Background
Acquired resistance to chemotherapeutic agents remains a major obstacle for the effective treatment of many advanced and metastatic cancers. Several mechanisms are thought to be involved in the development of multidrug resistance (MDR), defined by simultaneous cross-resistance to a variety of anticancer drugs that differ in their chemical structures, modes of action, and molecular targets [
1‐
3]. Emergence of MDR is often associated with over-expression of the
MDR1 gene product, P-glycoprotein (P-gp) [
4]. In certain cancers, such as chronic or acute myeloid leukemia and breast cancer, over-expression of
MDR1 gene is a prognostic indicator for clinical outcome and correlates with a poor response to chemotherapy [
5‐
8]. Therefore, inhibition of P-gp function or expression can reverse P-gp-mediated MDR and improve the efficacy of chemotherapy [
9].
Previously, we have reported that an increased expression of DNA-dependent protein kinase (DNA-PK) participates in the development of MDR, and inhibition of DNA-PK leads to increase of drug sensitivity in MDR cells [
10]. DNA-PK comprises a catalytic subunit (DNA-PK
cs) with a DNA- binding Ku70 and Ku80 heterodimer acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets [
11]. Recently, it has been demonstrated that DNA-PKcs-catalyzed RNA Helicase A phosphorylation enhanced the transcription of the
MDR1 gene through the CAAT-like element of the
MDR1 gene promoter and thus DNA-PKcs played an important role in regulation of P-gp expression by
MDR1 promoter activation [
12].
The phosphoinositide 3-kinase (PI3K)/Akt pathway is also frequently implicated in tumorigenesis and chemotherapeutic resistance [
13]. Recent studies have shown that there is a significant correlation between the phosphorylated, activated Akt and P-gp expression, and inhibition of the PI3K/Akt signaling pathway can reverse P-gp-mediated MDR [
14‐
16]. Akt phosphorylation on Ser473 (S473) is required for activation of Akt, and a major Akt S473 kinase activity was found to be DNA-PK, a member of the PI3K-related kinase subfamily of protein kinases [
17]. DNA-PKcs has been shown to colocalize with Akt and enhance Akt phosphorylation [
18]. One of the downstream targets of pro-survival Akt is GSK-3β, which is inactivated by phosphorylation on Ser9 by Akt. The inactivation of GSK-3β through Akt-mediated phosphorylation leads to down-regulate its pro-apoptotic activity and inhibit the induction of cell death. Death receptor-induced extrinsic apoptotic signaling is also modulated by GSK-3β activity [
19].
Recently, it has been shown that the extrinsic death receptor pathway represents a suitable target for cancer treatment [
20]. Since TNF-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in various tumor cells, but only rarely in non-transformed cells, TRAIL is currently assessed in clinical trials [
21]. The extrinsic apoptotic signaling cascade is a vital process initiated by activation of death receptors, DR4/DR5. Stimulation of these death receptors causes receptor trimerization, followed by recruitment of FADD (Fas associated with death domain protein) and caspase-8 (or caspase-10) to form the death-inducing signaling complex (DISC). DISC formation promotes autoactivation of caspase-8/10 and the subsequent activation of effector caspases, primarily caspase-3, -6 and -7, which implement the cell death program. Cellular FLICE inhibitory protein (c-FLIP) is expressed as long form (c-FLIP
L) and short form (c-FLIP
S) and inhibits caspase-8 binding to FADD and prevents DISC formation and apoptosis and splice forms [
22]. Elevated Akt activity up-regulates c-FLIP and inhibits TRAIL-induced apoptosis in cancer cells [
23].
It has been reported that MDR cells over-expressing P-gp are more susceptible to TRAIL than their drug-sensitive counterparts through various mechanisms such as a reduced expression of endogenous Akt [
24] or enhancement of TRAIL binding to DR5 by P-gp [
25]. However, the mechanism underlying the increased susceptibility of MDR cells to TRAIL mediated cell death was not understood well. Here, we demonstrated that TRAIL sensitized MDR cells to MDR-related drugs by inhibition of DNA-PKcs/Akt/GSK-3β pathway, activation of caspases and subsequent down-regulation of P-gp.
