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Intracellular regulation of tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis in human multiple myeloma cells

Nicholas Mitsiades, Constantine S. Mitsiades, Vassiliki Poulaki, Kenneth C. Anderson and Steven P. Treon

Article Figures & Data

Figures

  • Fig. 1.

    Immunoblot analysis for caspases and PARP in myeloma cells exhibiting differential sensitivities to TRAIL.

    MM.1S, ARH-77, and IM-9 cells were treated with TRAIL (300 ng/mL) for 0.5, 1, and 5 hours. Rapid cleavage of caspase-8, caspase-3, and PARP was observed in the TRAIL-sensitive MM.1S cells. Due to lower caspase-8 protein levels in TRAIL-resistant cells, longer exposures were used to ensure adequate visualization of the bands. Caspase-10 was not cleaved, excluding it as an apical caspase in the TRAIL pathway for MM. In the moderately TRAIL-resistant ARH-77 cells, cleavage of caspases and PARP was significantly inhibited, though eventually low-level cleavage of caspase-3 (on longer exposure of the film) and PARP was seen at 5 hours of treatment. No cleavage of any caspases or PARP was detected in highly TRAIL-resistant IM-9 cells.

  • Fig. 2.

    Assessment of caspase-3 enzymatic activity in TRAIL-sensitive and -resistant cells.

    Caspase-3 enzymatic activity was measured with a caspase-3 intracellular activity assay kit in TRAIL-sensitive (MM.1S, panel A), moderately resistant (ARH-77, panel B), and highly resistant (IM-9, panel C) cells treated with (shaded curves) or without (unshaded curves) TRAIL (300 ng/mL) for 5 hours. A large population of cells was found to be positive for caspase-3 activity in MM.1S, but not ARH-77 or IM-9 cells treated with TRAIL.

  • Fig. 3.

    Immunoblot analysis for caspase-6, caspase-9, and DFF45 in myeloma cells exhibiting differential sensitivities to TRAIL.

    Immunoblot analysis for caspase-6, caspase-9, and DFF45 was performed for TRAIL-sensitive (MM.1S), moderately resistant (ARH-77), and highly resistant (IM-9) cells treated with TRAIL (300 ng/mL) for 0.5 and 1 hour. Rapid cleavage of caspase-6, caspase-9, and DFF45 was observed in MM.1S, but not ARH-77 or IM-9 cells. A band corresponding to cleaved caspase-9 was constitutively present in ARH-77 or IM-9 cells, but remains unchanged after TRAIL treatment.

  • Fig. 4.

    Modulation of TRAIL-induced cytotoxicity in MM.1S myeloma cells by selective inhibition of caspases.

    (A) TRAIL-sensitive MM.1S myeloma cells were pretreated 1 hour before overnight incubation with TRAIL (100, 200, and 300 ng/mL) with or without the pancaspase inhibitor ZVAD-FMK, the caspase-8 inhibitor IETD-FMK, the caspase-3 and -7 inhibitor DEVD-FMK, the caspase-9 inhibitor LEHD-FMK, or the caspase-6 inhibitor VEID-FMK. Cell survival was determined using the MTT colorimetric assay. Bars represent mean ± SD values. (B-D) Caspase-3 enzymatic activity was measured by a caspase-3 intracellular activity assay in TRAIL-sensitive MM.1S cells as in Figure 2. (B) Control MM.1S cells. (C) MM.1S cells were treated with TRAIL (300 ng/mL) for 5 hours. (D) MM.1S cells were treated with TRAIL in the presence of DEVD-FMK (1 hour before treatment). DEVD-FMK inhibits caspase-3 activity, confirming that it is indeed internalized by MM cells and is functional.

  • Fig. 5.

    Expression of intracellular regulators of apoptosis in TRAIL-sensitive and TRAIL-resistant myeloma cells.

