Anthracycline drugs are potent anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand with promising anti-cancer effects. However, some tumor types develop resistance to TRAIL. We examined the effect of aclarubicin (ACR), an anthracycline, in combination with TRAIL. The combination of TRAIL and ACR synergistically induced apoptosis in human acute lymphoblastic leukemia Jurkat cells and human lung cancer A549 cells. In contrast, another anthracycline, doxorubicin (DOX), only slightly sensitized Jurkat cells and A549 cells to TRAIL-induced apoptosis, with weaker enhancement of death receptor 5 (DR5) expression than ACR. The RNase protection assay, real time RT-PCR and western blot demonstrated that ACR upregulated the expression of a TRAIL receptor, DR5. Caspase inhibitors and dominant negative DR5 efficiently reduced the apoptotic response to the treatment with ACR and TRAIL, indicating that the combined effect depends on caspase activities and the interaction between TRAIL and its receptor. ACR but not DOX increased the activity of the DR5 gene promoter in Jurkat cells carrying a mutation in the p53 gene, suggesting that ACR upregulates DR5 expression through p53-independent transcription. These results suggest the combination of TRAIL and ACR to be a promising treatment for malignant tumors. (Cancer Sci 2012; 103: 282–287)
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, kills many types of cancer cells in vitro and in vivo, but has little or no toxicity in normal cells.(1,2) Five TRAIL receptors have been reported, death receptor (DR) 5, also called TRAIL-R2,(3–5) DR4, decoy receptor (DcR) 1, DcR2 and osteoprotegerin.(6,7) DR4 and DR5 can mediate TRAIL-induced apoptosis, but the others act as dominant negative receptors by competing with DR to interact with TRAIL. After the interaction of DR4 and DR5 with TRAIL, DR forms a death-inducing signaling complex (DISC) with a Fas-associated death domain and pro-caspase-8 and pro-caspase-10.(6,7) Caspase-8 and caspase-10 are auto-activated following the formation of DISC, and finally activate caspase-3, resulting in apoptosis. Bid is cleaved by caspase-8 and caspase-10 and mediates apoptotic signaling to mitochondria.(8) Activation of caspase-9 at downstream of mitochondria leads to the cleavage and activation of effector caspases.
Recombinant human TRAIL and agonistic antibodies for TRAIL receptors have been developed as anti-cancer agents, and phase I/II clinical trials are underway in patients with non-Hodgkin lymphomas and solid malignant tumors.(9–11) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-deficiency promoted tumor initiation and metastasis in an animal model,(12) and activation of the TRAIL signaling pathway is considered an attractive option for cancer treatment and prevention. However, many malignant tumor cell lines are resistant to TRAIL,(13) and even TRAIL-sensitive cancer cells acquire resistance when treated with inadequate doses of TRAIL.(14) Therefore, it is important to overcome the resistance in TRAIL-based cancer treatment.
Doxorubicin (DOX), also known as adriamycin, is an anthracycline drug reported to overcome TRAIL resistance in malignant tumor cells.(15–18) Aclarubicin (ACR), another anthracycline, is a clinical anti-cancer agent with less cardiotoxicity than DOX.(19,20) However, ACR has not been tried in combination with TRAIL.
In the present study, we found that ACR but not DOX sensitized cancer cells to TRAIL by acting on the DR5 promoter, suggesting that clinically less toxic ACR might be a promising sensitizer for TRAIL-induced apoptosis.
Materials and Methods
Reagents. Aclarubicin (ACR) and doxorubicin (DOX) were purchased from Sigma (St. Louis, MO, USA) and dissolved in DMSO. Soluble recombinant human TRAIL/Apo2L was obtained from PeproTech (London, UK). The human recombinant DR5 (TRAIL-R2)/Fc chimera and caspase inhibitors, zVAD-fmk, zDEVD-fmk, zIETD-fmk and zAEVD-fmk, were purchased from R&D Systems (Minneapolis, MN, USA).
