High Concentration of CXCR4 Antagonist AMD3100 Is Able to Surrender t(4;11) Leukemia Cells

Abstract 4257

Interactions between leukemia stem cells and the microenvironment protect these cells from the effects of chemotherapy. Functional CXCR4 is necessary for the homing of leukemia cells to the marrow microenvironment. The CXCR4 inhibitor, AMD3100, has being tested in leukemia and multiple myeloma (MM) to disrupt the interaction of tumor cells with the microenvironment, mobilize leukemia cells into the peripheral blood and inhibit engraftment and enhance the sensitivity to therapy in xenograft model. We are interested in how CXCR4 antagonist affects a special type of childhood leukemia with t(4;11), which presents frequently in infant leukemia.

AMD3100 induces proliferation of t(4;11) leukemia cells at low concentration

We investigated the AMD3100’s effect on cell proliferation of t(4;11) leukemia cells. As shown inFigure 1B, leukemia cell numbers did not decrease when AMD3100 was at the concentrations below 0.1mg/mL. AMD3100 at 0.1mg/mL significantly increased the viable cells comparing with the control. It indicates that AMD3100 facilitates leukemia cells proliferation at this concentration. When we used Annexin-V and PI for apoptosis assay, it showed that both PI and Annexin-V positive populations decreased at AMD3100 concentrations of 1 μ g/mL and 10 μ g/mL comparing with the control (Figure 1E). Homogeneous caspases assay did not show increase at these two concentrations (Figure 1F). This indicated that AMD3100 at lower concentrations can protect leukemia cells from apoptosis. Leukemia cells transmigrated through stromal cells with higher numbers of cells when AMD3100 presents at 1 μ g/mL and 10 μ g/mL although without significant statistic differences (Figure 1D).

View largeDownload slide

View largeDownload slide

Close modal

AMD3100 above 500μ g/mL induces apoptosis of t(4;11) leukemia cells

When AMD3100 was included in the culture of t(4;11) leukemia cells, the cell viability percentage started to significantly decrease on day 2 at 1mg/mL (Figure 1A). After normalizing the cell count, it showed that AMD3100 decreased the cell number from concentration 0.5mg/ml and did so significantly at 1.0mg/ml (Figure 1B). Cell culture with AMD3100 did not change the expression level of its receptor CXCR4 (Figure 1C), but decreased the migration level through stromal cells (Figure 1D). To figure out if the reason of low transmigration was because of inhibiting migration or cell killing, we did two apoptosis related assay. First, AMD3100 at concentrations above 0.5 mg/mL significantly increased the apoptosis population showing with Annexin-V positive and cell death population with PI positive by flow cytometry (Figure 1E). Second, the homogneous caspases assay also showed that activated caspases level significantly increased after one day’s culture with AMD3100 at 0.5 mg/mL and 1 mg/mL (Figure 1F). These results confirm that AMD3100 induced apoptosis at higher concentrations.

Downstream kinases assay

To investigate the mechanism, we used Western blot to study the phosphorylation of key kinases in the downstream of SDF-1-CXCR4 pathway. We used two concentrations of AMD3100 to culture with leukemia cells at a series of time points. The results showed that low concentration of AMD3100 (50μ M) increased the phosphorylation of Stat3 at two time points of 10 minute and 6 hours (Figure 2A). Whereas, high concentration of AMD3100 (625μ M) increased the phosphorylation of Stat3 at the first half an hour then always decreased after that. The big difference of AMD3100 cultured overnight (15 hours) with the t(4;11) leukemia cells was that low concentration significantly increased the phosphorylation level of p44/42 mitogen-activated protein kinase (MAPK), i.e. extracellular-related kinase (Erk1/2). But high concentration of AMD3100 significantly decreased phosphorylation level of Erk1/2 (Figure 2B). The cleaved caspase-3 (p11) was increased at high concentration which was consistent with the result of previous apoptosis detection and homogneous caspases assay. There was no big difference of MMP9 change between the two concentrations.

View largeDownload slide

View largeDownload slide

Close modal

In summary, our study showed that AMD3100 at low concentrations increased proliferation and cell survival through activation of phosphorylation of Erk1/2. While at high concentrations, AMD3100 induced apoptosis through inhibiting the phophorylation of Stat3 and Erk1/2.