    Immunoblot analysis was used to examine cell lysates from myeloma cells that are moderately resistant (ARH-77), highly resistant (HS-Sultan, IM-9), or sensitive (MM.1S, MM.1R, RPMI 8226, RPMI-Dox 40, ARP-1) to TRAIL for expression of apoptosis-inducing (procaspase-8 and FADD) or apoptosis-blocking (FLIP, cIAP1, cIAP2, XIAP, and survivin) proteins. Equal protein loading was ensured by demonstration of uniform tubulin expression.

  • Fig. 6.

    Increased procaspase-8 expression sensitizes TRAIL-resistant myeloma cells to TRAIL.

    Cell survival of highly TRAIL-resistant IM-9 cells that were either untransfected or transfected with an empty vector or a procaspase-8 complementary DNA (cDNA)–containing vector, were treated with or without TRAIL (white bars) or vehicle (black bars) for 18 hours and assessed for survival using the MTT assay. Bars represent mean ± SD values.

  • Fig. 7.

    CHX and BIM down-regulate FLIP and cIAP2 expression and overcome TRAIL resistance.

    (A) Cell survival of IM-9 cells pretreated with CHX (0.1 μg/mL) or BIM (20 μM) for 6 hours and then treated with 0 ng/mL (black bars), 100 ng/mL (white bars), or 200 ng/mL (gray bars) TRAIL for an additional 18 hours. Cell survival was determined using the MTT colorimetric assay. Bars represent mean ± SD values. (B) Immunoblotting for FLIP, cIAP1, cIAP2, Bcl-2, and Bcl-xL of cell lysates from IM-9 cells treated with CHX (0.1 μg/mL) or BIM (20 μM) for 6 hours. Levels of tubulin are shown for confirmation of equal loading.

  • Fig. 8.

    Inhibition of FLIP expression with antisense oligonucleotides sensitizes TRAIL-resistant MM cells to TRAIL.

    Cell death of highly TRAIL-resistant IM-9 cells transfected with either FLIP antisense (ASO) or control (CO) oligonucleotides, and then treated with or without TRAIL (200 ng/mL) for 18 hours. FLIP and tubulin protein levels are shown for comparison.

  • Fig. 9.

    Expression of TRAIL receptors and intracellular apoptosis regulator proteins in myeloma cells treated with the NF-κB inhibitor SN50.

    Immunoblot analysis for TRAIL receptors (DR4, DR5, DcR1, DcR2), procaspase-8, procaspase-10, procaspase-3, FLIP, cIAP1, cIAP2, XIAP, survivin, Bcl2, and Bcl-xL in TRAIL-sensitive (MM.1S) and moderately resistant (ARH-77) cells treated with and without the NF-κB inhibitor SN50 (30 μg/mL) for 6 hours. Levels of tubulin are shown for confirmation of equal protein loading.

  • Fig. 10.

    Flow cytometric analysis for TRAIL receptor expression on myeloma cells treated with the NF-κB inhibitor SN50.

    Flow cytometric analysis was performed to determine the cell surface expression of TRAIL receptors DR4, DR5, DcR1, and DcR2 (shaded peaks) on MM.1S cells treated with and without the NF-κB inhibitor SN50 (30 μg/mL) for 6 hours. Isotype control antibody staining appears as unshaded peaks.

  • Fig. 11.

    Demonstration of caspase-8 activation in TRAIL-resistant myeloma cells preincubated with CHX, BIM, or SN50 before TRAIL treatment.

    (A) Caspase-8 activity was determined by use of the ApoAlert caspase-8 colorimetric kit in highly TRAIL-resistant IM-9 cells that were incubated with CHX (0.1 μg/mL), BIM (20 μM), or SN50 (30 μg/mL) for 6 hours before treatment with TRAIL (300 ng/mL) for 30 minutes. (B) Immunoblot analysis for caspase-8 cleavage in IM-9 cells preincubated with BIM (20 μM) or SN50 (30 μg/mL) for 6 hours before treatment with TRAIL (300 ng/mL) for 1 hour.