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Clinical Cancer Research Vol. 11, 3075-3083, April 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Role of Intracellular Glutathione in Cell Sensitivity to the Apoptosis Induced by Tumor Necrosis Factor {alpha}–Related Apoptosis-Inducing Ligand/Anticancer Drug Combinations

Olivier Meurette, Luz Lefeuvre-Orfila, Amélie Rebillard, Dominique Lagadic-Gossmann and Marie-Thérèse Dimanche-Boitrel

Authors' Affiliation: Institut National de la Santé et de la Recherche Médicale U620, Détoxication et Réparation Tissulaire, Faculté de Pharmacie, Université Rennes 1, Rennes, France

Requests for reprints: Marie-Thérèse Dimanche-Boitrel, Institut National de la Santé et de la Recherche Médicale U620, 2 Av du Pr Léon Bernard, 35043 Rennes Cedex, France. Phone: 33-2-2323-4837; Fax: 33-2-2323-4794; E-mail: marie-therese.boitrel{at}rennes.inserm.fr.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We have recently shown that combination of tumor necrosis factor {alpha}–related apoptosis-inducing ligand (TRAIL) with anticancer drugs induced an apoptotic cell death pathway involving both caspases and mitochondria. The present work further explores the role of intracellular reduced glutathione (GSH) level in cell sensitivity to this cell death pathway.

Experimental Design: Intracellular GSH level was measured by high-performance liquid chromatography. Cell death was detected by immunofluorescence after Hoechst 33342/propidium iodide staining. Reactive oxygen species production was evaluated by flow cytometry after dihydroethidium probe labeling. Western blot analysis was done to study stress-activated protein kinase/c-jun NH2-terminal kinase (SAPK/JNK) phosphorylation. The Student's t test was used to determine significance of the results. Three to six experiments were done.

Results: GSH depletion enhanced apoptosis induced by TRAIL/cisplatin (CDDP) or TRAIL/5-fluorouracil (5-FU) combinations in both human HT29 colon carcinoma and HepG2 hepatocarcinoma cells, whereas it enhanced cytotoxicity induced only by TRAIL/CDDP in human primary hepatocytes. Our results further suggested that GSH depletion enhanced SAPK/JNK phosphorylation upon TRAIL/5-FU exposure and likely reduced the detoxification mechanisms of CDDP in HT29 cells. Resistance of Bcl-2–expressing HT29 and HepG2 cells to combined treatment was not overcome by GSH depletion, thus indicating that Bcl-2–mediated antiapoptotic effect occurs independently of intracellular GSH level.

Conclusion: GSH depletion could be useful to increase the therapeutic efficacy of cancer treatment by TRAIL/anticancer drug combinations. Furthermore, TRAIL/5-FU combination might be a potential anticancer treatment of human tumors, being ineffective on human primary hepatocytes and thus could be of interest in clinical cancer treatment. Nevertheless, Bcl-2 expression remains an important resistance factor.

Key Words: tumor cells • GSH • Bcl-2 • TRAIL/5-FU • hepatocytes

Cisplatin (CDDP) and 5-fluorouracil (5-FU) are used in the treatment of many cancers (1, 2). However, resistance to these anticancer drugs represents a major drawback to their clinical effectiveness. A new approach to overcome cancer hemoresistance consists of triggering extrinsic death pathway by cytokines belonging to the tumor necrosis factor superfamily. In this way, tumor necrosis factor {alpha}–related apoptosis-inducing ligand (TRAIL) may be a potential anticancer agent, because it induces apoptosis in many cancer cells and not in normal cells (3). TRAIL administration in nude or severe combined immunodeficient mice reduced growth of colon carcinoma or mammary adenocarcinoma xenografts without any toxic effects (3, 4). In this context, TRAIL seems a promising cytokine for treating various carcinomas including colon cancers. Furthermore, the combination of TRAIL with a wide range of anticancer agents leads to a synergistic cytotoxic effect (5). However, overexpression of Bcl-2 can delay the chemotherapy-dependent potentiation of TRAIL-induced apoptosis in many cancer cells (6–8). Bcl-2 expression has been shown to interfere with the relocalization of cytochrome c from mitochondria to cytosol or with the binding of cytochrome c to Apaf-1, thus preventing the apoptosome formation (9). This points out the major antiapoptotic action of Bcl-2 on the mitochondria-dependent death pathway. Moreover, an antioxidant action of Bcl-2 has also been proposed (10–12).

