Selective TRAIL-triggered apoptosis due to overexpression of TRAIL death receptor 5 (DR5) in P-glycoprotein-bearing multidrug resistant CEM/VBL1000 human leukemia cells

Abstract

The death-inducing cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), holds enormous promise as a cancer therapeutic due to its highly selective apoptosis-inducing action on neoplastic versus normal cells. Our results revealed that TRAIL selectively triggered apoptosis in the P-glycoprotein (P-gp, ABCB1) and DR5 overexpressing CEM/VBL1000 multidrug resistant leukemia cell line, but not in the parental CEM cells. Moreover, TRAIL treatment reduced P-gp expression in these cells. Mechanistic analysis of TRAIL-induced apoptosis revealed that TRAIL hypersensitivity is due to robust upregulation of the TRAIL receptor DR5 at the protein and mRNA levels during development of MDR in the CEM/VBL1000 variant. DR5 upregulation was independent of the level of expression of endoplasmic reticulum stress regulator C/EBP homologous transcription factor (CH0P/GADD153). TRAIL-triggered apoptosis was associated with increased expression of FADD; activation of caspases-3, -8, -9, and -10; and cytochrome c release from mitochondria. Therefore, both the extrinsic and intrinsic apoptosis pathways are involved in this process. These findings for the first time reveal that TRAIL treatment selectively causes apoptosis in P-gp-overexpressing CEM/VBL1000 cells through strong upregulation of DR5. Moreover, this hypersensitivity to TRAIL and its effect on reducing P-gp expression in these cells hold significant clinical implications for using TRAIL to eradicate MDR malignant cells.

Keywords: P-glycoprotein; TRAIL; TRAIL death receptor 5 (DR5); apoptosis; caspases; death receptors.

Figure 1.

Detection P-gp in CEM/WT and CEM/VBL1000 cells. (A) Western blot analysis. P-gp was detected using 1 μg/ml anti-P-gp monoclonal C219 (1:1,000), and the blot was developed as described in the Materials and Methods. (B) Confocal microscopic analysis. Cells were incubated with P-gp monoclonal antibody UIC2 (1:500), and subsequently labeled with Texas Red-conjugated secondary antibodies (1:1,000).

Figure 2.

Analysis of TRAIL sensitivity in CEM/WT and CEM/VBL1000 cells. (A-B) Analysis of chemotherapeutic agents and TRAIL sensitivity. Cells were exposed to the indicated concentrations of vinblastine (5 ng/ml), vincristine (5 ng/ml), paclitaxel (0.1 μM), and TRAIL (5 ng/ml, or 0.1-5 ng/ml) for 24 h. After these treatments, the percentage of apoptotic cells was determined by annexin V staining followed by flow cytometry. (C) Effect of TRAIL on the viability of CEM/WT and CEM/VBL1000 cells. Cells were treated with 0.1-5 ng/ml TRAIL for 24 h, and viability was assessed by MTT assay. Viability results are given as a percentage of control cells. The results represent the mean ± SD of three independent experiments. (D) Western blot analysis of P-gp showing that TRAIL downregulates P-gp expression.

Figure 3.

Flow cytometric analysis of TRAIL binding in TRAIL-treated CEM/WT and CEM/VBL1000 cells. Cells were treated with TRAIL for 24 h. Cells were then incubated with anti-TRAIL antibody (1:100 v/v) for 1 h and subsequently labeled with 5 μg/ml FITC conjugated secondary antibody for 1 h. Dotted line: CEM/WT. Solid line: CEM/VBL1000.

Figure 4.

Evaluation of surface expression of TRAIL receptors and the effect of neutalizing anti-TRAIL receptors antibodies on TRAIL-induced apoptosis. (A) Confocal microscopic analysis and (B) flow cytometry. Cells were incubated with anti-TRAIL-R1 (DR4) or anti-TRAIL-R2 (DR5) antibody (1:500), and subsequently labeled with FITC-conjugated secondary antibodies (1:1,000) (C) Effect of neutralizing antibodies to DR4 and DR5 on TRAIL-induced apoptosis. Cells were pretreated with the anti-DR4 or anti-DR5 antibodies 3 h before treatment with 1 and 5 ng/ml TRAIL for 24 h. Mouse IgG_2a was used as the control isotype antibody. Apoptosis was detected by annexin V binding assay, as described in the Materials and Methods.

Figure 5.

Expression of DR5 at mRNA and protein levels. (A) The mRNA levels of DR5 and (β-actin in the CEM/WT cell line and its MDR variant CEM/VLB10000 were determined by RT-PCR as described in the Materials and Methods. (B) Western blot analysis of DR5 using 1 μg/ml anti-DR5 polyclonal antibody (1:1,000) or mouse anti-CADD153 monoclonal antibody (1:1,000) as described in the Materials and Methods.

Figure 6.

Effect of TRAIL on activation of caspases, cytosolic accumulation of cytochrome c, and cleavage of PARP. Cells were exposed to 1 and 5 ng/ml TRAIL for 24 h. Subsequently, cells were lysed and proteins used for Western blot analysis of the indicated proteins using specific antibodies to caspases-8, -9, -3, and PARP. To detect cytochrome c released from the mitochondria, the cytosolic fractions were prepared and used as described in the Materials and Methods.