Báo cáo khoa học: Etoposide upregulates Bax-enhancing tumour necrosis factor-related apoptosis inducing ligand-mediated apoptosis in the human hepatocellular carcinoma cell line QGY-7703 pdf

Eur J Biochem 270, 2721–2731 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03639.x Etoposide upregulates Bax-enhancing tumour necrosis factor-related apoptosis inducing ligand-mediated apoptosis in the human hepatocellular carcinoma cell line QGY-7703 Lin Miao, Peng Yi, Yi Wang and Mian Wu Department of Molecular and Cell Biology, Key Laboratory of Structural Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted much attention because of its ability to kill tumour cells In this study, we demonstrated that treatment of QGY-7703 cells with the combination of TRAIL and etoposide resulted in synergistic cytotoxic effects In dissecting the mechanism underlying this synergistic effect, we found that treatment with etoposide alone resulted in the upregulation of Bax, while the level of truncated Bid (tBid) was unchanged In contrast, while treatment with TRAIL alone significantly increased the level of tBid, the expression of Bax remained unaffected The enhanced apoptosis was accompanied by an increased release of cytochrome c and second mitochondria-derived activator of caspase/direct IAP binding protein with low pI (DIABLO) from mitochondria, leading to the activation of cellular caspase-8, -9, -3 and -7, as well as poly ADP-ribose polymerase This enhanced release of cytochrome c and second mitochondria-derived activator of caspase/DIABLO was inhibited by the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone The RT–PCR and Western blotting results demonstrated that the levels of both mRNA and protein for death receptor-4, death receptor-5 and decoy receptor-2 remained unchanged in response to etoposide, indicating that the synergistic effect of TRAIL and etoposide is not a result of increasing the expression for TRAIL receptors, but rather is associated with amplification of the mitochondrial signal pathway Chemotherapeutic agents are used widely in the treatment of different types of cancer Hetapocellular carcinoma, one of the most common tumours in adults, remains largely incurable despite intensive multimodality treatment, including surgical eradication, irradiation and chemotherapy Besides the difficulties of complete surgical removal, resistance to chemotherapy and irradiation is a major hindrance for the successful treatment of liver cancers Defects in signalling pathways leading to the activation of caspases are common in most types of malignancies Accumulating data suggest that two major signal pathways are involved in apoptosis The first is the mitochondrial pathway (intrinsic), usually triggered by DNA damage, and the second is the receptor-mediated pathway (extrinsic), initiated by death receptor activation Resistance to chemotherapeutic agents may be caused by repression of the mitochondrial pathway In order to achieve effective treatment of those drug-resistant tumour cells, new methods, which could bypass the resistance to chemotherapeutic drugs through activation of the death receptor-mediated pathway, need to be developed Tumour necrosis factorrelated apoptosis-inducing ligand (TRAIL), a type II membrane protein, is a member of the tumour necrosis factor death-ligand family [1,2] and it selectively induces apoptosis in a number of transformed cells in vitro [1–3] and tumours in vivo, while leaving normal tissues intact [4,5] Four cognate TRAIL death receptors – death receptor (DR)4 [6,7], DR5 [7,8], decoy receptor-1 (DcR-1)/TRID (i.e TRAIL decoy receptor lacking an intracellular domain) [9–11] and DcR-2/TRUNDD (i.e TRAIL decoy receptor containing a truncated death domain) [12–14] – have been identified to date Both DR4 and DR5 contain an intracellular Ôdeath domainÕ that recruits effector molecules, such as Fas-associated death domain protein (FADD) [15] and death-associated protein (DAP-3) [16] to activate initiator caspase-8 and subsequently the effector caspases leading to apoptosis [6,7] In contrast, DcR-1/TRID and DcR-2/ TRUNDD contain a truncated or a null intracellular death Correspondence to M Wu, Department of Molecular and Cell Biology, School of Life Sciences, University of Sciences and Technology of China, Hefei, Anhui, China, 230027 Fax: + 86 551 360 6264, Tel.: + 86 551 360 6264, E-mail: wumian88@yahoo.com Abbreviations: DcR, decoy receptor; DIABLO, direct IAP binding protein with low pI; DR, death receptor; FADD, Fas-associated death domain protein; FITC, fluorescein isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly ADP-ribose