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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 - Cancer Res Treat (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Internal Medicine, Chungnam National University College of Medicine, Daejeon, Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: To examine whether SDF-1 and AMD3100 affect the proliferation of myeloma cells, the two myeloma cells and primary CD138+ cells from three patients with multiple myeloma were incubated in serum-free X-VIVO medium in the absence or presence of AMD3100 (10-7 to 10-5 M) or T140 (10-7 M to 10-5 M) for up to 3 days and analyzed by a CCK-8 assay. SDF-1 alone did not affect the proliferation of the myeloma cells (data not shown). AMD3100, but not T140, dose-dependently increased the number of myeloma cells and the primary CD138+ cells. After a 3-day incubation, 10-5 M AMD3100 increased the proliferation of RPMI8226 cells, U266 cells and primary CD138+ cells by 1.8-, 1.4- and 1.3-fold, respectively, compared to the control (all p<0.05; Fig. 2A and 2B). These proliferation-enhancing effects were more prominent in the presence of IL-6 in U266 cells, but not in RPMI8226 cells (Fig. 2C). Pretreating the cells with PTX for 2 hours abrogated the enhancement (Fig. 2D). AMD3100 did not affect the proliferation of hepatocellular carcinoma PLC/PRF5 or Hep3B cells (data not shown), indicating that the proliferation-enhancing effect of AMD3100 is not common to all cells.


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 - Cancer Res Treat (2010)

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.
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Figure 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.
Mentions: To examine whether SDF-1 and AMD3100 affect the proliferation of myeloma cells, the two myeloma cells and primary CD138+ cells from three patients with multiple myeloma were incubated in serum-free X-VIVO medium in the absence or presence of AMD3100 (10-7 to 10-5 M) or T140 (10-7 M to 10-5 M) for up to 3 days and analyzed by a CCK-8 assay. SDF-1 alone did not affect the proliferation of the myeloma cells (data not shown). AMD3100, but not T140, dose-dependently increased the number of myeloma cells and the primary CD138+ cells. After a 3-day incubation, 10-5 M AMD3100 increased the proliferation of RPMI8226 cells, U266 cells and primary CD138+ cells by 1.8-, 1.4- and 1.3-fold, respectively, compared to the control (all p<0.05; Fig. 2A and 2B). These proliferation-enhancing effects were more prominent in the presence of IL-6 in U266 cells, but not in RPMI8226 cells (Fig. 2C). Pretreating the cells with PTX for 2 hours abrogated the enhancement (Fig. 2D). AMD3100 did not affect the proliferation of hepatocellular carcinoma PLC/PRF5 or Hep3B cells (data not shown), indicating that the proliferation-enhancing effect of AMD3100 is not common to all cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Internal Medicine, Chungnam National University College of Medicine, Daejeon, Korea.

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.

No MeSH data available.


Related in: MedlinePlus