Discussion
Although targeted drugs are being developed or used in some leukemia, chemotherapeutic drugs are still useful for the treatment of leukemia. However, acquired resistance against MDR-related drugs is a serious problem in the management of leukemic patients. Altered expression of various kinds of protein and enzymes could be seen in MDR-type cancer cells [
2,
9]. In the present study, we suggest a new molecular mechanism that TRAIL down-regulates P-gp through inhibition of DNA-PKcs/Akt/GSK-3β pathway and activation of caspases and thereby sensitize MDR cells to MDR-related drugs.
TRAIL is emerging as most promising agent for cancer therapy, because it induces apoptosis in a variety of cancers and transformed cells without any toxicity to normal cells [
20]. But, it has been reported that a majority of human leukemic cells such as CEM, K562 and Molt-4 cells are relatively resistant to TRAIL-induced apoptosis [
36,
37]. In our study, interestingly, MDR variants derived from human lymphoblastic leukemia CEM cells showed a hypersensitive response to TRAIL compared with parental CEM cells. MDR variants, CEM/VLB
10-2, CEM/VLB
55-8 and CEM/VLB
100 cells with gradually increased levels of P-gp were gradually more susceptible to TRAIL-induced apoptosis and cytotoxicity than CEM cells. This result was supported by the findings that the expression of DR5 was gradually up-regulated in the CEM/VLB
10-2, CEM/VLB
55-8 and CEM/VLB
100 cells, and conversely, the expression of c-FLIPs was gradually down-regulated in the MDR variants as compared with those of CEM cells. Therefore, modulation of TRAIL receptor pathway including up-regulation of DR5 and down-regulation of c-FLIPs might contribute to TRAIL sensitization of MDR cells. It has been reported that TRAIL responsiveness correlates with a reduced expression of endogenous Akt in MDR-U2OS human osteosarcoma cell line [
24], and P-gp enhances TRAIL-triggered apoptosis by interacting with the death receptor DR5 in the
MDR1-transfected MCF-7 breast cancer cell line [
25]. Therefore, it could be suggested that a marked sensitivity to TRAIL of MDR cells might be mediated by complex mechanisms, not a single mechanism. In the present study, hypersensitivity to TRAIL of CEM/VLB
100 cells, MDR variant of CEM cells, was accompanied by the activation of the mitochondrial apoptotic pathway by the cleavage of bid as well as the activation of caspase-8 and -10, which are apoptotic characteristics of the type II cells and caspase-3 and -9 [
38]. We also observed an increase in cell surface expression of DR4/DR5 and down-regulation of c-FLIP by TRAIL in MDR-variant of CEM cells. These results suggest that there might be a positive feedback regulation in TRAIL receptor signaling leading to intensification of sensitivity to TRAIL in MDR-variant of CEM cells.