Oxidative stress has been long recognized a common feature of apoptosis (13). Aerobic cells generate reactive oxygen species (ROS) that can affect numerous cell processes, ultimately leading to cell death under some circumstances. In this regard, ROS have been implicated in apoptosis (14) and more specifically in TRAIL-induced apoptosis (15). Mitogen-activated protein kinases (MAPK) are key proteins involved in signal transduction pathways in mammalian cells. Among them, extracellular signal–regulated kinases (ERK1/2) are generally related to cell proliferation, whereas stress-activated protein kinase/c-jun NH2-terminal kinase (SAPK/JNK) and p38 MAPK are mostly implicated in stress response and cell death (16). For example, oxidative stress activates MAPKs which may serve as a primary transducer of cell cytoplasmic oxidative signals to the nucleus where stress-related genes are induced (17, 18). Furthermore, p38 MAPK and SAPK/JNK have also been described as stimulated during TRAIL-induced apoptosis (19, 20) related or not to oxidative stress.

Among intracellular antioxidant molecules, reduced glutathione (GSH) is the most important thiol in cells, thereby protecting them from toxic oxygen products (21). Furthermore, GSH exhibits a large panel of actions in controlling gene expression, apoptosis mechanisms, or membrane transport (22). Especially, GSH has a prominent role in resistance to chemotherapy (23) and also to TRAIL-induced apoptosis (19). The antioxidant property of Bcl-2 could be related to GSH because Bcl-2 expression or overexpression increases GSH level in diverse cells (24–26). Moreover, Bcl-2 would also be capable of inducing GSH relocalization into the nucleus, thus protecting cells from DNA fragmentation (27). In favor of the hypothesis of a Bcl-2–conferred protection via GSH level regulation, GSH depletion in Bcl-2–expressing cells has been shown to restore apoptosis induction (25, 28).

Interest for clinical trials in modulating GSH level to increase tumor cell response to anticancer drug treatment is still growing (29). L-S,R-buthionine sulfoximine (BSO) is a potent specific inhibitor of {gamma}-glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis, and has been used to deplete intracellular GSH (30) and to reverse drug resistance in tumor cells (31, 32). GSH depletion has also been shown to activate p38 MAPK (33) or to inhibit Akt phosphorylation (34), which sensitizes cells to TRAIL-induced apoptosis, thus suggesting a role for glutathione level in this cell death pathway. Besides, a recent work has indicated that GSH depletion increased cell sensitivity to Fas-induced apoptosis (35), but the relation with MAPK transduction pathways was not investigated. Relationship between intracellular GSH level and activation of the MAPK pathways in death receptor-induced apoptosis still remains to be defined. Moreover, GSH depletion seems more efficient in cancer than in normal tissues thus allowing a specific targeting of cancers (36, 37).