polymerase; PI, phosphatidylinositol; Smac, second mitochondria-derived activator of caspase; tBid, truncated Bid; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand; TRID, TRAIL decoy receptor lacking an intracellular domain; TRUNDD, TRAIL decoy receptor containing a truncated death domain; zVAD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone Enzymes: alkaline phosphatase (EC 3.1.3.1) (Received 13 January 2003, revised 26 March 2003, accepted 28 April 2003) Keywords: p53; Bax; tBid; mitochondrial pathway; death receptor Ó FEBS 2003 2722 L Miao et al (Eur J Biochem 270) domain, respectively, and are unable to transduce the death signal [6,7] Mutation in p53 initiates oncogenesis and accounts for more than 50% of different tumour types [17–19] p53 acts as a Ôgenome guardianÕ to scrutinize DNA injury in cell cycle regulation and cell death control by modulating expression of a number of target genes in response to DNA-damaging agents, hypoxia or oncogene activation A number of chemotherapeutic drugs, acting as DNA-damaging agents, enhance the expression of p53 The downstream target genes of p53 include p21, Bax, DR4, DR5, DcRs and Bcl-2 Upregulation of p53, resulting in the enhanced expression of DR4 and/or DR5 in some cancer cell lines, is believed to be one of the mechanisms by which the susceptibility of the tumour cells to TRAIL is synergistically increased when treated with chemotherapeutic agents and TRAIL together [20,21] However, some research groups have reported that the upregulation of DR4 and DR5 is not necessarily p53dependent [22,23] Many chemotherapeutic drugs can promote mitochondrial membrane permeabilization and release of caspaseactivating factors, in particular cytochrome c and second mitochondria-derived activator of caspase (Smac)/DIABLO from mitochondria to the cytosol Bax is a pro-apoptotic factor belonging to the Bcl-2 family and stimulates mitochondria to release cytochrome c and Smac/ DIABLO Bax, together with its homologue Bak, plays a vital role in the TRAIL-mediated mitochondrial apoptosis [24] The Bid protein, a member of the Bcl-2 family, may stand at the cross-roads of the mitochondria and the death receptor, as Bid is cleaved by active caspase-8 to form truncated Bid (tBid), which, in turn, stimulates the mitochondria to release cytochrome c [25] In this study, we examined the apoptotic effects of the cytokine TRAIL, in the presence and absence of a chemotherapeutic agent, on hepatocellular carcinoma QGY-7703 cells to determine whether co-operative killing could be achieved and, if so, what possible mechanism might be underlying this effect We found that TRAIL plus etoposide acts synergistically to kill human liver tumour cells and this synergistic effect involves the upregulation of Bax and tBid, but not of DR4 or DR5 The interaction between Bax and tBid results in the amplification of mitochondrial release of cytochrome c and Smac/DIABLO, leading to augmented cell death through enhanced activation of cellular caspases Materials and methods Regents and antibodies Recombinant human (rh)TRAIL was purchased from R & D Systems The general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVADFMK), was purchased from BIOMOL Research Laboratories Inc Most drugs used in this study, including etoposide, cisplatin, doxorubincin, 5-fluorouracil, methotrexate, cytarabine, cyclophophamide and daunorubicin, were purchased from Sigma Some other drugs of GCP (good clinical practice) grade were ordered from a local pharmaceutical company Antibodies used in this study were as follows Polyclonal antibodies: anti-caspase-7, anti-actin, anti-DR4, anti-DR5, anti-DcR-2, anti-Bid, antiBax (Santa Cruz Biotech Inc., Santa Cruz, CA, USA), anti-caspase-9 (Immunotech), anti-caspase-3/CPP32 (BD Biosciences) and anti-poly ADP-ribose polymerase (antiPARP) (Upstate Biotechnology) Monoclonal antibodies (mAbs): anti-cytochrome c (R & D Systems), anti-caspase-8 (BD Biosciences) and anti-Smac/DIABLO (Calbiochem) All the secondary antibodies [anti-goat, anti-mouse, antirabbit (H + L)] were purchased from Promega Cell culture The human hepatocellular carcinoma cell line, QGY-7703, was kindly provided by D Lu (Dept of Neurology, Stanford University, Palo Alto, CA, USA) The cells were maintained in RPMI-1640 containing 10% heat-inactivated bovine serum, mM L-glutamine, 100 ml)1 penicillin and 100 lgỈml)1 streptomycin (Life Technologies, Inc Grand Island, NY, USA) at 37 °C under an atmosphere of 5% CO2 in air Cell death assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine tumour cell viability Briefly, cells were plated at · 104 cells per well in 96-well