High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde
Tumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL.
PC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer.
This study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and combination of TRAIL therapy with agent that inhibits the DNA-PKcs/Akt signaling pathway might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.
KeywordsCell Surface Expression Metastatic Cell Metastatic Cancer Cell KM12 Cell Trail Treatment
tumor necrosis factor-related apoptosis-inducing ligand
DNA-dependent protein kinase
small interfering RNA
3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Despite the improvement of therapeutic strategies against cancer, the acquisition of invasive/metastatic capabilities and the development of resistance to therapy in cancer cells are still critical problems for successful cancer therapy because recurrent or metastatic cancers that appear after the initial radiotherapy or chemotherapy are generally refractory to secondary therapies . Some metastatic cancers are more resistant to chemotherapeutic drugs than their poorly metastatic counterparts as a result of their acquisition of anti-apoptotic mechanisms [2, 3, 4, 5]. Therefore, it is necessary to elucidate the therapy-resistance mechanisms of metastatic cells for development of effective therapeutic modalities against metastatic cancers, since the molecular basis for the association of an aggressive metastatic phenotype with resistance to apoptosis is still unclear.
The death-inducing cytokine tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) holds enormous promise as an anti-cancer agent due to its highly selective apoptosis-inducing action on neoplastic versus normal cells [6, 7]. The apoptosis signaling cascade is initiated through the engagement of the cell surface death receptors DR4 and/or DR5 by their ligand TRAIL. The binding of TRAIL to the death receptors leads to their trimerization and the recruitment of Fas-associated protein with death domain (FADD). Subsequently, FADD recruits the initiator procaspase-8 or -10, leading to the assembly of the death-inducing signaling complex (DISC), where the initiator caspases are autoactivated by proteolysis. Activated caspase-8 or -10 then cleaves the effector caspase-3, resulting in the cleavage of the death substrates .
c-Myc is deregulated and over-expressed in many cancer cells. The deregulation of c-Myc confers a selective advantage on cancer cells by promoting proliferation, cell survival, and genetic instability, which can contribute to metastasis . By contrast, the activation of c-Myc dramatically sensitizes cells to the apoptotic action of TRAIL by up-regulating the cell surface level of DR5 and activating DISC, thereby playing an important role in determining cellular sensitivity to TRAIL . The decision of a cell to undergo apoptosis or to promote cell survival by c-Myc depends on the specific cell type and the physiological status of the cell . c-Myc-dependent priming of the mitochondrial pathway is critical for the capacity of TRAIL-induced caspase-8 signals to activate effector caspases and for the establishment of a lethal caspase feedback amplification loop in human cells [12, 13].
Recently, we demonstrated that metastatic cancer cells have an increased level and activity of DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), compared with their primary cells , and An et al. reported that DNA-PKcs regulates the stability of c-Myc through the Akt/GSK3β/c-Myc ubiquitination signal pathway . In addition, it has been shown that the level of phosphorylated Ser-473 Akt was much higher in metastatic cancer cells than in non-metastatic cancer cells, and siRNA against Akt blocked cell migration, indicating that Akt activation is necessary for the metastasis of these cultured cells .
Therefore, we studied sensitivity of metastatic cancer cells to TRAIL and demonstrated the differential TRAIL responsiveness of two pairs of primary (PC3 and KM12) and metastatic (PC3-MM2 and KM12L4A) cells. PC3-MM2 and KM12L4A cells were more susceptible to TRAIL than their poorly metastatic primary counterparts. This susceptibility was correlated with the up-regulation of cell surface DR5 and the prominent activation of caspases (caspase-8, -9 and -3) by TRAIL treatment, leading to the inactivation of the DNA-PKcs/Akt pathway through the cleavage of DNA-PKcs and the up-regulation of proapoptotic Bax.
