Article Figures & Data
Figures
- Figure 1.
HIF-1α expression in multiple myeloma cells. A, after 24 h of serum starvation, the myeloma cell lines were treated with interleukin 6 (50 ng/mL) or IGF-1 (100 ng/mL) for 4 h, and nuclear extracts were prepared for the analysis of HIF-1α protein expression. B, CD138+ cells were isolated from bone marrow samples of myeloma patients (patients 1 and 2 in Table 1). CD19+ normal B lymphocytes were purified from the peripheral blood of healthy volunteers. Purified cells were then divided into two groups and cultured with or without IGF-1 (100 ng/mL) for 24 h. Normal B lymphocytes were also cultured under hypoxic conditions (1% O2) for 24 h. HIF-1α expression was monitored using immunofluorescence microscopy. Green, HIF-1α blue, 4', 6-diamidino-2-phenylindole nuclear staining. C, pGL3-HRE-LUC reporter plasmids were transfected with pRL-TK Luc plasmid into myeloma cell lines. After 24 h in culture, the transfected cells were treated with 100 ng/mL of IGF-1 (gray column) or left untreated (white column) for an additional 24 h and then harvested to determine luciferase activity. The reporter activities were normalized to an internal control. Each column is the average and SE of the three independent experiments. *, P < 0.05.
- Figure 2.
IGF-1 induces HIF-1α through PI3-K and MAPK activation. A, myeloma cells were serum starved for 24 h and then stimulated with IGF-1 (100 ng/mL) for 10 min. Whole cell lysates were prepared, and the activation of AKT and MAPK was analyzed by Western blotting using antiphospho antibodies for each protein. B, myeloma cells were cultured for 4 h with or without inhibitors (10 μmol/L of LY294002 or 10 μmol/L of U0126) and the expression of HIF-1α was analyzed by Western blotting. C, after 24 h of serum deprivation, myeloma cells were left untreated or cultured with U0126 (10 μmol/L) or LY294002 (10 μmol/L) for 1 h. Thereafter, the cells were treated with IGF-1 (100 mg/mL) for 4 h, and HIF-1α expression was monitored by Western blotting.
- Figure 3.
Stable HIF-1α–silencing KMM-1 cells showed increased sensitivity to melphalan-induced apoptosis. A, two independent stable HIF-1α knockdown KMM-1 cell lines were established. Parental KMM-1 cells and two HIF-1α knockdown clones (1 and 12) were cultured without serum for 24 h and then stimulated with IGF-1 (100 ng/mL) for 4 h. Nuclear extracts were prepared, and the expression levels of HIF-1α and β-subunit were examined by Western blotting. The membrane was reprobed with an anti-TFIIH antibody. B, parental KMM-1 and two independent HIF-1α knockdown clones were cultured with various concentrations of melphalan for 24 h, and Annexin V staining was done to analyze the percentage of apoptotic cells. Each column is the average of three independent experiments and their SEs. White column, parental KMM-1; shaded column, clone 1, and black column, clone #12. **, P < 0.01. C, parental KMM-1 cells and HIF-1α knockdown clones were cultured with 10 μmol/L melphalan with or without 24 h of IGF-1 pretreatment. Annexin V staining was used to detect apoptotic cells. Average numbers of apoptotic cells in three independent experiments are presented as columns with SE. White columns, control; shaded columns, melphalan; gray column, IGF-1 pretreatment plus melphalan. **, P < 0.01. N.S., not significant.
- Figure 4.
