TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3beta pathway and activation of caspases

Abstract

Background: The development of new modulator possessing high efficacy, low toxicity and high selectivity is a pivotal approach to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in cancer treatment. In this study, we suggest a new molecular mechanism that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) down-regulates P-glycoprotein (P-gp) through inhibition of DNA-PKcs/Akt/GSK-3beta pathway and activation of caspases and thereby sensitize MDR cells to MDR-related drugs.

Results: MDR variants, CEM/VLB10-2, CEM/VLB55-8 and CEM/VLB100 cells, with gradually increased levels of P-gp derived from human lymphoblastic leukemia CEM cells, were gradually more susceptible to TRAIL-induced apoptosis and cytotoxicity than parental CEM cells. The P-gp level of MDR variants was positively correlated with the levels of DNA-PKcs, pAkt, pGSK-3beta and c-Myc as well as DR5 and negatively correlated with the level of c-FLIPs. Hypersensitivity of CEM/VLB100 cells to TRAIL was accompanied by the activation of mitochondrial apoptotic pathway as well as the activation of initiator caspases. In addition, TRAIL-induced down-regulation of DNA-PKcs/Akt/GSK-3beta pathway and c-FLIP and up-regulation of cell surface expression of death receptors were associated with the increased susceptibility to TRAIL of MDR cells. Moreover, TRAIL inhibited P-gp efflux function via caspase-3-dependent degradation of P-gp as well as DNA-PKcs and subsequently sensitized MDR cells to MDR-related drugs such as vinblastine and doxorubicin. We also found that suppression of DNA-PKcs by siRNA enhanced the susceptibility of MDR cells to vincristine as well as TRAIL via down-regulation of c-FLIP and P-gp expression and up-regulation of DR5.

Conclusion: This study showed for the first time that the MDR variant of CEM cells was hypersensitive to TRAIL due to up-regulation of DR5 and concomitant down-regulation of c-FLIP, and degradation of P-gp and DNA-PKcs by activation of caspase-3 might be important determinants of TRAIL-induced sensitization of MDR cells to MDR-related drugs. Therefore, combination of TRAIL and chemotherapeutic drugs may be a good strategy for treatment of cancer with multidrug resistance.

Figure 1

High susceptibility of MDR cells to TRAIL and up-regulation of death receptor and down-regulation of c-FLIP in the cells. (A) mRNA levels of mdr1 in CEM cells and its MDR variants, CEM/VLB_10-2, CEM/VLB_55-8 and CEM/VLB₁₀₀ cells were determined by RT-PCR (insert). To determine the growth inhibitory effect of TRAIL, CEM cells and its multidrug-riesistant variants were treated with graded single doses of TRAIL, and the percentage of cell survival was determined after 5 days incubation using the MTT assay. (B) CEM cells and its MDR variants were treated with or without TRAIL (10 ng/ml) for 24 h. Thereafter, the percentage of apoptotic cells in each cell population was determined by Annexin V staining and flow cytometry. Each bar represents the mean ± S.D. of triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.005 versus TRAIL untreated control cells. (C) mRNA levels of DR4/DR5 and c-FLIP_L/S in CEM cells and its MDR variants were determined by RT-PCR analysis. β-Actin (Actin) was used as a loading control.

Figure 2

Sensitization of MDR cells to TRAIL is mediated through caspase-dependent mitochondrial apoptotic pathway. The cell lysates obtained from CEM or CEM/VLB₁₀₀ cells after exposure to graded doses of TRAIL (1~10 ng/ml) for 24 h were subjected to western blot analysis to monitor levels of caspase-8, -10, -9 and -3, Bid, truncated Bid (tBid), Bax and Bcl-2. The levels of PARP and its cleavage fragment (CF) in TRAIL-treated cells were also determined.

Figure 3

Expression of p-gp in MDR cells is associated with the enhanced expression of c-Myc and DR5 and activation of DNA-PKcs/Akt/GSK-3β pathway. The protein levels of P-gp, c-Myc, DNA-PKcs, pAkt, GSK-3β, phosphorylated Akt (pAkt) at Ser 473, total Akt (tAkt), phosphorylated GSK-3β (pSK3β) at Ser9, total GSK-3α/β, DR4 and DR5 were determined by western analysis. Actin was used as a loading control.

