± SD. For indicated comparisons, *P < 0.05, **P < 0.01.

Figure 2

WZ811 inhibits chronic lymphocytic leukemia cells proliferation and aggressive effectively. A: Cells were exposed to indicated concentrations of WZ811 (1, 5, 10, 20, 40 μM) for 24 h and 48 h, respectively. Cell viability was determined by MTS proliferation assay. The data are presented as mean ± SD. The values are expressed as percentage of viable cells normalized to percentage of viable cells in 0.5% DMSO-treated cells. *P < 0.05 and **P < 0.05 compared with the DMSO-treated group. B: TF-1 and UT-7 cells were treated with the indicated concentrations of WZ811 for 6 h. The migration activity of the cells in the WZ811n group was reduced obviously compared with the control cells. *P < 0.05 and **P < 0.05 compared with the control group. C: The effects of WZ811 on the colony forming ability of TF-1 and UT-7 cells. Images are representative of three independent experiments. *P < 0.05 and **P < 0.05 compared with the control group.

Figure 3

WZ811 increases the sensitivity to docetaxel in TF-1 and UT-7 cells. A: The results of flow cytometry showed that CLL cells treated with WZ811 exhibited an increased rate of the G1-phase and a decrease in the S-phase. The data are presented as mean ± SD. B: In accordance with the inhibition of the cells survival by WZ811, cells in the WZ811 group exhibited an increased proportion of apoptosis compared with the control group. The values are expressed as percentage of apoptotic cells normalized to percentage of viable cells in 0.5% DMSO-treated cells. For indicated comparisons, **P < 0.05 compared with the DMSO-treated group. C: Chemo-sensitivity assays showed that WZ811 can obviously increase the sensitivity of TF-1 and UT-7 cells to docetaxel (P < 0.05; n = 3) with a lower IC50 values in WZ811 group than control group (P < 0.05; n = 3).

Figure 4

WZ811 inhibits aggressiveness markers and induces apoptosis in chronic lymphocytic leukemia cells. A. WZ811 suppressed the activation of CXCR4 induced by PI3K-AKT signaling pathway in TF-1 and UT-7 cells by western blot analysis. B. WZ811 down-regulated the expression of Bcl-2, Bax and caspase-3 in both TF-1 and UT-7 cells by western blot analysis. β-Tublin was shown as loading control. C. Fluorescence microscopic images of TF-1 and UT-7 cells treated with WZ811 for 24 h. Nuclear brightness were observed.

Figure 5

WZ811 suppresses the lymphocytic leukemia cells growth on mouse xenograft models. A: Immunosuppressed mice with established TF-1 cells were given WZ811 (40 mg/kg) by oral gavage or vehicle (control). The mice treated with WZ811 showed marked reduction in tumor growth compared with the mice treated with vehicle. B: The volume of the tumors was significantly lower in mice treated with WZ811 than in the control group mice. **P < 0.01 compared with the control group. Each data point represents the Mean ± SD of 6 mice. C: The weight of the tumors was significantly decreased in WZ811-treated mice than in vehicle-treated mice (**P < 0.01). D: Body weight was measured and plotted as absolute value from 0-25 days post-treatment. E: Tumor sections were analyzed by immunohistochemistry for detection of Ki67 expression in each group of nude mice. Apoptotic cells were examined by TUNEL staining. Each image was representative of six independent mice.

Figure 6

WZ811 suppresses CXCR4/PI3K/AKT signaling pathway in mouse xenograft model of lymphocytic leukemia. A: Immunosuppressed mice with established TF-1 cells were given WZ811 (40 mg/kg) by oral gavage or vehicle (control). Tumor sections were analyzed by immunohistochemistry for detection of CXCR4, PI3K, and AKT expression in each group of nude mice. Each image was representative of six independent mice. B: The proteins were extracted from tumor xenografts and were subjected to western blot for measuring protein levels of phosphor-PI3K, phosphor-AKT, phosphor-mTOR, phosphor-GSK-3β, phosphor-NF-κB p65, and Bcl-2, Bax as well as Caspase-3 expression respectively.