microtitre plates overnight Cells were then treated with 100 lL of fresh medium containing the drug to be tested, cultured for 20 h, then for a further h with 10 lL of mgỈmL)1 MTT After incubation, the medium was removed and replaced with 100 lL of dimethylsulfoxide and the data were analysed by using an ELX800 Universal Microplate Reader (BIO-TEK Instruments, Inc.) at a wavelength of 570 nm with the reference wavelength set at 630 nm The effect of drug treatment was expressed as a percentage of growth inhibition using untreated cells as the uninhibited control Assessment of apoptosis by Annexin V staining An Annexin V–fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Pharmingen, San Diego, CA, USA) was used in this assay After treatment with the test drugs, cells were harvested and resuspended in binding buffer [0.01 M Hepes/ NaOH (pH 7.4), 0.14 mM NaCl, 2.5 mM CaCl2] at a concentration of · 106 cellsỈmL)1 One-hundred microlitres of this resuspended solution (1 · 105 cells) was transferred to a 5-mL culture tube After incubation with lL of Annexin V–FITC and 10 lL of phosphatidylinositol (PI) (50 lgỈmL)1) for 15 at room temperature in the dark, the cells were analysed by flow cytometry in a FACSCalibur using CELL QUEST software (Becton Dickinson) Western blot analysis Cells were lysed in 50 lL of lysis buffer [50 mM Tris/HCl (pH 7.5), 250 mM NaCl, mM EDTA, 50 mM NaF, 0.5% Nonidet P-40] supplemented with a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) on ice for 30 Fifty micrograms of each sample was separated by SDS/PAGE (12% or 15% gel) and transferred to nitrocellulose membrane (Amersham Ó FEBS 2003 Augmented apoptosis induced by TRAIL and etoposide (Eur J Biochem 270) 2723 Pharmacia Biotech) Filters were blocked with NaCl/Tris (TBS) containing 5% nonfat milk and 0.1% Tween-20 for h at room temperature and then incubated (for a further h) with primary antibodies Blots were then probed with appropriate alkaline phosphatase-conjugated secondary antibodies and the proteins visualized by using Western Blue stabilized substrate for alkaline phosphates (Promega) RT–PCR Total cellular RNA was extracted from QGY-7703 cells using the SV total RNA Isolation kit (Promega) The yield and purity of the RNA sample were determined by ultraviolet spectrometry using the DU 640 Nucleic Acid and Protein Analyser (Beckman Coulter) Two micrograms of total RNA was reverse transcribed using the TaKaRa One Step RNA PCR kit (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s instructions In both RT and PCR steps, the reaction reagents were prepared as master mixtures and then aliquotted PCR primers were designed to amplify the sequence for the intracellular domain of the TRAIL/Apo2L receptor The housekeeping gene, b-actin, was amplified as an internal control The mRNA levels were then normalized to actin mRNA expression Equal amounts of RT–PCR products, loaded onto an agarose gel, were quantified by using the Eagel Eye Jr Still Video System (Stratagene) Differences in mRNA levels as a result of treatment with etoposide for different incubation times are represented as relative units over basal actin mRNA levels The sequences of the primers used in this study are as follows: DR4 forward, 5¢-CGGAATTCGGAGGGGACCCCAAGTGCAT-3¢; DR4 reverse, 5¢-CGGGATCCTCACTCCAAGGACA CGGCA-3¢; DR5 forward, 5¢-CGGAATTCTGCA AGTCTTTACTGTGGAA-3¢; DR5 reverse, 5¢-CGGA TCCTTAGGACATGGCAGAG-3¢; DcR2 forward, 5¢-CGGAATTCCGCGGAAGAAATTCATTTCT-3¢; DcR2 reverse, 5¢-CGGGATCCTCACAGGCAGGACG TAGCAG-3¢; Bax forward, 5¢-GCGAATTCCATGG ACGGGTCCGGGGAG-3¢; Bax reverse, 5¢-CGCTC GAGTCAGCCCATCTTCTTCCAG-3¢; Bid forward, 5¢-CGGGATCCCCATGGCGATGGACTGTGAGGT-3¢; Bid reverse, 5¢-CGGAATTCTCAGTCCATCCCATTTC TGG-3 The primers for b-actin were kindly provided by Z Tian (Department of Immunology, University of Science and Technology of China, Hefei, Anhui, China) Cytochrome c release assay Cell were harvested, as described above, and lysed in icecold lysis buffer (20 mM Hepes, pH 7.4, 10 mM KCl, mM EDTA, mM MgCl2, with mM dithiothreitol and protease inhibitor cocktail added before use) for 15 on ice Cell lysates were centrifuged at 16 000 g for and the supernatants mixed with · Laemmli buffer and resolved by SDS–PAGE (15% gel) Released cytochrome c and Smac/DIABLO were subjected to analysis by Western blot Statistical analysis All determinations were made in triplicate, and the results were expressed as the mean value ± SD Statistical signi- ficance was determined by the Student’s t-test A P-value of < 0.05 was considered significant Calculations of synergistic cytotoxicity were determined by isobolographic analysis, as described by