High susceptibility of metastatic cancer cells to TRAIL is mediated via DR5 and down-regulation of c-FLIP and Mcl-1
Increased expression of c-Myc in metastatic cancer cells is associated with the increased susceptibility to TRAIL
Activation of pro-caspases is accelerated after treatment with TRAIL in metastatic cancer cells over-expressing c-Myc
The amplified TRAIL-induced caspase activation by overexpressed c-Myc counteract the anti-apoptotic activity of DNA-PKcs by increasing proteolytic cleavage in the metastatic cancer cells
Since c-Myc could control the TRAIL sensitivity, and DNA-PKcs is a substrate of caspase-3 , we determined whether depletion of c-Myc could block degradation of DNA-PKcs. When PC3-MM2 cells were transfected with c-Myc siRNA, TRAIL-induced cleavage of DNA-PKcs was reduced compared with transfection with scrambled siRNA. These results were followed by prevention of cleavage of DNA-PKcs as well as PARP in the TRAIL-treated cells by pretreatment with Z-DEVD-FMK, a caspase-3-specific inhibitor (Figure 7B). These results suggest that the amplified TRAIL-induced caspase activation by over-expressed c-Myc may curtail the anti-apoptotic activity of DNA-PKcs by increasing its proteolytic cleavage in the metastatic cancer cells.
Suppression of DNA-PKcs is associated with hypersensitivity to TRAIL-induced cytotoxicity
Combination of DMNB and TRAIL renders PC3 and KM12 cells highly susceptible to TRAIL-induced apoptosis
It is unclear why some cells are sensitive to TRAIL-induced apoptotic stimuli, whereas other cell types survive after exposure to TRAIL. Therefore, it is necessary to characterize the molecular mechanisms underlying this apoptotic sensitivity. Furthermore, the molecular determinants regulating TRAIL sensitivity in metastatic cancer cells are still poorly understood. In this study, we demonstrated the differential TRAIL responsiveness of primary human prostate adenocarcinoma PC3 and colorectal carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines. Interestingly, PC3-MM2 and KM12L4A cells were more susceptible to TRAIL than their primary counterparts. We found that DR5 could be the major receptor involved in TRAIL-mediated apoptosis of these highly metastatic cells. Previously, we and other group reported that the expression of DR5 in MDR cells was up-regulated following TRAIL treatment [28, 29]. In case of our results, cell surface expression of DR5, but not DR4, in PC3-MM2 and KM12L4A cells was up-regulated after TRAIL treatment whereas DR4/DR5 expression in primary PC3 and KM12 cells was not modulated by TRAIL treatment (data not shown). Moreover, pre-treating PC3-MM2 and KM12L4A cells with a neutralizing antibody against the DR5 receptor strongly reduced the degree of apoptosis induced by TRAIL. However, DR4 neutralizing antibody did not significantly affect TRAIL-triggered apoptosis in these highly metastatic cells, indicating that TRAIL induced apoptosis occurs preferentially via DR5, and therefore, DR5 could be the major receptor involved in increased susceptibility of metastatic cells to TRAIL. We also found that the levels of c-FLIPL/S and Mcl-1, the major factors for resistance to TRAIL-induced apoptosis, were significantly decreased in PC3-MM2 and KM12L4A cells compared to their primary counterparts. Therefore, our data suggest that high susceptibility to TRAIL of metastatic cancer cells is associated with up-regulation of DR5 and concurrent down-regulation of c-FLIPL/S and Mcl-1.