We have previously shown that CDDP or 5-FU sensitized human colon cancer cells to TRAIL-induced apoptosis (6, 38). In the present study, we have modulated the intracellular GSH level by depletion/repletion in human tumor cells (HT29 colon cancer cells and HepG2 hepatocarcinoma cells) and normal human primary hepatocytes to study the role of this variable in cell sensitivity to combined treatment with TRAIL and CDDP or 5-FU. The involvement of stress-induced kinases, SAPK/JNK and p38 MAPK, and the relationship between GSH level and Bcl-2 expression, were also investigated. Our results indicate that GSH depletion could be useful to increase the therapeutic efficacy of cancer treatment by combining TRAIL with anticancer drugs. In the case of TRAIL/5-FU combination, an increased phosphorylation of JNK2 upon GSH depletion is related to increased cancer cell sensitivity, whereas in the case of TRAIL/CDDP combination, a decrease in detoxification mechanisms of CDDP is probably involved. Furthermore, TRAIL/5-FU combination might be less toxic than TRAIL/CDDP in human primary hepatocytes. Nevertheless, Bcl-2 expression remains an important resistant factor.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and antibodies. The cytotoxic drugs, cisplatin and 5-FU, were obtained from Merck (Lyon, France). The recombinant human soluble Flag-tagged TRAIL was from Alexis Biochemicals (Coger, Paris, France, http://www.alexis-corp.com). The anti-Flag M2, menadione, monobromobimane, propidium iodide, N-ethyl maleimide, DL-BSO, and glutathione monoethyl ester were from Sigma-Aldrich (Saint-Quentin Fallavier, France). The anti-Flag M2 was used to cross-link the ligand TRAIL leading to the formation of oligomers that are more effective to induce cell death. Briefly, 100 ng/mL of the soluble Flag-tagged TRAIL were incubated with 2 µg/mL anti-Flag M2 for 5 minutes at room temperature before cell treatment. When TRAIL was combined with anticancer drugs, TRAIL has also been crosslinked with anti-Flag M2. Dihydroethidium was from Molecular Probes (Eugene, OR). Mouse antiphosphophorylated JNK monoclonal IgG1 antibody and polyclonal rabbit anti–total JNK antibody were from Calbiochem (VWR International S.A.S., France). Mouse antihuman Bcl-2 IgG1 antibody was from Becton Dickinson (Le Pont de Claix, France). Mouse anti–heat shock protein constitutive 70 (HSC70) IgG1 antibody was from Santa Cruz (Tebu, Le Perray en Yvelines, France). p38 MAPK inhibitor (SB 203580) was from Calbiochem and JNK inhibitor (L-i-JNK) was from Alexis Biochemicals.

Cell lines and apoptotic assay. The HT29 human colon carcinoma cell line and the HepG2 human hepatocarcinoma cell line were obtained from American Type Culture Collection (Rockville, MD, Etats Unis) and cultured in Eagle's MEM (Eurobio, Les Ulis, France) or Williams' E medium (Life Technologies, Cergy Pontoise, France), respectively, supplemented with 10% (v/v) FCS (Life Technologies), glutamine (2 mmol/L; HT29 and HepG2), and bovine serum albumin (0.1 mg/mL), bovine insulin (1 µg/mL), and hydrocortisone (0.25 µg/mL; HepG2) at 37°C under a 5% CO2 atmosphere. The HT29 neo/Bcl-2 cells were previously described (6). The HepG2 neo/Bcl-2 cells were a kind gift of Dr. P. Despres (Institut Pasteur, Paris, France).

Microscopic detection of apoptosis and necrosis was carried out in both floating and adherent cells recovered after treatment using nuclear chromatin staining with 1 µg/mL Hoechst 33342 and 1 µg/mL propidium iodide for 15 minutes at 37°C. Cells with apoptotic nuclei (i.e., condensed or fragmented) were counted in comparison with total population (n = 300 cells). Under our experimental conditions, we never detected red necrotic cells.

Human primary hepatocyte cultures and cytotoxic assay. Human hepatocytes from adult donors undergoing resection for primary and secondary tumors were obtained by perfusion using a collagenase solution as described previously (39). Cells were seeded at a density of 3 x 104 cells/cm2 in Williams' E medium supplemented with glutamine (2 mmol/L), bovine serum albumin (0.2 mg/mL), bovine insulin (10 µg/mL), and 10% (v/v) FCS. The medium was discarded 24 hours after cell seeding and hepatocytes were thereafter maintained in serum-free medium supplemented with hydrocortisone (10–7 mol/L). Hepatocytes from six adult donors were isolated and treatments were done in triplicate under the same conditions as described above for human cancer cell lines.