Cell culture. Human acute lymphoblastic leukemia Jurkat cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Human lung cancer A549 cells were maintained in DMEM supplemented with 10% FBS, 4 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
Cell viability assay. The number of viable cells was determined using Cell Counting Kit-8 according to the manufacturer’s instructions (Dojindo, Kumamoto, Japan). After the incubation of Jurkat cells for 72 h with the indicated concentrations of ACR or DOX, the kit reagent WST-8 was added to the medium and the cells were incubated for a further 4 h. The absorbance of samples (450 nm) was determined using a scanning multi-well spectrophotometer (DS Pharma Biomedical, Osaka, Japan). Cell viability was also measured using the ViaCount Assay according to the manufacturer’s instructions (Guava Technologies, Hayward, CA, USA).
Detection of apoptosis. For detection of the sub-G1 population, cells were harvested from culture dishes, washed with PBS and suspended with PBS containing 0.1% Triton-X100 and RNase A (Sigma). Nuclei were stained with propidium iodide. DNA content was measured using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and cells in the sub-G1 phase were counted as apoptotic cells. For each experiment, 10 000 events were collected. The data was analyzed using Cell Quest software (Becton Dickinson).
Western blot analysis. Stepwise extraction of the cytosolic fraction and organelle/membrane fractionation was performed using a subcellular proteome extraction kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. The cell lysate was prepared as described previously.(21) Cell lysate containing 30 μg of protein was resolved on a 10 or 12.5% SDS-polyacrylamide gel for electrophoresis, and blotted onto PVDF membranes (Millipore, Bedford, MA, USA). A rabbit polyclonal anti-DR5 antibody (Prosci, Poway, CA, USA), mouse monoclonal anti-Bid, and anti-caspase-8, anti-caspase-9 and anti-caspase-10 antibodies (MBL, Nagoya, Japan), a mouse monoclonal anti-pro-caspase-3 antibody (Immunotech, Marseile, France), a rabbit monoclonal cleaved caspase-3 antibody (Cell Signaling, Beverly, MA, USA) and a mouse monoclonal anti-β-actin antibody (Sigma) were used as the primary antibodies. The signal was detected with an ECL western blot analysis system (GE Healthcare, Piscataway, NJ, USA).
RNA analysis. Total RNA from the cells was extracted using Sepasol-RNA I (Nacalai Tesque, Kyoto, Japan), according to the manufacturer’s instructions. The RNase protection assay was done using the RPA III Ribonuclease Protection Assay Kit (Ambion, Austin, TX, USA) and labeled RNA probes generated with hAPO3d template sets (BD PharMingen, San Diego, CA, USA), according to the manufacturer’s instructions as described previously.(21) For quantitative real-time RT-PCR, total RNA (2 μg) was reversely transcribed to cDNA in 20 μL reaction volume with Moloney Murine Leukemia Virus (MMLV)-reverse transcriptase (Promega, Madison, WI), using oligo (dT) primers (TOYOBO, Osaka, Japan), according to the manufacturer’s instructions. Quantitative real-time RT-PCR was carried out using an RT-PCR system GeneAmp7300 (Applied Biosystems, Foster, CA, USA). Real-time quantitative RT-PCR primer-probe sets for DR5 mRNA and Human 18S rRNA were purchased from Applied Biosystems. The expression level of DR5 mRNA was normalized against the level of 18S rRNA in the same sample.
Luciferase assay. The DR5 promoter luciferase plasmid pDR5PF was generated as described previously.(22,23) The transfection of plasmids and luciferase assay were performed as described previously.(24) Luciferase activity was standardized against the protein concentration of each sample.
Statistical analysis. Data represent means ± SD of three determinations. Data was analyzed using a Student’s t-test and differences were considered significant from controls at P < 0.05.