Berenbaum [26] The isobolographic analysis can detect whether the dose-dependent effects of two compounds in a mixture are more or less effective than the expected effects based on tests of the compounds individually Simply put, the point representing the dose combination lying on, below, or above the straight line represents additive, synergistic, or antagonistic effects, respectively Results and discussion Effect of TRAIL on the liver carcinoma cells QGY-7703 To investigate the apoptotic function of TRAIL containing the extracellular domain (amino acids 114–281) of the human liver cancer cell line, QGY-7703 [27], we treated the cells with increasing concentrations of TRAIL and then analysed cell viability by using the MTT assay After incubation with TRAIL at a concentration of 10 ngỈmL)1 for 30 h, QGY-7703 cells were found to show morphological changes, from a spindle-like to a rounded shape, a characteristic feature of apoptosis (Fig 1A,c) As controls, untreated cells (Fig 1A,a) and cells treated with medium (Fig 1A,b) remained healthy and viable Although TRAIL can induce the apoptosis of QGY-7703 cells, we found that the cells were relatively insensitive to TRAIL In the dose– response experiment (Fig 1B), even though the QGY-7703 cells were treated with TRAIL at a high concentration of 100 ngỈmL)1 for 24 h, only 23% cell death was detected To further verify whether the TRAIL-induced cell death represents apoptosis, we utilized the AnnexinV–FITC staining method to quantify the apoptotic cell numbers by flow cytometry, which relies on the property of cells to lose membrane asymmetry in the early phase of apoptosis As shown in Fig 1C,a, only 1.4% of untreated QGY-7730 cells were Annexin-V positive and PI negative (early apoptotic cells) In contrast, approximately 7% of TRAIL (10 ngỈmL)1)-treated QGY-7730 cells were Annexin-V positive and PI negative (Fig 1C,c) Approximately 6% of untreated cells were double-positive for Annexin-V and PI (Fig 1C,a) We noted that the percentage of these AnnexinV- and PI-positive cells remained similar after treatment with TRAIL (Fig 1C,c; 7.3%), etoposide (Fig 1C,b; 6.2%), or both (Fig 1C,d; 5%), indicating that those double-positive cells may represent either late-stage apoptotic cells or necrosis cells, which are independent of apoptotic induction Etoposide, in conjunction with TRAIL, results in synergistic effects in inducing apoptosis The resistance of tumours to conventional chemotherapeutic drugs, and the potential toxicity of conventional chemotherapeutic drugs to patients, is a major problem in the treatment of malignant liver tumours Despite its potent cytotoxic effect on malignant cells, TRAIL causes little, if any, damage to normal adult tissues [28] To examine whether treatment with the combination of TRAIL and chemotherapeutic drugs was able to trigger enhanced 2724 L Miao et al (Eur J Biochem 270) Ó FEBS 2003 Fig The effects of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) on human liver cancer cells QGY-7703 (A) QGY-7703 cells treated with TRAIL (10 ngỈmL)1) for 30 h and the morphological changes associated with apoptosis were photographed under an inverted light microscope In contrast to the untreated cells (a) and those treated with medium only (b), viability loss was noted at 30 h in cells treated with TRAIL (c) (B) Cytotoxicity of TRAIL (1–100 ngỈmL)1) on QGY-7703 cells was determined by the MTT assay (C) Flow cytometry analysis of TRAILinduced apoptosis QGY-7703 cells treated with medium (a), lgỈmL)1 etoposide (b), 10 ngỈmL)1 TRAIL (c), or 10 ngỈmL)1 TRAIL plus lgỈmL)1 etoposide in the presence (e) or absence (d) of 100 lM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-FMK) for 12 h were stained with fluorescein isothiocyanate (FITC) conjugated to Annexin V and phosphatidylinositol (PI) Ten-thousand events were analysed The percentage of apoptotic cells is indicated in the respective boxes Ó FEBS 2003 Augmented apoptosis induced by TRAIL and etoposide (Eur J Biochem 270) 2725 Fig The synergistic cytotoxicity of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and etoposide on QGY-7703 cells (A) The cytotoxicity of TRAIL (1–100 ngỈmL)1) and etoposide (8, 16, 32, 64 lgỈmL)1), coincubated with QGY-7703 cells for 24 h, was measured by an MTT assay All determinations were made in triplicate, and the results are expressed as the mean value ± SD The data shown are representative of three independent experiments Bars, SD (B) Synergistic cytotoxicity of TRAIL and etoposide was assessed by isobolographic analysis apoptotic induction in the hepatocellular carcinoma cells, QGY-7703, we treated these cells with