Cancer Res Treat.  2010 Dec;42(4):225-234.

The CXCR4 Antagonist AMD3100 Has Dual Effects on Survival and Proliferation of Myeloma Cells In Vitro

• Kim HY
• Hwang JY
• Kim SW
• Lee HJ
• Yun HJ
• Kim S
• Jo DY

Abstract

PURPOSE
AMD3100, an antagonist of the CXCR4 chemokine receptor is soon to be used clinically for the peripheral mobilization of hematopoietic stem cells (HSCs) in patients with multiple myeloma. AMD3100 has been shown to activate a G protein coupled with CXCR4 and thus acts as a partial CXCR4 agonist in vitro. Thus, we explored whether AMD3100 affected the survival and proliferation of myeloma cells in vitro.
MATERIALS AND METHODS
The effects of AMD3100 on survival and proliferation of two myeloma cell lines (RPMI8226 and U266) as well as CD138+ cells obtained from several patients with multiple myeloma were analyzed by flow cytometry using annexin V and a colorimetric cell proliferation assay (CCK-8 assay).
RESULTS
AMD3100, but not T140, another CXCR4 antagonist, stimulated the proliferation of myeloma cell lines and CD138+ primary human myeloma cells (-2-fold increase) in a dose-dependent manner in serum-free culture for up to 5 days, which was inhibited by pretreating the cells with pertussis toxin. AMD3100 enhanced the proliferation of U266 cells induced by interleukin-6 and partially reversed AG490-mediated growth inhibition and apoptosis induced by serum deprivation in RPMI8226 cells. AMD3100 induced the phosphorylation of Akt and MAPK p44/p42 in U266 cells and MAPK p44/p42 in RPMI8226 cells. In contrast, AMD3100 markedly increased the cell apoptosis and reduced the number of RPMI8226 cells after 5 to 7 days of culture under serum-free conditions.
CONCLUSION
AMD3100 exerts dual effects, initially enhancing and subsequently inhibiting the survival and proliferation of myeloma cells, signaling via CXCR4 in vitro.

Keyword

AMD3100; CXCR4 protein; Multiple myeloma; Cell proliferation

MeSH Terms

Annexin A5
Apoptosis
Cell Line
Cell Proliferation
Flow Cytometry
GTP-Binding Proteins
Hematopoietic Stem Cells
Heterocyclic Compounds
Humans
Interleukin-6
Multiple Myeloma
Oligopeptides
Pertussis Toxin
Phosphorylation
Annexin A5
GTP-Binding Proteins
Heterocyclic Compounds
Interleukin-6
Oligopeptides
Pertussis Toxin

Figure

• 

  Fig. 1 AMD3100 and T140 inhibit the chemotaxis of myeloma cells induced by stromal cell-derived factor-1 (SDF-1) and induce the internalization of surface CXCR4 in myeloma cells. (A) The myeloma cell lines RPMI8226 and U266 and CD138+ primary bone marrow myeloma cells were loaded into the upper chamber of a 24-well Transwell plate and were allowed to migrate into the lower chamber containing 100 ng/mL SDF-1 for 4 hours. AMD3100 and T140 were added at 10-5 M and 10-6 M in the upper chamber, respectively. The data are the mean±SD of the migration index from three independent experiments. (B) U266 cells were incubated with or without 10-5 M AMD3100 and 10-6 M T140, respectively, for 3 hours and then subjected to flow cytometry. To detect cytoplasmic CXCR4, the cells were permeabilized with saponin-based reagents before labeling. *p<0.05 compared to the controls (migration toward SDF-1).

• 

  Fig. 2 AMD3100 stimulates the proliferation of myeloma cells. Cells were incubated in 96-well plates in serum-free X-VIVO medium, and cell proliferation was measured using a colorimetric assay. (A) RPMI8226 cells were incubated in serum-free medium in the presence of AMD3100 for up to 72 hours. A representative result from three independent experiments is shown. (B) Myeloma cell lines and CD138+ primary myeloma cells were incubated in the presence of AMD3100 or T140 for 3 days. Data are the mean±SD of the proliferation index from three independent experiments. (C) The proliferation-enhancing effect of AMD3100 was increased further in the presence of interleukin-6 (IL-6). RPMI8226 cells (upper) and U266 cells (lower) were incubated in the presence or absence of AMD3100 (10-5 M), SDF-1 (100 ng/mL) and/or IL-6 (20 ng/mL) for 3 days. Data are the mean±SD of the proliferation index from three independent experiments. (D) Pertussis toxin (PTX) blocked the proliferation of myeloma cells induced by AMD3100. RPMI8226 cells were incubated in the presence or absence of AMD3100 (10-5 M). As indicated, the cells were pretreated with PTX (100 to 200 ng/mL) for 2 hours before the incubation. The relative proliferation index represents the fold-increase in the OD compared to that of the control (medium only) at the beginning of incubation. Data are the mean±SD of the three independent experiments. *p<0.05 compared to the control.