It has been reported that TRAIL-resistant cells can be resensitized by c-Myc, which induces DR5 expression, and represses the transcription of both c-FLIP and Mcl-1 . Consistently, in our data the expression of c-Myc was significantly increased in PC3-MM2 and KM12L4A cells with high metastatic potency compared to their primary cells. c-Myc is deregulated in a wide range of human cancers, including breast, colon, cervical and small-cell lung carcinomas, osteosarcomas, glioblastomas, melanoma and myeloid leukaemias, and c-Myc over-expression is frequently correlated with aggressive metastasis, poor differentiation and poor prognosis . Paradoxically, in addition to promoting proliferation, c-Myc inhibits survival signals, resulting in apoptosis, if the apoptotic signal is sufficiently strong . On the other hand, activation of c-Myc renders primary and non-transformed cells sensitive to TNF-α, CD95L, and TRAIL-induced apoptosis [13, 18, 19, 32, 33]. It is known that c-Myc can alter the expression of several key players of the death receptor pathway, such as up-regulation of DR5  and down-regulation of c-FLIP , promoting the activation of death receptors and DISC and, thus, renders cells sensitive to TRAIL-induced apoptosis . In addition, it was demonstrated that c-Myc suppresses Mcl-1 transcription, and is constitutively bound to the Mcl-1 promoter . Therefore, our results suggest that increased sensitivity to TRAIL in metastatic cancer cells may be in part due to increased expression of c-Myc in the cells, which is associated with up-regulation of DR5 cell surface expression and down-regulation of c-FLIP and Mcl-1. In addition to enhancement of death-receptor signaling, c-Myc also amplifies the death signal at the mitochondria for synergistic induction of apoptosis by activating Bak, enabling TRAIL to fully activate the caspase machinery in human cells [10, 12]. We found that after treatment with TRAIL activity of caspases including caspase-8, -9, and -3 were higher, and up-regulation of Bax and down-regulation of Bcl-2 were more significant in PC3-MM2 and KM12L4A cells than in their primary cells. Since c-FLIPL and Mcl-1 were known as substrates of caspases [34, 35], it could be possible that TRAIL-induced potent activation of caspases in metastatic cancer cells led to accelerated down-regulation of c-FLIPL and Mcl-1. Therefore, these increased proapoptotic responses to TRAIL in the highly metastatic cancer cells might be attributed to the increased level of c-Myc.
Moreover, PC3-MM2 and KM12L4A cells showed the enhanced constitutive expression of DNA-PKcs as well as c-Myc compared to the corresponding primary cells. DNA-PK, a complex consisting of the regulatory subunits Ku70/80 and the catalytic subunit DNA-PKcs, plays a central role in the repair of DNA double-strand breaks (DSB). Over-expression of DNA-PKcs was reported in various human tumors [36, 37, 38], and the activity and protein/mRNA levels of DNA-PKcs were significantly higher in tumor tissues than in normal tissues . Previously we demonstrated that the DNA-PK activity is remarkably increased in metastatic cancer cells . Although DNA-PKcs was primarily defined as a component of the DNA DSB repair complex, DNA-PKcs is implicated, directly and indirectly, in various cellular metabolic processes, since DNA-PKcs may be able to phosphorylate the oncoproteins c-Myc, c-fos, and c-abl . DNA-PKcs activity may contribute to the overexpression of c-Myc, probably via its critical role in maintaining the stability of c-Myc [15, 40]. DNA-PKcs also activates Akt via phosphorylation of Ser473, which in turn inactivates GSK-3β via phosphorylation of Ser9, resulting in the stabilization of c-Myc . We showed a prominent interaction between DNA-PKcs and c-Myc and an increased level of phosphorylated c-Myc levels in PC3-MM2 cells compared to PC3 cells, suggesting the possibility that the overexpressed DNA-PKcs might contribute to the overexpression c-Myc in metastatic cancer cells via its stabilization. As previously described, c-Myc can participate in cell death as a pro-apoptotic factor [10, 12]. In our experiment, we demonstrated that siRNA-mediated depletion of c-Myc in metastatic cancer cells suppressed TRAIL-induced up-regulation of DR5 and activation of caspase, and these phenomena could be associated with decreased cytotoxic effect of TRAIL on PC3-MM2 and KM12L4A cells.