Inhibition of HIF-1 by echinomycin enhances the sensitivity of myeloma cells to melphalan. A, pGL3-HRE-LUC reporter plasmids were transfected with the pRL-TK Luc plasmid into myeloma cell lines. The transfected cells were cultured with the indicated concentrations of echinomycin for 48 h and then harvested to determine luciferase activity. The reporter activities were normalized to an internal control. Each column is the average and SEs of three independent experiments.*, P < 0.05; **, P < 0.01. B, each myeloma cell line was cultured with the indicated concentrations of melphalan either with (gray column) or without (white column) echinomycin (1 nmol/L for KMM-1 and RPMI8226, 10 nmol/L for U266) for 24 h, and the ratio of apoptotic cells was monitored by Annexin V staining. The average of the percentages of apoptotic cells from three independent experiments is shown together with the SE. *, P < 0.05; **, P < 0.01. C, KMM-1, U266, and RPMI8226 cell lines were cultured with echinomycin (1 nmol/L for KMM-1 and RPMI8226, 10 nmol/L for U266), melphalan (10 μmol/L), or echinomycin plus melphalan for 24 h, and total cell lysates were then prepared. Caspase-3 activation was examined by Western blotting using an anti–cleaved caspase-3 antibody. As an internal control, the membrane was reprobed with an anti–β-actin antibody. D, myeloma cells were cultured with or without IGF-1 (100 ng/mL) for 24 h, then treated with melphalan (10 μmol/L) or melphalan plus echinomycin (1 nmol/L for KMM-1 and RPMI8226, 10 nmol/L for U266) for an additional 24 h. The ratio of apoptotic cells was analyzed by flow cytometry using Annexin V staining. The columns are average ratios of apoptotic cells ±SEs of three independent experiments. *, P < 0.05; **, P < 0.01.
- Figure 5.
Echinomycin enhances melphalan-induced apoptosis in isolated primary CD138+ myeloma cells to suggest a schematic model of the HIF-1 role in myeloma cells. Primary CD138+ myeloma cells were isolated from bone marrow samples from multiple myeloma patients 3 through 8, as shown in Table 1. CD19+ normal B lymphocytes were purified from peripheral blood from healthy volunteers (control 1 and 2). The cells were divided into four different groups and cultured under the indicated conditions for 24 h. Hoechst staining was done to monitor the apoptotic cell ratio. At least 50 cells were examined for each set of conditions, and the ratio of apoptotic cells was calculated and shown as a column. (control; white, 1 nmol/L echinomycin; gray, 10 μmol/L melphalan; shaded, 10 μmol/L melphalan plus 1 nmol/L echinomycin; black).
- Figure 6.
Survivin is a target of HIF-1 in multiple myeloma cells. A, multiple myeloma cells were cultured with or without echinomycin (1 nmol/L for KMM-1 and RPMI8226 cells and 10 nmol/L for U266 cells) for 24 h, and total cell lysates were prepared. Expression rates of survivin, MCL-1, BCL-xL, and BCL-2 were analyzed by Western blotting. B, myeloma cells were cultured with or without echinomycin for 12 h, and total RNA was prepared. The mRNA level of survivin was analyzed by quantitative RT-PCR. Each column is an average of survivin mRNA levels normalized to 18S rRNA (internal control) ±SE of three experiments (white columns, without echinomycin; shaded columns, with echinomycin). **, P < 0.01. C, the parental KMM-1 cells and two HIF-1α knockdown clones were serum starved for 24 h and subsequently cultured with 100 ng/mL IGF-1 for 24 h. Total cell lysates were prepared, and Western blotting was done to analyze survivin, MCL-1, BCL-xL, and BCL-2 expression. As an internal control, the membrane was reprobed with an anti–β-actin antibody. D, after 24 h of serum starvation, myeloma cells were treated with the indicated concentrations of echinomycin for 1 h and then cultured with IGF-1 for an additional 24 h. Whole cell lysates were prepared for analysis of the survivin expression level.
Tables
- Table 1.
Clinical features of patients with multiple myeloma
Patient no. Age/Sex Disease status, Durie-Salmon stage Treatment Paraprotein type 1 58/M Refractory VAD, endoxan BJP, κ 2 69/M Refractory MP IgG, λ 3 73/M Newly diagnosed, stage II — IgM, κ 4 65/M Newly diagnosed, stage II — IgG, κ 5 79/M Newly diagnosed, stage II — IgG, κ 6 78/M Newly diagnosed, stage II — IgG, κ 7 76/F Newly diagnosed, stage III — IgA, κ 8 59/M Newly diagnosed, stage III — IgG, κ Abbreviations: VAD, vincristine, adriamycin, dexamethasone; MP, melphalan + prednisolone; M, male; F, female; Ig, immunoglobulin; BJP, Bence Jones protein.
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Volume 8, Issue 8