Aclarubicin is a more effective enhancer of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis than doxorubicin in Jurkat cells. It has been reported that DOX has a synergistic effect with TRAIL,(15–18) but ACR, with fewer side effects than DOX,(19,20) has not been tested in combination with TRAIL. Therefore, we investigated the cytotoxic effect of TRAIL and ACR in human acute lymphoblastic leukemia Jurkat cells. First, to investigate the antiproliferative effects of each agent alone, we assessed the viable cell number of Jurkat cells after 72 h of treatment with ACR or DOX. As shown in Figure 1a, ACR and DOX were similarly effective against cell proliferation. Next, we tried to compare ACR and DOX at the same dose in terms of the induction of sensitivity to TRAIL in the loss of cell viability. Using a Guava ViaCount assay, we investigated the combination effect on cell viability. As shown in Figure 1b, the loss of cell viability from the combined effect of TRAIL and ACR was more remarkable than that of TRAIL and DOX. Next, we quantified apoptotic cells by measuring the sub-G1 population to compare ACR and DOX in combination with TRAIL. Treatment with TRAIL alone weakly induced apoptosis in less than 10% of cells (Fig. 1c). As previously reported, Jurkat cells were resistant to even high concentrations of TRAIL.(21) Only ACR or DOX treatment very weakly caused apoptosis in Jurkat cells. However, TRAIL treatment for an additional 12 h in combination with ACR markedly induced apoptosis, whereas that with DOX moderately induced apoptosis (Fig. 1c), indicating that ACR sensitizes Jurkat cells to TRAIL-induced apoptosis more effectively than DOX. We then examined the dose-dependent effects of ACR combined with TRAIL. ACR treatment (7.5–240 nM) did not cause apoptosis, but co-treatment with TRAIL resulted in significant apoptosis, beyond just an additive effect (Suppl. Fig. S1). Thus, it was confirmed that ACR possesses the ability to sensitize Jurkat cells to TRAIL-induced apoptosis.
Next, to understand why the combined effect of TRAIL and ACR was more remarkable than that of TRAIL and DOX, we examined DR5, a TRAIL receptor. Interestingly, ACR induced the expression of DR5 more than DOX (Fig. 1d). In addition, we found that ACR but not DOX significantly increased the activity of the DR5 promoter (Fig. 1e). These results indicate that ACR upregulates DR5 expression and sensitizes Jurkat cells to TRAIL-induced apoptosis through a different mechanism from that of DOX.
Aclarubicin induces death reactor 5 expression in Jurkat cells. To investigate the mechanisms by which sensitivity to TRAIL is enhanced by ACR, we analyzed whether ACR regulates gene expression related to the TRAIL pathway by conducting RNase protection assays. As shown in Figure 2a, ACR significantly upregulated the mRNA expression of DR5, Fas, caspase-8 and receptor-interacting protein, but not DR4, DcR1 and DcR2. Furthermore, we analyzed the DR5 mRNA level after the treatment with 120 nM ACR or 120 nM DOX for 24 h by quantitative real-time RT-PCR (Fig. 2b). DOX treatment increased DR5 mRNA expression approximately 2.3-fold. Therefore, DOX might upregulate the stability of DR5 at the mRNA level in Jurkat cells. Next, we examined the effects of ACR on the expression of DR5 at the protein level for 12, 24 and 36 h. As shown in Figure 2c, the time course study showed that DR5 protein was drastically increased 24 h after the treatment with 120 nM ACR. Furthermore, the upregulation of DR5 expression by ACR occurred both in the membrane fraction and in the cytosolic fraction (Fig. 2d).
Involvement of caspases in the enhancement of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by aclarubicin. To determine whether the sub-G1 population reflects caspase-dependent apoptosis, we analyzed the effect of caspase inhibitors on the cytotoxicity. As shown in Figure 3a, the apoptosis induced by the combination of TRAIL and ACR was almost completely inhibited by the general caspase inhibitor zVAD-fmk and the caspase-8, caspase-10, caspase-9 and caspase-3 inhibitors. These results indicate that the ACR-mediated sensitization to TRAIL occurred in a caspase-dependent manner and are consistent with previous reports on the role of caspases in TRAIL-mediated apoptosis.(6,7) Furthermore, we performed western blot analysis of caspase-8, caspase-10, caspase-9, caspase-3 and Bid in cells treated with both ACR and TRAIL or each agent alone at a suboptimal concentration (Fig. 3b). Bid is a substrate of caspase-8 and is cleaved during the activation of TRAIL signaling.(8) Combined treatment with TRAIL and ACR clearly induced the cleavage of caspases and Bid. Moreover, the DR5/Fc chimeric protein, which has a dominant negative effect on DR5 and inhibits TRAIL-mediated apoptosis, effectively blocked the cleavage of Bid and caspases induced by co-treatment with TRAIL and ACR. These results indicate that the combination of TRAIL and ACR induces apoptosis dependent on caspase and TRAIL-DR5 interaction.