It is known that the inhibition of Akt in gliomas enhances their susceptibility to TRAIL  and TRAIL down-regulates Akt levels by caspase-dependent degradation . In addition, DNA-PKcs, an upstream regulator of Akt, is cleaved by caspase-3 and, thus, the inactivation of these molecules occurs during apoptosis [43, 44]. Our data showed that the cleavage of DNA-PKcs and the subsequent reduction of DNA-PKcs and pAkt levels occurred in the PC3-MM2 and KM12L4A cells after TRAIL treatment, but not in their primary counterparts, even though the metastatic cells exhibited higher basal levels of DNA-PKcs and pAkt than their primary counterparts. This TRAIL-induced suppression of DNA-PKcs/Akt signaling pathway in metastatic cancer cells seemed to be at least in part due to overexpression of c-Myc, since it was prevented by knockdown of c-Myc. Therefore, our results suggest that the amplified TRAIL-induced caspase activation by overexpressed c-Myc may suppress the anti-apoptotic activity of DNA-PKcs by increasing its proteolytic cleavage and consequently promote apoptosis in the metastatic cancer cells. Finally, we found that suppression of DNA-PKcs by siRNA and DNMB significantly increased TRAIL-induced growth inhibition and apoptosis in TRAIL-insensitive PC3 or KM12 cells, which was accompanied by the inhibition of Akt S473 phosphorylation, the activation of caspases (caspase-8, -9, and -3), and the up-regulation of the cell surface expression of DR5. Indeed, it has been reported that inhibitors of DNA-PK have a great potential to enhance the efficacy of chemotherapy and radiotherapy [45, 46, 47].
This study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis, which indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc. In addition, DMNB, a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive KM12 and PC3 cells and therefore functioned as a TRAIL sensitizer, which might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.
Cell culture and Reagents
The poorly metastatic primary human colorectal carcinoma KM12 cells were originally derived from a Dukes' B2 colon cancer. The highly liver-metastatic KM12L4A cells were derived from KM12 cells. Human androgen-independent PC3 prostate adenocarcinoma cells and PC3-MM2 cells, a variant of PC3 selected for their highly metastatic potential were used in this study. These four cell lines were provided by Professor IJ Fidler (MD Anderson, TX, USA). The cells were maintained in culture (5% CO2 and 95% at 37°C) as adherent monolayers in DMEM supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, L-glutamine, and vitamin solution. The recombinant human soluble TRAIL was obtained from R&D Systems (Minneapolis, MN, USA). 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB) was purchased from Merck KgaA (Darmstadt, Germany).
Cell Proliferation Assay (MTT assay)
Cell proliferation was measured by counting viable cells by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Company, St. Louis, MO, USA) colorimetric dye-reduction method. Exponentially growing cells (5 × 103 cells/well) were plated in a 96-well plate and incubated in growth medium treated with the indicated concentration of TRAIL and/or DMNB at 37°C. After 96 h, 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 medium was proportional to the number of viable cells. Inhibition of proliferation was evaluated as a percentage of control growth (no drug in the medium). All experiments were repeated in at least two experiments in triplicate.
Flow cytometry analysis
Cells (5 × 105 cells/well) treated with or without TRAIL (or DMNB) were centrifuged at 500 × g, washed with phosphate-buffered saline (PBS), and resuspended in 500 μl PBS. The cells were then incubated with 5 μl mouse IgG, or anti-DR4 or -DR5 monoclonal mouse antibody (1:100; R&D Systems, Minneapolis, MN, USA) for 2 h. After washing with PBS, FITC-conjugated rabbit anti-mouse IgG (1:200; Sigma Chemical Co., St. Louis, MO, USA) was added to the cell suspension and incubated for 2 h on ice followed by washing with PBS. After rinsing, the samples were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed using the CellQuest program.
To determine whether TRAIL-induced apoptosis occurs through the death receptors, cells were pretreated with the TRAIL-R1 (anti-DR4) or TRAIL-R2 (anti-DR5) antibody (0.5 μg/ml, R & D Systems, Minneapolis, MN) 3 h before treatment with 2 and 10 ng/ml TRAIL for 6 h. In control experiments, cells were treated normal goat IgG (Sigma-Aldrich Corp., St Louis, MO, USA) before TRAIL treatment. Apoptosis was the measured by annexin V assay.