Cell viability was assessed by a methylene blue colorimetric assay as previously described (40). Briefly, hepatocytes were seeded in 96-well flat-bottomed plates at a density of 30,000 cells per well. After treatment, cells were washed thrice in PBS and fixed for 30 minutes in 95% ethanol. Following removal of ethanol, fixed cells were dried and colored for 5 minutes in methylene blue. After three washes in tap water, 100 µL of 0.1 N HCl per well were added. Plates were analyzed with a spectrometer at 620 nm.

Western blot analysis. Cells were sonicated in radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl, 0.1% (v/v) SDS, 0.5% (v/v) sodium deoxycholate, 100 µmol/L paramethylsulfonide, 1 µg/mL pepstatin, 2 µg/mL leupeptin] at 4°C and boiled for 3 minutes. Proteins (50 µg) were separated on a polyacrylamide sodium dodecylsulfate-containing gel and transferred to a nitrocellulose membrane (Amersham, Orsay, France). After blocking nonspecific binding sites for 1 hour at room temperature by 8% (w/v) skimmed milk in TTBS [TBS with 0.1% (v/v) Tween 20], membranes were incubated for 2 hours with mouse anti–phosphorylated JNK, polyclonal rabbit anti–total JNK, mouse antihuman Bcl-2, or mouse anti-HSC70 antibodies. Membranes were then washed twice with TTBS and incubated for 1 hour with peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies. Revelation was done by chemiluminescence.

Measurement of total intracellular glutathione level by high-performance liquid chromatography. GSH measurement was done by high-performance liquid chromatography method modified from Reed et al. (41). Briefly, cells (2 x 106) were sonicated in 0.1 N HCl, 1 mmol/L EDTA and centrifuged at 10,000 rpm for 15 minutes. The supernatant was then neutralized by adding an equal volume of 0.1 N NaOH and buffered with 0.1 mol/L Tris-HCl, 1 mmol/L EDTA. Then, 0.1 mmol/L DTT was added to reduce the oxidized form and left to react 1 hour at room temperature. GSH was finally conjugated with monobromobimane for 15 minutes at room temperature. The reaction was blocked by addition of acetate 5%. Fifty microliters of samples were injected in a silice uptisphere 5-µm column (Interchim, Montluçon, France). The flow was 1 mL min–1 with an aqueous phase of 0.1 mmol/L K+ acetate (pH 5.5) and an organic phase of acetonitrile (0-20% in 15 minutes then 20-100% until 18 minutes). A Bradford assay was used to determine the protein concentration and the GSH amount was expressed in function of the protein concentration. The results were expressed as percentage of the untreated or control cells.

Measurement of intracellular superoxide anion by flow cytometry. Dihydroethidium was used to detect intracellular superoxide anion production. After treatment, floating and adherent cells (0.5 x 106 cells) were recovered and incubated in medium without FCS containing 5 µmol/L DHE for 15 minutes at 37°C. Dye oxidation (increase in FL-2 fluorescence) was measured using a Becton Dickinson FACScan flow cytometer with excitation and emission settings of 488 and 530 nm, respectively. A positive control was obtained by incubating cells with menadione (1 mmol/L). Superposition of control and menadione histograms allowed to define a gate for calculating the percentage of cells producing superoxide anion (indicated on each histogram).

