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Article Figures & Data

Figures

  • Fig. 1.

    Caspase-8 mediates IMiD-induced apoptosis.

    (A) Quantification of caspase-8 (black bars) and caspase-9 (white bars) activity was performed using the ApoAlert caspase colorimetric assay kit in MM.1S cells treated with IMiD1 (1 μM for 72 hours in 1% serum) after normalization for cellular protein. Treatment with Dex (1 μM for 48 hours) activated caspase-9 and served as a positive control (gray bar). Data shown (mean ± SD) are representative of 3 experiments. (B) The caspase-8–specific inhibitor IETD-FMK, but not the caspase-9 inhibitor LEHD-FMK (both used at 20 μM), protected MM.1S cells from cell death induced by IMiD1 (1 μM for 72 hours in 1% serum), as quantified by the MTT assay (black bars). LEHD-FMK attenuated apoptosis induced by Dex (1 μM for 48 hours; white bars) and served as a positive control. Data shown (mean ± SD) are representative of 3 experiments. (C) The caspase-8–specific inhibitor IETD-FMK (20 μM) protected OCI-My-5, S6B45, and primary MM patient cells from cell death induced by IMiD1 (1 μM for 72 hours in 1% serum), as quantified by the MTT assay (black bars: no inhibitor; white bars: caspase-8 inhibitor). Data shown (mean ± SD) are representative of 3 experiments. (D) The caspase-8–specific inhibitor IETD-FMK (20 μM) protected MM.1S cells from cell death induced by Thal (100 μM for 72 hours in 1% serum) or IMiD3 (1 μM for 72 hours in 1% serum), as quantified by the MTT assay (black bars: no inhibitor; white bars: caspase-8 inhibitor). Data shown (mean ± SD) are representative of 3 experiments.

  • Fig. 2.

    IMiD1 enhances Fas-mediated apoptosis.

    Pretreatment of MM.1S cells with (black bars) or without (white bars) 1 μM IMiD1 for 24 hours increased their sensitivity to apoptosis induced by Fas cross-linking antibody CH11 (12.5-25 ng/mL for 18 hours). Percentage specific cell death was calculated with the formula: % specific cell death = 100 − (absorbance in cells treated with IMiD1 and CH11)/(absorbance in cells treated with IMiD1 alone). Data shown (mean ± SD) are representative of 3 experiments.

  • Fig. 3.

    IMiD1 enhances TRAIL/Apo2L–mediated apoptosis and down-regulates the expression of the antiapoptotic proteins cIAP-2 and FLIP.

    (A) Combined proapoptotic effect of IMiD1 with TRAIL/Apo2L in MM.1S cells. Cells were pretreated with or without 1 μM IMiD1 for 4 hours, and a low concentration of TRAIL/Apo2L (50 ng/mL) was then added for an additional 24 hours. Data shown (mean ± SD) are representative of 3 experiments. IMiD1 potentiated the apoptotic effect of TRAIL/Apo2L. (B) Immunoblotting analysis for the antiapoptotic molecules cIAP-2, FLIP, Bcl-2, ICAM-1, and tubulin in MM.1S cells. Treatment with IMiD1 (1 μM for 72 hours) decreased the protein levels of caspase-8 inhibitors cIAP-2, FLIP, and ICAM-1, but not Bcl-2.

  • Fig. 4.

    IMiD1 inhibits NF-κB activity and enhances the effectiveness of Dex and PS-341 in MM.

    (A) Quantification of the DNA binding activity of the transcriptional factor NF-κB in MM.1S cells treated with or without 1 μM IMiD1 for 48 hours, 1 μM Dex for 24 hours, or both, after normalization for cellular protein. Data shown (mean ± SD) are representative of 3 experiments. (B) Combined proapoptotic effect of IMiD1 with Dex in MM.1S cells. Cells were pretreated with or without 1 μM IMiD1 for 24 hours, and then Dex (0.25 μM) was added for an additional 48 hours. IMiD1 potentiated the apoptotic effect of Dex. Data shown (mean ± SD) are representative of 3 experiments. (C) Combined proapoptotic effect of IMiD1 with PS-341 in MM.1S cells. Cells were pretreated with or without 1 μM IMiD1 for 48 hours, and subtoxic concentrations of PS-341 (3 or 5 nM) were added for an additional 24 hours. IMiD1 potentiated the apoptotic effect of PS-341. Data shown (mean ± SD) are representative of 3 experiments. (D) Combined proapoptotic effect of IMiD1 with PS-341 in primary MM cells. Cells were pretreated with or without 1 μM IMiD1 for 48 hours, and a subtoxic concentration of PS-341 (3 nM) was added for an additional 24 hours. IMiD1 potentiated the apoptotic effect of PS-341. Data shown (mean ± SD) are representative of 3 experiments.

  • Fig. 5.

    IMiD3 enhances MM cell death induced by TRAIL/Apo2L and PS-341.

    (A) Combined proapoptotic effect of IMiD3 with TRAIL/Apo2L in MM.1S cells. Cells were pretreated with or without 1 μM IMiD3 for 4 hours, and a low concentration of TRAIL/Apo2L (50 ng/mL) was then added for an additional 24 hours. IMiD3 potentiated the apoptotic effect of TRAIL/Apo2L. Data shown (mean ± SD) are representative of 3 experiments. (B) Combined proapoptotic effect of IMiD3 with PS-341 in MM.1S cells. Cells were pretreated with or without 1 μM IMiD3 for 48 hours, and then a subtoxic concentration of PS-341 (3 nM) was added for an additional 24 hours. IMiD3 potentiated the apoptotic effect of PS-341. Data shown (mean ± SD) are representative of 3 experiments.

  • Fig. 6.

    IMiD1 blocks the antiapoptotic effect of IGF-1 in MM.

    (A) DNA binding activity of the transcriptional factor NF-κB was quantified in MM.1S cells pretreated with or without 1 μM IMiD1 for 3 hours, and then treated for an additional 4 hours with or without 200 ng/mL IGF-1 or 50 ng/mL TNF-α. IMiD1 inhibited activation of NF-κB induced by IGF-1 and TNF-α in MM cells. (B) Immunoblotting analysis for the antiapoptotic molecules cIAP-2 and FLIP in MM.1S cells. Treatment with IGF-1 (200 ng/mL for 24 hours) increased the protein levels of the caspase-8 inhibitors cIAP-2 and FLIP; IMiD1 (1 μM) inhibited this effect.