Quantitative real-time RT-PCR
Quantitative analysis of FLIPL/S mRNA levels was performed by the SYBR Green real time PCR method using 2X Power SYBR® Green PCR Master Mix (Applied Biosystems, CA, USA) in a Bio-Rad iCycler & iQ Real-time PCR systems (Bio-Rad, Hercules, CA, USA). An increase in the fluorescence of the reporter dye, SYBR green, during quantitative real-time RT-PCR is due to the SYBR green binding to double-stranded DNA. The sequences of forward and reverse primers for FLIPL, FLIPS and β-actin (as loading control) are the following: FLIPL (forward) 5'-TTCCAGGCTTTCGGTTTCTT-3' and (reverse) 5'-GTCCGAAACAAGGTGAGGGT-3'; FLIPS (forward) 5'-ACCCTCACCTTGTTTCGGAC-3' and (reverse) 5'-CTTTTGGATTGCTGCTTGGA-3'; and β-actin (forward) 5'-CAGAGCAAGAGAGGCATCCT-3' and (reverse) 5'-TTGAAGGTCTC AAACATGAT-3'. After initial denaturation step at 95°C for 10 min, each amplification step was repeated 60 cycles with the following condition: denaturation at 95°C for 30 sec, annealing at 55°C for 60 sec and extension at 60°C for 60 sec. Each sample was tested in triplicate, and gene expression levels were normalized to β-actin mRNA.
Western blot and immunoprecipitation analysis
Protein samples were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Hybond-ECL; GE Healthcare, Buckinghamshire, UK). The membrane was incubated with antibody as specified, followed by a secondary antibody conjugated with horseradish peroxidase. Specific antigen-antibody complexes were detected using enhanced chemiluminescence (PerkinElmer Life Sciences, MA, USA). Western blot analysis was performed with the following antibodies: Bax, caspase-3, PARP and Bcl-2, (Santa Cruz Biotechnology, CA, USA), Akt, phospho-Akt (Ser 473), caspase-8, caspase-9 (Cell signaling Technology, MA, USA), DNA-PKcs (Thermo Fisher Scientific, CA, USA), c-Myc, phospho-c-Myc (pMyc, Epitomics, CA, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were obtained from GE Healthcare (Buckinghamshire, UK). For immunoprecipitation, whole cell or nuclear extracts from PC3 and PC3-MM2 cells were incubated with anti-DNA-PKcs or c-Myc antibody overnight at 4°C, and then protein G-Sepharose beads (Sigma-Aldrich Corp., St Louis, MO, USA) were added and constantly mixed for 4 h. The beads were collected by centrifugation for 5 min at 15,000 × g and 4°C, and washed 3 times with cold extraction buffer. The beads with immune complexes were boiled and electrophoresed on 8% SDS-PAGE gels and analyzed by Western blotting using antibodies against c-Myc, pMyc or DNA-PKcs.
The DNA-PKcs (5'-CAGUCUUAGUCCGGAUCAUdTdT-3'), c-Myc (5'-GACAGUGUCAGAGUCCUGAdTdT-3'), and control scrambled (5'-CUUCCCGAAAACUUGAGACdTdT-3') siRNAs were used in this study. Cells were transfected with 0.1 μM siRNA for 48 h using oligofectamine according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). In brief, the siRNA/oligofectamine complex was added to cells (2 × 105 cells/well) that were seeded in 6-well plate. The cells were incubated for 4 h at 37°C in serum free DMEM medium and then FBS was added. After 48 h, the cells were treated with TRAIL and collected for Western blot analysis to determine the levels of DNA-PKcs, c-Myc, and the indicated proteins.
Untransfected control cells or transfected with the various siRNAs cells (2 × 105 cells/ml) were treated with or without TRAIL and/or DMNB for the indicated times. The cells were centrifuged and resuspended in 500 μl of a staining solution containing Annexin V fluorescein (FITC Apoptosis Detection Kit; BD Pharmingen, San Diego, CA, USA) and propidium iodide in PBS. After incubation at room temperature for 15 min, the cells were analyzed by flow cytometry. Annexin V binds to cells that express phosphatidyl serine on the outer layer of their cell membrane, and propidium iodide stains the cellular DNA of cells with 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 Annexin V and propidium iodide).
The results obtained were expressed as the mean ± standard error (SE) from at least three independent experiments. The data were analyzed using the Student's t-test. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant in all experiments.
This study was supported by the Mid-career Researcher Program through an NRF grant funded by the MEST (No. 2009-0084444) and the National R&D Program for Cancer Control, Ministry for Health, Welfare, and Family affairs, Republic of Korea (0920050).
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