Statistical analysis. The statistical analyses were carried out using the unilateral Student's t test considering the variances as unequal. The significance is shown as follows: *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.01.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Combined treatment of tumor necrosis factor {alpha}–related apoptosis-inducing ligand with anticancer drugs induces apoptosis and superoxide anion production in HT29 and HepG2 cells. We have tested the ability of the alkylating agent cisplatin and the antimetabolite 5-FU to increase TRAIL-induced apoptosis in both human HT29 colon cancer and HepG2 hepatocarcinoma cell lines, shown to be constitutively TRAIL resistant (see Fig. 1A). HepG2 cells being more sensitive to CDDP than HT29 cells, we used a lower concentration of CDDP (2 µg/mL) in combination with TRAIL. A 24-hour treatment with CDDP, 5-FU, or TRAIL elicited only limited apoptotic effect towards both cell lines (<10% of apoptotic cells detected by Hoechst staining; Fig. 1A). However, TRAIL-induced apoptosis was significantly enhanced by cotreatment with CDDP or 5-FU (40-60% of apoptotic cells; Fig. 1A). This increase in cell death was not accompanied by necrosis because no propidium iodide staining was observed in cotreated cells (data not shown). We analyzed superoxide anion production by staining cells with the dihydroethidium probe after combined treatment of TRAIL with chemotherapeutic agents. Treatment with CDDP, 5-FU, or TRAIL for 24 hours weakly increased superoxide anion production (<18% positive cells in HT29 and <8% positive cells in HepG2; Fig. 1B). However, combined treatment with TRAIL and CDDP or 5-FU increased such a production in both HT29 (25-30% positive cells) and HepG2 cells (18-22% positive cells; Fig. 1B). Note that this increase in superoxide anion production was only additive between TRAIL and the anticancer drugs, whereas the increase in apoptotic effect was synergistic, thus suggesting a partial contribution of ROS production in this cell death pathway.



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  		Fig. 1. Combined treatment with TRAIL and cisplatin or 5-FU induces apoptosis and uperoxide anion production in HT29 and HepG2 cells. A, cells were exposed to CDDP (2 µg/mL for HepG2 or 5 µg/mL for HT29 cells), 5-FU (100 µg/mL), TRAIL (100 ng/mL cross-linked with 2 µg/mL anti-Flag M2) or a combination of CDDP/TRAIL (C + T) or 5-FU/TRAIL (F + T) for 24 hours before identifying apoptotic cells by staining nuclear chromatin with Hoechst 33342; 300 cells per point were counted. Columns, mean (n = 3); bars, ±SD. B, superoxide anion production was studied by flow cytometry in HT29 and HepG2 cells after the same treatments as described above and after staining cells with dihydroethidium. An increase in FL-2 fluorescence indicates superoxide anion production. Numbers indicate the percentage of cells producing superoxide anion. One representative of three independent experiments.

 
Depletion of reduced glutathione level sensitizes HT29 and HepG2 cells towards combined treatment with tumor necrosis factor {alpha}–related apoptosis-inducing ligand and anticancer drugs. We hypothesized that modulation of GSH level would modify the cell response to apoptosis induced by combined treatment with TRAIL and CDDP or 5-FU. Following a 4-hour treatment with BSO, the intracellular GSH level was reduced by >60% and 50% in HT29 and HepG2 cells, respectively (Fig. 2A). In addition, we used GSH monoethyl ester to replete intracellular GSH level after BSO treatment. Following a 3-hour GSH monoethyl ester treatment (BSO + GSH), an ~4- to 5-fold increase in intracellular GSH level was detected in both cell lines (Fig. 2A).



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  		Fig. 2. GSH depletion increases both apoptosis and superoxide anion production after combined treatment with TRAIL and CDDP or 5-FU in HT29 and HepG2 cells. A, GSH level was measured by high-performance liquid chromatography in HT29 and HepG2 cells after a 4-hour BSO (0.2 mmol/L) treatment (BSO) followed or not by a 3-hour glutathione monoethyl ester incubation (10 mmol/L; BSO + GSH). The values are expressed as percentage of GSH level measured in untreated cells (NT). Columns, mean (n = 3); bars, ±SD. *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.01. B, cells are treated as in Fig. 1 after GSH depletion (BSO) or repletion (BSO + GSH) and percentage of apoptotic cells was estimated as in Fig. 1. Columns, mean (n = 3); bars, ±SD. *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.01. C, superoxide anion production was analysed as in Fig. 1 in HT29 cells after similar treatments as above. Numbers indicate the percentage of cells producing superoxide anion. One representative of three independent experiments.