Figure 4

TRAIL inhibited P-gp expression and DNA-PKcs/Akt/GSK-3β signaling pathway and up-regulation of death receptors and down-regulation of c-FLIP in MDR cells. (A) Cell lysates obtained from CEM or CEM/VLB₁₀₀ cells treated with indicated dose of TRAIL for 24 h were subjected to western blot analysis to monitor levels of P-glycoprotein (P-gp), DNA-PKcs, pAkt (Ser473), tAkt, pGSK3β (Ser 9), GSK-3α and -3β, Mcl-1. (B) CEM/VLB₁₀₀ cells treated with TRAIL (10 ng/ml) for 24 h were incubated on ice in the presence of DR4- and DR5-specific antibodies (1:500), and subsequently labeled with FITC-conjugated secondary antibody (1:1000). The fluorescence intensity was analyzed with flow cytometry. The thin line indicates that the cells only incubated with Goat IgG2a that was used as a control isotype antibody; the thick line indicates the specific labeling (left). The cells treated with or without TRAIL, and changed mRNA level of c-FLIP_L/S was determined by RT-PCR analysis (right).

Figure 5

TRAIL inhibited P-gp efflux function in MDR cells by P-gp cleavage via caspase-3 activation. (A) The cell lysates obtained from CEM/VLB₁₀₀ cells treated with or without TRAIL (10 ng/ml) for 3 ~ 9 h (left) and the cell lysates of the MDR cells treated with TRAIL (5 ng/ml) for 6 h or pretreated with 50 μM Z-DEVD-FMK, a specific caspase-3 inhibitor, for 3 h and then with TRAIL for 6 h (right) were subjected to western blot analysis to monitor levels of P-gp, DNA-PKcs, caspase-3 and PARP and their its cleavage fragment (CF). (B) flow cytometric assay of P-gp efflux activity in TRAIL-treated MDR cells was based on extrusion of the fluorescent P-gp substrate, rhodamine123 (Rho123). The efflux activity of P-gp is highly temperature sensitive because functions optimally 37°C but is inactive at 4°C. Cell suspension from CEM and CEM/VLB₁₀₀ cells treated with or without 10 ng/ml TRAIL for 6 h was incubated with Rho 123 and further incubated at 37°C for 3 h (TREATED 37°C as TRAIL-treated cells or CTRL37°C as TRAIL-untreated control) to allow P-gp-mediated drug efflux or on ice as control (CTRL 4°C).

Figure 6

TRAIL enhanced the cytotoxicity of MDR-related drug in MDR cells by the down-regulation of DNA-PKcs and P-gp via caspase-3 activation. (A) CEM/VLB₁₀₀ cells were treated with graded single doses of doxorubicin (DOX) or vinblastine (VLB) after pretreatment of low dose of TRAIL (1 ng/ml) for 6 h. The percentage of cell survival after combined treatment of TRAIL with MDR-related drug was determined after 5 days incubation using the MTT assay. Each bar represents the mean ± S.D. of triplicate experiments. ***p < 0.005 versus TRAIL alone treated cells at the same dose point. (B) The cell lysates obtained from CEM/VLB₁₀₀ cells co-treated with TRAIL (1 ng/ml) and indicated dose of DOX were subjected to western blot analysis to monitor levels of P-gp, DNA-PKcs, caspase-3, PARP, and actin as a loading control.

Figure 7

Supprerssion of DNA-PKcs up-regulated surface expression of DR5 and down-regulated the expression of c-FLIPs. (A) CEM/VLB₁₀₀ cells were transfected with a siRNA against DNA-PKcs or scrambled siRNA as a control. After 48 h, the total RNA extracted from transfectant of CEM/VLB₁₀₀ cells performed RT-PCR analysis to monitor the mRNA levels of DNA-PKcs, DR4/5, c-FLIP_L/S , MDR1, and actin as a loading control. (B) The transfectant incubated with an anti-DR4 or -DR5 (1:500), and subsequently labeled with FITC-conjugated secondary antibodies (1:1000) to determine the surface expression of DR4 and DR5. Goat IgG2a was also used as control isotype antibody.

Figure 8

Potentiated suppression of DNA-PKcs expression by TRAIL leads to a severe inhibition of both Akt/GSK-3β phosphorylation and P-gp and c-FLIPs expression and enhances the cytotoxicity of TRAIL and of MDR-related drug in MDR cells. (A) CEM/VLB₁₀₀ cells were transfected with a siRNA against DNA-PKcs or scrambled siRNA as a control. After 48 h, the transfectant was treated with or without TRAIL (1- and 10 ng/ml for 24 h), and the changed level of DNA-PKcs, pAkt (Ser473), tAkt, pGSK3β (Ser9), GSK-3α/β, P-gp, PARP, c-FLIP_L/S and actin was performed by western blot analysis. (B) Each transfectant was treated with indicated doses of TRAIL or vincristine (VCR). After 5 days, the percentage of growth inhibition was determined incubation using the MTT assay. Data represent means ± S.D. of triplicate experiments. **p < 0.001, ***p < 0.005 versus cells transfected with scrambled siRNA at the same dose point.