The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis

Abstract

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.

Figure 1

P-gp confers resistance to DOX-mediated caspase-dependent DNA fragmentation. (a) CEM cell lines (P-gp^low CCRF, 2H6 and 4G9, P-gp^high A7⁺, 2G10, and IC10) were labeled with ¹²⁵IUdR for 1 h, washed in growth media, and incubated for 48 h in 96-well plates (2 × 10⁴ cells/well) with DOX. (b) In some wells CCRF or A7⁺ cells also were preincubated with anti-P-gp mAb (MRK-16) or isotype control W6/32 mAb (50 μg/ml, final concentration) for 30 min, and DOX was added. Neither mAb caused more than 5% ¹²⁵IUdR release above background in the absence of DOX. (c) In some wells CCRF or A7⁺ cells were preincubated for 30 min with 20 μM (final concentration) ZVAD-fmk or control ZFA-fmk inhibitor, and DOX was added. These data are calculated as the mean ± SE of triplicate samples and are representative of at least two different experiments. DOX concentrations (μg/ml) are shown on the x-axes of each part.

Figure 2

DOX- and Fas-mediated DNA fragmentation and membrane lysis is caspase dependent and reduced by P-gp function. CEM cell lines, CCRF (P-gp^low) or A7⁺ (P-gp^high) were labeled with ⁵¹Cr (lysis; a and c) and ¹²⁵IUdR (DNA fragmentation; b, d, and e) for 1 h, washed in growth media, and incubated for 16–48 h in 96-well plates (2 × 10⁴ cells/well) with cell death stimuli at final concentrations as follows: (a and b) DOX (0.1 μg/ml, 48 h); (c and d) anti-human Fas IgM mAb, CH-11 (0.01 μg/ml, 16 h). (a–d) Some A7⁺ cells were preincubated for 30 min with anti-P-gp mAb (MRK-16) (50 μg/ml, final concentration) and/or followed by 20 μM (final concentration) ZVAD-fmk or control ZFA-fmk inhibitor for 30 min. Cell death stimuli then were added as indicated. (e) A7⁺ cells were preincubated with increasing concentrations of MRK-16 mAb (1–100 μg/ml, M1 to M100), UIC2 mAb (0.1–5 μg/ml, U0.1 to U5) or verapamil (0.5–10 μM, V0.5 to V10), then treated with 1 or 10 ng/ml anti-Fas mAb for 16 h. These data are calculated as the mean ± SE of duplicate samples and are representative of at least two different experiments.

Figure 3

Caspase-independent forms of membrane lysis unaffected by P-gp overexpression. CEM cell lines, CCRF (P-gp^low) or A7⁺ (P-gp^high) were labeled with ⁵¹Cr (lysis; a, c, and e) and ¹²⁵IUdR (DNA fragmentation; b, d, and f) for 1 h, washed in growth media, and incubated for 4 h in 96-well plates (2 × 10⁴ cells/well) with cell death stimuli at final concentrations as follows: (a and b) pfp (300 units); (c and d) pfp (30 units) and gB (0.5 μg/ml); and (e and f) NK cells at effector/target ratios as shown. In some wells CCRF or A7⁺ cells were preincubated for 30 min with anti-P-gp mAb (MRK-16) (50 μg/ml, final concentration) and/or followed by 20 μM (final concentration) ZVAD-fmk or control ZFA-fmk inhibitor for 30 min. Cell death stimuli then were added as indicated for 4 h (NT = not tested). gB alone at these concentrations was without effect. These data are calculated as the mean ± SE of duplicate samples and are representative of at least two different experiments.

Figure 4

P-gp affects caspase-3-dependent DNA fragmentation in drug-resistant cell lines. CEM cell lines [CCRF (P-gp^low) and A7⁺ (P-gp^high)] were labeled with ¹²⁵IUdR for 1 h, washed in growth media, and incubated for 16–48 h in 96-well plates (2 × 10⁴ cells/well) with cell death stimuli at final concentrations as follows: (a) DOX (0.1 μg/ml, 48 h); (b) anti-human Fas IgM mAb CH-11 (0.01 μg/ml, 16 h). In some wells cells were preincubated for 30 min with anti-P-gp mAb (MRK-16) (50 μg/ml, final concentration) and/or followed by 20 μM (final concentration) ZDEVD-fmk or ZYVAD-fmk inhibitor for 30 min. Cell death stimuli then were added as indicated for 16–48 h. These data are calculated as the mean ± SE of duplicate samples and are representative of at least two different experiments. Protein lysates from CCRF (c) or A7⁺ (d) cells treated with anti-Fas IgM (0.01 μg/ml, 4 h) in the presence or absence of MRK-16 (50 μg/ml, final concentration) or verapamil (5 μM) were separated by SDS/PAGE, and Western blots were performed by using an anti-caspase-3 mAb. The addition of anti-Fas IgM, MRK-16, and verapamil is indicated by the table above the blots, and the position of molecular weight markers in kDa is indicated on the left. The expression of pro-caspase-3 and an active cleaved product is indicated by arrows.

Figure 5

P-gp affects caspase-dependent DNA fragmentation in drug-resistant K562 cell lines. K562 cell lines [K562 (P-gp^low) or KVIN (P-gp^high)] were labeled with ⁵¹Cr (a and c) and/or ¹²⁵IUdR (b and d) for 1 h, washed in growth media, and incubated for 4–48 h in 96-well plates (2 × 10⁴ cells/well) with cell death stimuli at final concentrations as follows: (a and b) VIN (0.1 μg/ml, 48 h) and (c and d) pfp (60 units) and gB (0.5 μg/ml, 4 h). In some wells cells were preincubated for 30 min with anti-P-gp mAb (MRK-16) (50 μg/ml, final concentration) and/or followed by 20 μM (final concentration) ZVAD-fmk or control ZFA-fmk inhibitor for 30 min. Cell death stimuli then were added as indicated for 4–48 h. These data are calculated as the mean ± SE of duplicate samples and are representative of at least two different experiments.