Oncogene c-Myc is known to act as an important regulator for TRAIL sensitivity in cancer cells. It has been shown that c-Myc induces and represses the transcription of DR5 [
39] and c-FLIP [
40], respectively, therefore enhancing the sensitivity of cancer cells to TRAIL-induced apoptosis. Recently, it has been reported that abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway [
28]. Previously, we have demonstrated that the increased expression of DNA-PKcs is associated with the development of drug resistance in MDR variants of CEM cells [
10]. In addition, the c-Myc is known to be involved in regulating expression of P-gp, the product of
MDR1 gene [
29,
30]. It has been reported that elevated P-gp expression in MDR cells is accompanied by increased level of pAkt [
41]. Once phosphorylated, activated Akt inactivate GSK-3β through phosphorylation at Ser9, resulting in stabilization and activation of β-catenin that enhanced P-gp expression [
42]. In the present study, the gradually increased level of P-gp, was well correlated with the gradually increased levels of c-Myc, DNA-PKcs, pAkt and pGSK-3β in MDR variants, CEM/VLB
10-2, CEM/VLB
55-8 and CEM/VLB
100 cells, suggesting that the molecular changes are not dependent on the each subline type, but implicate the causal relationships between the molecules, which have been changed during the process of MDR acquisition. And the increased level of DR5 and decreased level of c-FLIPs in the MDR-variants of CEM cells also might be associated with the up-regulated c-Myc since it has been reported that c-Myc up-regulated the DR5 receptor and down-regulated c-FLIP [
39,
40]. We also found that the expression of up-regulated molecules in CEM/VLB
100 cells including P-gp, DNA-PKcs, pAkt and pGSK-3β were suppressed after treatment with TRAIL. Akt and GSK-3β are signaling molecules downstream to DNA-PKcs. We showed that the phosphorylated form of Akt and GSK-3β would be decreased in TRAIL-treated CEM/VLB
100 cells since DNA-PKcs was down-regulated by TRAIL treatment. Therefore, our data indicated that TRAIL caused the down-regulation of P-gp in MDR cells by the inactivation of DNA-PKcs/Akt/GSK-3b pathway. Since these molecules are related with drug-resistance, down-regulation of P-gp, DNA-PKcs, pAkt, and pGSK-3β after treatment with TRAIL might lead to the hypersensitivity to MDR-related drugs of MDR-variant of CEM cells. Indeed, inhibition of Akt enhances susceptibility to TRAIL by up-regulation of death receptors [
43] and down-regulation of c-FLIP [
44] and down-regulates P-gp expression in multidrug-resistant human T-acute leukemia [
14].
Our study also showed that anti-apoptotic Bcl-2 and Mcl-1 proteins were over-expressed in CEM/VBL
100 cells and the levels of these proteins and Bax were significantly decreased and increased by Bcl-2 and Mcl-1, the antiapoptotic Bcl-2 family proteins, were over-expressed in CEM/VBL
100 cells in comparison with CEM cells, and the levels of these anti-apoptotic proteins and Bax were significantly decreased and increased by treatment of TRAIL in CEM/VBL
100 cells, respectively, suggesting that TRAIL-induced apoptosis of MDR cells was mediated through mitochondria-dependent pathway as well as caspase activation. Bcl-2 and Mcl-1 are often highly expressed in chemotherapy-resistant cancers and prevents apoptosis by inactivating pro-apoptotic Bax and Bak [
45,
46]. The increased expression of Bcl-2 or Bcl-xL was the common feature of P-gp-related drug-resistant human leukemic ce1l lines [
47]. Over-expression of Mcl-1 decreased sensitivity of leukemia cells to cytotoxic chemotherapeutic agents [
45] and specific down-regulation of Mcl-1 via RNA interference sensitized multidrug-resistant leukemia cells towards chemotherapy and induced apoptosis [
48]. Therefore, the reduction of Bcl-2 and Mcl-1 after exposure to TRAIL may be in part a cause of TRAIL-induced sensitization of CEM/VLB
100 cells to MDR-related drugs.
Moreover, our data showed the cleavage of P-gp and DNA-PKcs by treatment with TRAIL. Recently, it has been shown that the cleavage of P-gp (170 kDa) is dependent on caspase-3 during apoptotic cell death induced by LY294002, H
2O
2, and Z-LEHD-FMK in MDR variant of CEM cells [
33]. DNA-PKcs is also a substrate of caspase-3 [
35]. Here, we demonstrated that the degradation of P-gp and DNA-PKcs during treatment of CEM/VLB
100 cells with TRAIL was a caspase-3 dependent manner. This result was followed by the significant reduction of rhodamine123 efflux and the increased sensitivity to MDR-related drugs such as VLB and DOX after exposure to TRAIL in CEM/VLB
100 cells, suggesting that the degradation of P-gp as well as the down-regulation of Bcl-2 and Mcl-1 could be involved in sensitization of MDR cells to MDR-related drug after treatment with TRAIL. Since the expression of DNA-PKcs regulates
MDR1 gene [
12] and cellular c-Myc protein levels [
28], which can affect TRAIL sensitivity in cancer cells as described above, we showed that knockdown of DNA-PKcs with specific siRNA led to the increased expression of DR5 and the decreased expression of c-FLIP and caused the reduction of pAkt, pGSK-3β and P-gp levels in CEM/VBL
100 cells. These results resembled the effects of TRAIL on the CEM/VBL
100 cells. Therefore, DNA-PKcs may play an important role on TRAIL sensitivity in MDR-variant of CEM cells.