eight different agents, at increasing concentrations, with and without TRAIL, then analysed apoptosis using the MTT cell death assay and plotted the results of the cytotoxic effect The inhibition rate was calculated as follows: Inhibition rate ẳ ẵ1 À ðabsorbance of drug-treated cells/ absorbance of control cells)]Â100 Of the eight drugs tested, etoposide was found to be able to enhance the TRAIL-induced apoptosis in QGY-7703 cells (Fig 2A) and was found, by isobolographic analysis, to act synergistically (Fig 2B) The remaining seven chemotherapeutic agents (cisplatin, doxorubincin, 5-fluorouracil, methotrexate, cytarabine, daunorubicin and cyclophospha- nide) did not show any synergistic effect when applied together with TRAIL (data not shown) As shown in Fig 2A, only about 25% cell death was detected as a result of treatment with etoposide alone at a very high concentration (64 lgỈmL)1) The QGY-7703 cells were relatively resistant to etoposide As shown in Fig 1C,b, treatment of QGY-7703 cells with etoposide (8 lgỈmL)1) alone for 12 h caused the induction of early apoptosis in 4.8% of cells, which is only a slightly increase compared to cells treated with medium However, when QGY-7703 cells were treated with a combination of TRAIL and etoposide, a significant potentiation of cytotoxicity was achieved For example, when combined with TRAIL at a very low concentration of ngỈmL)1, lgỈmL)1 etoposide was sufficient to induce the same cytotoxicity induced by 64 lgỈmL)1 etoposide As shown in Fig 1C,d, the percentage of early apoptotic cells induced by TRAIL (10 ngỈmL)1) plus etoposide (8 lgỈmL)1) for 12 h was significantly increased, from 1.4 to 31% This effect, however, can be prevented by the general caspase inhibitor, zVAD-FMK (Fig 1C,e) In the presence of zVAD-FMK, the percentage of early apoptotic cells was reduced to 0.59%, indicating complete abrogation of the synergistic effects resulting from co-treatment with TRAIL and etoposide It is also worthy of note that the top three cell death curves, shown in Fig 2A become indistinguishable when the TRAIL concentration used was > 10 ngỈmL)1; this saturation phenomenon could be explained by the fact that TRAIL-induced apoptosis involves ligand/receptor (TRAIL/DR4 or TRAIL/DR5) interaction Myen et al and Maccon et al demonstrated that cisplatin and etoposide dramatically augment TRAILinduced apoptosis in both LNCap and PC3 prostate cancer cells [29] and malignant breast cells [30] Taken together, our results may have provided some clinical significance in the killing of tumour cells, as combined treatment will help to achieve more effective therapy with less toxicity by using a lower dose of chemotherapeutic drugs The synergistic effect of augmented apoptosis is involved in the upregulation of Bax and tBid, but not of DR4 or DR5 Etoposide is a DNA-damaging agent that affects chromosomal DNA [31] It is well known that the transcription factor, p53, is essential for the apoptosis caused by DNA damage [32] We therefore studied the expression level of p53 in hepatocellular carcinoma cells, in response to etoposide or TRAIL As shown in Fig 3A, treatment with etoposide resulted in a time-dependent accumulation of p53, as expected Treatment with TRAIL alone did not stimulate the production of endogenous p53, which is consistent with the results reported by Ashkenazi & Rieger, who claimed that TRAIL-induced apoptosis is p53-independent [14,33] Tumour-suppressor p53 modulates apoptosis through regulating its target genes involved in apoptosis We performed RT–PCR analysis to determine which genes regulated by p53 could account for the synergistic effect of etoposide and TRAIL The QGY-7703 cells were first treated with etoposide (8 lgỈmL) for the indicated times, and the subsequent RT–PCR results are shown in Fig 3B The quantitative results from RT–PCR are shown in Fig 3C It is interesting to note that only Bax mRNA 2726 L Miao et al (Eur J Biochem 270) Ó FEBS 2003 Fig Etoposide induces the accumulation of p53 and upregulates the mRNA level for Bax, but not for death receptor (DR)4, DR5, decoy receptor-2 (DcR-2) or Bid (A) Cells were treated with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) (10 ngỈmL)1) or etoposide (8 lgỈmL)1) for 0, 8, 16 and 24 h, and 50 lg of cell lysate was then used to detect the protein level of p53 (B) Total RNA was extracted from QGY7703 cells treated with etoposide (8 lgỈmL)1) for the indicated time-periods Two micrograms of total RNA of each sample was used in RT–PCR to evaluate the level of mRNA for DcR-2, DR4, DR5, Bax and Bid The housekeeping gene, b-actin, was used as internal control for ensuring