Aclarubicin induces death reactor 5 expression and promotes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in other human malignant tumor cells in a synergistic fashion. We next investigated whether ACR induces DR5 protein and enhances TRAIL-induced apoptosis in a synergistic fashion in other human malignant tumor cells. Because ACR has been used for lung cancer in a clinical setting, we used human lung cancer A549 cells. We then found that ACR significantly increased DR5 protein expression in a dose-dependent manner in A549 cells (Fig. 4a), whereas DOX did not (data not shown). Next, we found that the combination of ACR and TRAIL induced apoptosis more strongly than treatment with either TRAIL or ACR alone, whereas the sensitization to TRAIL by DOX was much weaker in A549 cells (Fig. 4b). These results indicate that ACR strongly enhanced TRAIL-induced apoptosis in a synergistic fashion not only in Jurkat cells but also in other human malignant tumor cells.
Anthracycline DOX is reported to sensitize cancer cells to TRAIL-induced apoptosis and upregulate DR5 expression.(15–18) In the present study, we first found that another clinically used anthracycline, ACR, with fewer side effects, enhanced the expression of DR5 much more markedly than DOX. Consequently, ACR with TRAIL synergistically and more remarkably induced apoptosis than DOX. Interestingly, our data also showed that ACR but not DOX increased DR5 gene promoter activity in Jurkat cells, resulting in higher levels of DR5 protein.
There are many reports that DOX regulates DR5 expression via p53.(25) In the present study, we have shown that ACR upregulates DR5 promoter activity independently of p53, because Jurkat cells carry a mutation in the p53 gene and the DR5 promoter region used does not contain a p53-binding site.(23,26)Figure 1c,d shows that DOX weakly induced expression of the DR5 protein without increasing DR5 promoter activity. The data is consistent with a report that DOX increased the stability of mRNA of porphobillinogen deaminase and GATA1 in K562 human erythroleukemic cell line.(27) In fact, in the present study, we have shown that DOX upregulates DR5 expression at the mRNA level in Jurkat cells (Fig. 2b).
In the previous reports, DOX enhanced TRAIL sensitivity, but the effect was not very strong in the present study. However, our data is consistent with the previous report that the induction of DR5 expression by DOX in Jurkat cells was very weak compared with that in HL-60 cells.(28) Furthermore, in our study, DOX did not induce DR5 protein expression in A549 cells (data not shown) and the combinational effect of DOX and TRAIL was much weaker than that of ACR and TRAIL. This suggests that the combinational effect of DOX and TRAIL could be different depending on cancer cell types.
Therefore, we at least propose that clinically less toxic ACR might be more effective than DOX in combination with TRAIL or stimulatory agents for the pathway, such as DR4 and DR5 agonistic antibodies.(29)
Chemoresistance in malignant tumors is a crucial problem in anti-tumor therapy. Although TRAIL is a promising anti-cancer agent, several malignant tumor cell lines remain resistant to TRAIL-induced apoptosis. Here, we showed for the first time that ACR, an anthracycline, could overcome TRAIL resistance in Jurkat leukemia and A549 lung cancer cells. As the mechanism overcoming TRAIL resistance, we showed that ACR specifically upregulated the expression of a TRAIL receptor, DR5. We have previously reported that combining agents based on molecular mechanisms is effective, and proposed the concept of “combination-oriented molecular-targeting cancer prevention and therapy.”(30) Using the model, we found the combination of TRAIL and DR5 inducers to be very effective and promising. The results of the present study are consistent with our concept. The present findings strongly suggest TRAIL and ACR to be a promising combination for patients with a variety of malignancies.
This work was supported in part by the Japanese Ministry of Education, Culture, Sports, Science and Technology.