Methods
Cell culture and Reagents
Human lymphoblastic leukemia CCRF-CEM (CEM) line and its the multidrug-resistant sublines, CEM/VLB
10-2, CEM/VLB
55-8, and CEM/VLB
100 [
10], were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, GIBCO BRL, Life Technologies, Inc.). The recombinant human soluble TRAIL was purchased from R&D System (Minneapolis, MN). Vinblastine, vincristine, doxorubicin and Rho123 were obtained from Sigma-Aldrich (St. Louis, MO).
Cell Proliferation Assay
Cell proliferation was measured either by counting viable cells by using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Company, St. Louis, MO) colorimetric dye-reduction method. Exponentially growing cells (5 × 103 cells/well) were plated in 96 well and incubated in growth medium treated with indicated condition of TRAIL and/or drug at 37°C. After 5 days, the medium was aspirated using centrifugation and MTT-formazan crystals solubilized in 100 μl DMSO. The optical density of each sample at 570 nm was measured using ELISA reader. The optical density of the media was proportional to the number of viable cells. Inhibition of proliferation was evaluated as a percentage of control growth (no drug in the sample). All experiments were repeated at least two experiments in triplicate.
Flow cytometric analysis of TRAIL receptors
CEM/VLB100 cells (2 × 106 cells) from the culture media were spun down at 500 × g, washed with phosphate-buffered saline (PBS) and resuspended in 500 μl PBS. The cells were then incubated with 5 μl of goat IgG2a, anti-DR4 or anti-DR5 polyclonal goat antibody (1:100, R&D, Minneapolis, MN) for 1 h. After washing with PBS, FITC-conjugated rabbit anti-goat polyclonal antibody (1:200, Sigma-Aldrich Co., St. Louis, MO) was added to the cell suspension and incubated for 1 h on ice. After rinsing with PBS, the samples were analyzed with a FACSort flow cytometer (Becton Dickinson, San Jose, CA). The data were analyzed using the CellQuest program.
RT-PCR analysis
Total cellular RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and the levels of RNA transcripts were assessed with The Titan One Tube RT-PCR System (MJ research Inc, NV, USA). One mg of total cellular RNA was reverse transcribed using Maloney murine leukemia virus reverse transcriptase (Invitrogen, Paisley, UK) with each dNTP and 1 μg oligo dT. Amplification of 1 μl of these cDNA by PCR was performed using the following gene-specific primers: DR4 (forward), 5'-CTGAGCAACGCAGACTCGCTGTCCAC-3' and (reverse), 5'-AAGGACACGGCAGAGCCT GTGCCAT-3'; DR5 (forward), 5'-CTGAAAGGCATCTGCTCAGGTG-3' and (reverse), 5'-CA GAGTCTGCATTACCTTCTA G-3'; FLIPL (forward), 5'-TTCCAGGCTTT CGGTTTCTT-3' and (reverse), 5'-GTCCGAAACAAGGTGAGGGT-3'; FLIPS (forward), 5'-ACCCTCACCTTG TTTCGGAC-3' and (reverse), 5'-CTTTTGGATTGCTGCTTGGA-3'; β-actin (forward), 5'-CAGAGCAAGAGAGGCATCCT-3' and (reverse), 5'-TTGAAGGTCTC AAACATGAT-3.'
The resulting total cDNA was used in PCR performed in total volume of 20 μl using Taq polymerase (Solgent Co., Korea) at 94°C for denaturation for 60 sec, 60°C for annealing for 60 sec, and 72°C for amplification for 90 sec for 30 cycles, followed by a final extension at 72°C for 12 min. The amplified fragments were separated on 1.5% agarose gel and visualized with ethidium bromide staining.