that an equal amount of template was used PCR was performed under the following conditions: 25 cycles (DR4 and DR5) or 30 cycles (DcR-2, Bax, Bid) of denaturation at 94 °C for 30 s, annealing at 56 °C or 59 °C (for Bax and Bid) for 30 s, and extension at 72 °C for 90 s The expected sizes of RT– PCR amplification fragments are indicated at the right of the panel The mRNA levels were quantified by densitometry and normalized to basal b-actin mRNA expression and the results are shown in (C) *, statistically significant result (P < 0.05) (D) Cells were treated with or without etoposide (8 lgỈmL)1) for 16 h and the cell lysates were then used to detect the protein level of DR4, DR5, DcR-2 and Bax Actin was used as the loading control was significantly increased at the 12-h time-point and reached a peak at 18 h, whereas mRNA levels for DR4, DR5, DcR-2 and Bid remained essentially unchanged at all time-points tested We further examined (by Western blotting) the protein expression of DR4, DR5, DcR-2 and Bax in QGY-7703 cells treated with and without etoposide As shown in Fig 3D, while no changes in the protein level for DR4, DR5 or DcR-2 were observed, the expression of Bax was found to be increased These Western blot results are in good accordance with those of RT–PCR (Fig 3B) DR4, DR5 and DcR-2 are receptors for TRAIL and their genes are downstream targets of p53 [34–37] The upregulation of TRAIL death receptors, especially DR5, by p53 was thought to be the bridge linking chemotherapeutic agents to the death receptor-elicited apoptotic pathway, contributing to the synergistic effect of TRAIL and chemotherapeutic agents [38,39] In this study, the mRNA for DR4 and DR5 appeared not to be regulated by p53, indicating that there exists another mechanism underlying the synergistic effect Liu et al reported that TRAIL and chemotherapy (such as doxorubicin) can significantly increase the apoptosis of Mesothelioma cell lines, which is a highly chemoresistant tumour, but showed no change in DR5 when treated with chemotherapy [40] Bax belongs to the Bcl-2 protein family and promotes apoptosis through increasing the release of cytochrome c from mitochondria Recently, Joanna et al reported that Bid can be regulated directly by p53 and contributes to chemosensitivity [41] Our data demonstrated that Bax, not Bid, was upregulated upon treatment with etoposide To examine whether TRAIL was also able to upregulate Bax, RT–PCR was performed and the result is shown in Fig 4A No effects on the expression of Bax were observed in QGY7703 cells treated with TRAIL Shi et al recently reported Ó FEBS 2003 Augmented apoptosis induced by TRAIL and etoposide (Eur J Biochem 270) 2727 rial pathway [45] We therefore examined (by Western blotting) the protein level of tBid in QGY-7703 cells treated with TRAIL or etoposide As shown in Fig 4B, the level of tBid was markedly increased with the incubation time when cells were treated with TRAIL alone, whereas treatment with etoposide alone did not affect the protein level of tBid We also examined the expression of tBid and Bax by combined treatment of TRAIL and etoposide; these results are shown in Fig 4C The protein levels of both tBid and Bax increased with the incubation time, and these results are in agreement with the results shown in Figs 3D and 4B Taken together, our results show that the increased level of active tBid resulting from treatment with TRAIL may link the death receptor pathway to the mitochondrial pathway by interaction with upregulated Bax Enhanced release of cytochrome c and Smac/DIABLO by combined treatment with TRAIL and etoposide Fig Activation of Bid in response to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), but not to etoposide (A) Total RNA was extracted from QGY-7703 cells treated with TRAIL (10 ngỈmL)1) for the indicated time-periods and then RT–PCR was performed to evaluate the levels of mRNA for Bax The housekeeping gene, b-actin, was used as an internal control for ensuring that an equal amount of template was used (B) and (C) QGY-7703 cells were exposed to TRAIL (10 ngỈmL)1) alone, etoposide (8 lgỈmL)1) alone, or TRAIL and etoposide together, for the indicated time-periods and the cell lysates were subject to Western blot analysis for detection of truncated Bid (tBid) or Bax that p53 was able to upregulate DR4 and DR5, but not Bax, in the human lung cancer cells cotreated with TRAIL and CD437 [38] Similarly, Kirsten also reported that the DNAdamaging agent, N-methyl-N¢-nitro-N-nitrosoguanidine (MNNG), did not affect the expression of Bax [42], implying that upregulation of Bax upon p53 activation may be cell-type