• 

  Fig. 3 AMD3100 protects RPMI8226 myeloma cells from serum deprivation-induced apoptosis, but not from dexamethasone (Dex)-induced apoptosis. Cells were incubated in RPMI-1640 medium without serum in the presence or absence of AMD3100 (10-5 M), interleukin-6 (IL-6; 20 ng/mL), or their combination for 24 hours. In parallel experiments, cells were treated with Dex alone or in combination with AMD3100 (10-5 M). To detect cell apoptosis, flow cytometric analysis was conducted after staining the cells with annexin V. (A, B) Representative flow cytometry profiles of the serum deprivation experiments. (C) The percentages of the annexin V-positive apoptotic cells are presented as the mean±SD from three independent serum deprivation experiments. (D) The percentages of the annexin V-positive apoptotic cells are presented as the mean±SD from three independent experiments of Dex-induced apoptosis. *p<0.05.

• 

  Fig. 4 AMD3100 has dual effects on the phosphorylation of signaling molecules. After a 12-hour serum deprivation, cells were incubated with stromal cell-derived factor-1 (SDF-1; 100 ng/mL), interleukin-6 (IL-6; 20 ng/mL), AMD3100 (10-5 M) or combinations thereof and then subjected to Western blot analysis, to detect phosphorylation of Stat3, Akt, MAPK p44/p42 and p38 MAPK. Representative results of three or more experiments are shown. (A) U266 cells were stimulated with AMD3100 over the indicated periods. (B) U266 cells were incubated with the agents for 10 minutes. The agents were added to the cells at the same time. (C) RPMI8226 cells were stimulated with AMD3100 over the indicated periods. (D) Hepatocellular carcinoma cells PLC/PRF5 (upper) and Hep3B (lower) were treated with AMD3100 for 10 minutes.

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  Fig. 5 Influence of signal-blocking agents on the proliferation of RPMI8226 cells induced by AMD3100. After a 3-day incubation of the cells in serum-free X-VIVO medium, cell proliferation was measured using a colorimetric assay. (A) Cells were treated with fixed concentrations of the agents (10-5 M AMD3100, 10 µM LY294002, 100 nM wortmanin, 50 µM PD98059, 10 µM AG490, 50 nM rapamycin, 10 µM SB203580). Wortmanin and PD98059 affected neither spontaneous nor AMD3100-induced proliferation. (B) The cells were treated with various concentrations of LY294002 and AG490 with or without 10-5 M AMD3100. Data are presented as mean±SD of the proliferation index from three independent experiments. *p<0.05 compared to the control (medium only).

• 

  Fig. 6 AMD3100 induced a marked inhibition of survival in myeloma cells during the later course in extended-period cultures. (A) RPMI8226 cells were incubated with or without AMD3100 or T140 in serum-free medium for up to 14 days. Data are the mean±SD of the relative proliferation index from three independent experiments. (B) RPMI8226 cells were incubated in the presence of stromal cell-derived factor-1 (SDF-1; 100 ng/mL), AMD3100 (10-5 M) or combinations thereof in serum-free medium for up to 14 days. A representative result is shown. (C) Effects of AMD3100 on the formation of tumor cell colonies were examined using RPMI8226 cells. Data are the mean±SD of the number of RPMI8226 cell colonies from three independent experiments in which AMD3100 was added at 10-5 M. (D) Representative profiles of the colony assay are shown. *p<0.05 compared to the control.

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  Fig. 7 AMD3100 exerts biphasic effects on apoptosis induced by serum deprivation in myeloma cells. RPMI8226 cells were incubated with or without AMD3100 in RPMI-1640 medium for up to 14 days. At indicated time points, apoptosis of the cells were analyzed by flow cytometry after staining with annexin V and propium iodide (PI). Representative results of three independent experiments showing consistent results are shown.

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