Western blot analysis
Protein samples were separated by SDS-PAGE and blotted to nitrocellulose membrane (Hybond-ECL, GE Healthcare). The membrane was incubated with antibody as specified, followed by secondary antibody conjugated with horseradish peroxidase. Specific antigen-antibody complexes were detected by enhanced chemiluminescence (PerkinElmer, Life science). Western blot analysis was performed with the following antibodies: anti-Bax, anti-caspase-3, anti-PARP, anti-Bcl-2 (Santa Cruz Biotechnology, CA), anti-Akt, anti-phospho-Akt (Ser 473), anti-caspase-8, anti-caspase-9 (Cell signal, Danvers, MA), anti-DNA-PKcs (Thermo Fisher Scientific, CA), anti-DR5, anti-caspase-10 (Calbiochem, Germany), anti-pGSK-3β (Ser8), anti-GSK-3β, anti-GSK-3α, anti-c-Myc (Epitomics, CA), anti-DR4 (R&D Systems, MN) and anti β-actin (Sigma-Aldrich) antibodies, Secondary antibodies were obtained from GE Healthcare.
Preparation of siRNA Transfection
The siRNA used for targeted silencing of DNA-PKcs were (5'-CAGUCUUAGUCCGGAUCAUdTdT-3'). CEM/VLB100 cells were transfected with 0.1 uM siRNA for 48 h by oligofectamine according to the manufacture's protocol (InVitrogen, Carlsbad, CA). In brief, CEM/VLB100 (2 × 105 cells/well) were seeded of 6-well plates and added to the siRNA/oligofectamine complex. Cells were incubated for 4 h at 37°C in serum free RPMI medium and then FBS was added. After 48 h, the cells were treated with TRAIL for another 24 h and collected for western blot analysis to determine the levels of DNA-PKcs and other indicated proteins.
Apoptosis assay
Cells (2 × 105 cells/ml) were treated with or without TRAIL and/or indicated drug for 24 h and the cells were centrifuged and resuspended in 500 μl of the staining solution containing Annexin V fluorescein (FITC Apoptosis detection kit; BD ParMingen San Diego, CA) and propidium iodide in PBS. After incubation at room temperature for 15 min, cells were analyzed by flow cytometry. Annexin V binds to those cells that express phosphatidyl serine on the outer layer of the cell membrane, and propidium iodide stains the cellular DNA of those cells that have a compromised cell membrane. This allows for the discrimination of live cells (unstained with either fluorochrome) from apoptotic cells (stained only with Annexin V) and necrotic cells (stained with both Annexin V and propidium iodide).
Flow-cytometric dye-efflux assay for multidrug resistance
The accumulation of rhodamine 123, a fluorescent substrate of P-gp, in CEM and CEM/VLB100 cells treated with or without TRAIL was measured using a FACS flowcytometer (FACScalibur, BD Biosciences, San Jose, CA) equipped with an ultraviolet argon laser (excitation at 488 nm and emission at 530 ± 15 nm). Cell suspension (500 μl) from CEM and CEM/VLB100 cells treated with or without 10 ng/ml TRAIL for 6 h was incubated with rhodamine 123 (0.5 μg/ml) at 37°C for 30 min. After incubation, the cells were washed with ice-cold PBS and further incubated at 37°C for 3 h to allow P-gp-mediated drug efflux or on ice (4°C) as control. Cells were pelleted by centrifugation at 500 × g and resuspended in PBS containing. Cellular fluorescence was analyzed immediately by using Flow cytometer.
Statistical analysis
The results obtained were expressed as the mean ± S.E. of at least three independent experiments. The statistical significance of differences assessed using the Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.005 was considered statistically significant in all experiments.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SBS, JGH, MJK, JWL, HBK and JHB designed and conducted experiments as well data analysis. DWK participated in discussion of the data and draft of the manuscript. SHK and CD K equally participated in experimental design, coordination, data analysis and draft of the manuscript. All authors read and approved the final manuscript.