dependent Recent studies indicated that cross-talk might exist between the two pathways by the Bid protein, a pro-apoptotic protein of the Bcl-2 family The BH3 domain of tBid (a cleaved form of Bid) is required to trigger Bax to release cytochrome c from mitochondria [43,44]; therefore, tBid is believed to be the linkage protein between the death receptor pathway and the mitochondrial pathway Kim and co-workers have demonstrated that TRAIL-induced translocation of Bax, subsequent to the cleavage of Bid, is vital in the TRAIL-induced mitochond- It is well established that mitochondria play a vital role in apoptosis and induce cell death by releasing cytochrome c [46,47] and Smac/DIABLO Our results also implied that the mitochondrial pathway is critical for contributing to the synergistic effect of TRAIL and etoposide in QGY7703 cells To test whether mitochondria are involved in the co-operative effect of TRAIL and etoposide, we treated QGY-7703 cells with and without etoposide (8 lgỈmL)1) in the presence or absence of TRAIL (10 ngỈmL)1) for 16 h to determine the release of cytochrome c and Smac/DIABLO As expected, little, if any, cytochrome c and Smac/DIABLO were detected in the cytoplasm when treated with TRAIL alone (Fig 5A), as there was no increase in the expression of Bax Treatment with etoposide alone (Fig 5A) effected some release of cytochrome c and Smac/DIABLO Furthermore, both cytochrome c and Smac/DIABLO showed a marked release from the mitochondria by combined treatment with TRAIL and etoposide (Fig 5A) Similarly to cytochrome c and Smac/DIABLO, the expression of Bax was also notably increased by cotreatment with TRAIL and etoposide compared with that of TRAIL or etoposide alone (Fig 5A) Smac/DIABLO promotes the activation of caspases, such as procaspase-9 and caspase-3, by binding to the inhibitor of apoptosis proteins and thus disrupts linkage of the caspase–inhibitor of apoptosis proteins complex during apoptosis [48–50] Recently, Deng et al reported that TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/ DIABLO [51] They have shown that the release of Smac/ DIABLO is required to remove the inhibitory effect of X-linked inhibitor of apoptosis protein (XIAP) and allow apoptosis to proceed, and thus mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis To our knowledge, ours is the first report to demonstrate that combined treatment of TRAIL and etoposide results in an enhanced release of Smac/DIABLO in the hepatocellular carcinoma cell system zVADFMK is the general caspase inhibitor and used to prevent caspase-dependent apoptosis We demonstrated that zVAD-FMK is able to prevent the early apoptosis induced by TRAIL plus etoposide (Fig 1C,e) To investigate whether the release of cytochrome c and Smac/DIABLO, 2728 L Miao et al (Eur J Biochem 270) Ó FEBS 2003 and Smac/DIABLO when compared with Bax expression in the absence of zVAD-FMK The exact mechanism underlying this discrepancy remains unclear Nevertheless, our results are similar to those reported by Adrain et al [52] They have proposed a model for explaining how the caspase inhibitor, zVAD-FMK, could inhibit the release of cytochrome c and Smac/DIABLO from mitochondria They claimed that release of Smac/DIABLO and cytochrome c requires downstream caspase activation Based on this model, the results shown in Fig 5B are plausible, as zVAD-FMK reduced the release of cytochrome c and Smac/DIABLO by inhibiting the downstream caspase activation Bax is upstream of cytochrome c and Smac/ DIABLO, therefore zVAD-FMK had little effect on its expression These results also confirmed that mitochondria might play a key role in contributing to the synergistic effect of TRAIL and etoposide in QGY-7703 cells Involvement of caspase activation in the enhancement of TRAIL-induced apoptosis by etoposide Caspases play key roles in apoptosis triggered by various pro-apoptotic signals To identify which caspase is involved in the process of apoptosis of QGY-7703 cells, and whether the activation of caspase is enhanced during the combined treatment with TRAIL and etoposide, QGY-7703 cells were incubated with TRAIL Fig Increased mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase (Smac)/DIABLO during the synergistic induction of apoptosis by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and etoposide (A) Cell lysates were isolated from QGY-7703 cells treated with etoposide (8 lgỈmL)1), with or without TRAIL (10 ngỈmL)1), for 16 h and assessed for Bax expression (cell lysate) and the contents of released cytochrome c and Smac/DIABLO (cytosolic fractions) by immunoblot analysis using respective antibodies Actin was used as the loading control (B) QGY7703 cells were exposed to the combination of etoposide (8 lgỈmL)1) and TRAIL (10 ngỈmL)1), with or without the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-FMK) (100 lM), for 16 h and both the cell lysate and the cytosolic fractions were subject to Western blot analysis for detection of the expression of Bax (cell lysate) and the release of cytochrome c and Smac/DIABLO (cytosolic fractions) Actin was used as the loading control +, treated; –, untreated or the expression of Bax, could also be inhibited by zVAD-FMK, QGY-7703 cells were exposed to TRAIL plus etoposide in the presence or absence of zVAD-FMK Whole-cell protein lysates or cytosolic protein fractions were subject to Western blot analysis As shown in Fig 5B, the release of cytochrome c and Smac/DIABLO were significantly decreased in the presence of zVADFMK However, the decrease of Bax in the presence of zVAD-FMK was not as marked as that of cytochrome c Fig The enhanced cleavage of caspases by combined treatment with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and etoposide Total cellular lysates were extracted from QGY-7703 cells treated with etoposide (8 lgỈmL)1), with or without TRAIL (10 ngỈmL)1), for 24 h Western blot analysis was performed to assess the processing of caspase-7, -9, -3, -8 and poly ADP-ribose polymerase (PARP) Human actin was used as the loading control +, treated; –, untreated Ó FEBS 2003 Augmented apoptosis induced by TRAIL and etoposide (Eur J Biochem 270) 2729 Fig Hypothetical model for illustrating the proposed apoptosis pathways in QGY-7703 cells cotreated with etoposide and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) The double line represents the cytoplasmic membrane The broken lines indicate that etoposide (or p53) has no effect on the expression of death receptor (DR)4, DR5, decoy receptor-2 (DcR-2) and Bid The mitochondrial pathway is symbolized by yellow arrows and the death receptor pathway by blue arrows The etoposide-activated mitochondrial pathway and the TRAIL-triggered DR pathway are converged on the mitochondria by the interactions of Bax with truncated Bid (tBid) which, in turn, amplifies the release of both cytochrome c and second mitochondria-derived activator of caspase (Smac)/DIABLO to activate downstream caspases, resulting in the augmented apoptosis observed in QGY-7703 hepatocellular carcinoma cells (10 ngỈmL)1) or etoposide (8 lgỈmL)1), or the combination of both, for 24 h (Fig 6) Etoposide alone and TRAIL alone slightly activated the initiators caspase-8 and -9, but their activation was much more enhanced when cells were cotreated with TRAIL plus etoposide Similar results were also obtained for effector caspases, caspase-3 and -7, as seen in Fig The activation of caspase-3 and -7 was further confirmed by the accelerated cleavage of PARP, a direct downstream substrate of caspase-3 and -7 As shown in Fig 6, cotreatment with TRAIL plus etoposide resulted in an enhanced cleavage of PARP compared to that obtained by treatment with either TRAIL or etoposide alone We noted that treatment with etoposide alone also resulted in some cleavage of procaspase-8; this observation has been documented previously [53,54], where it was concluded that caspase-8 can be processed by anticancer drugs, independently of death receptors In general, activation of the caspase cascade requires both initiator caspases such as caspase-8 and -9, and effector caspases, such as caspase-3 and -7 [55] The death ligands and the chemotherapeutic agents are two distinct classes of signals used to induce apoptosis and activate the caspase cascade Caspase-8 is known as the initiator caspase in the death receptor signal pathway, while caspase-9 is associated more with the mitochondrial pathway, which is activated by many chemotherapeutic drugs [56] Caspase-3 and -7 are the major effector caspases and act downstream of caspase-8 and -9 Our results have demonstrated that cotreatment with TRAIL and 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TRAIL on the liver carcinoma cells QGY-7703 To investigate the apoptotic function of TRAIL containing the extracellular domain (amino acids 114–281) of the human liver cancer cell line, QGY-7703. .. in the hepatocellular carcinoma cells, QGY-7703, we treated these cells with eight different agents, at increasing concentrations, with and without TRAIL, then analysed apoptosis using the MTT cell. .. Tumor necrosis factor-related apoptosisinducing ligand and chemotherapy cooperate to induce apoptosis in mesothelioma cell lines Am J Respir Cell Mol Biol 25, 111–118 Ó FEBS 2003 Augmented apoptosis