 
BSO treatment increased both apoptosis induction (10-20% more apoptotic cells; Fig. 2B) and superoxide anion production (37-41% positive cells; Fig. 2C) in HT29 and HepG2 cells cotreated with TRAIL and CDDP or 5-FU. Under such experimental conditions, the resulting cell death was mainly through apoptosis because we did not observe any necrosis (no increase in propidium iodide staining, data not shown). In contrast, a GSH monoethyl ester treatment induced a drastic inhibition of apoptosis induced by combined treatment with TRAIL and CDDP and reversed the effect of BSO on apoptosis induced by TRAIL/5-FU combination in both HT29 and HepG2 cells (Fig. 2B). According to this, a complete inhibition of superoxide anion production was only observed in HT29 cells cotreated with TRAIL and CDDP in the presence of GSH monoethyl ester (13.8% positive cells, Fig. 2C). Moreover, GSH repletion reversed only the effect of BSO on superoxide anion production following TRAIL/5-FU treatment (Fig. 2C).

These data show that decreasing cell ability to respond to an oxidative stress by GSH depletion results in an increase in apoptosis induced by both combinations. Furthermore, intracellular GSH level is a resistant factor only for combined treatment of TRAIL with CDDP. In this context, combination of TRAIL with 5-FU might thus be efficient in tumors with high intracellular GSH level and of interest for clinical cancer treatment.

Activation of stress-activated protein kinase/c-jun NH2-terminal kinase is enhanced by reduced glutathione depletion upon tumor necrosis factor {alpha}–related apoptosis-inducing ligand/5-fluorouracil treatment. We next studied the role of SAPK/JNK and p38 MAPK in apoptosis induced by TRAIL/anticancer agent upon GSH depletion. Using SB 203580, an inhibitor of p38 MAPK, and L-i-JNK, an inhibitor of JNK, we showed that p38 was not implicated in the potentiation of TRAIL/anticancer drug–induced apoptosis by BSO treatment, whereas JNK activation was involved in the effect of BSO on combined treatment of TRAIL with 5-FU. In fact, a 1-hour pretreatment of HT29 cells with 1 µmol/L L-i-JNK completely reversed the effect of BSO on the apoptosis induced by TRAIL/5-FU combination, whereas it had no effect on the apoptosis induced by TRAIL/CDDP combination after GSH depletion (Fig. 3A). In contrast, a 1-hour pretreatment of HT29 cells with 10 µmol/L SB 203580 remained ineffective whatever the combinations tested after GSH depletion (data not shown).



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  		Fig. 3. Activation of SAPK/JNK is enhanced by GSH depletion upon TRAIL/5-FU treatment. A, HT29 cells were exposed to CDDP (5 µg/mL), 5-FU (100 µg/mL), TRAIL (100 ng/mL cross-linked with 2 µg/mL anti-Flag M2), or a combination of CDDP/TRAIL (C + T) or 5-FU/TRAIL (F + T) for 16 hours after a 1-hour pretreatment or not with 1 µmol/L L-i-JNK. Percentage of apoptotic cells was estimated as in Fig. 1. Columns, mean (n = 3); bars, ±SD. *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.01. B, cells were treated as described above and western blot analysis of phospho-JNK2 (P-JNK2) was done as described in Materials and Methods. Antihuman HSC70 antibody was used as a control of protein loading. C, cells were treated with TRAIL/5-FU combination for 4, 8, or 24 hours after GSH depletion (BSO) or not, and Western blot analysis of phospho-JNK2 (P-JNK2) or of total JNK (JNK tot) was done as described in Materials and Methods. Anti-human HSC70 antibody was used as a control of protein loading.

 
Western blot analysis showed that JNK2 was phosphorylated in HT29 treated for 16 hours with TRAIL alone (Fig. 3B). The combination of TRAIL with CDDP or 5-FU did not increase JNK phosphorylation (Fig. 3B). However, BSO treatment enhanced phosphorylation of JNK2 after treatment of HT29 cells by TRAIL/5-FU combination but not by TRAIL/CDDP combination (Fig. 3B). As phosphorylation of JNK2 was increased after a 16-hour treatment with TRAIL/5-FU in presence of BSO, we did kinetics of JNK2 phosphorylation after TRAIL/5-FU treatment in presence or not of BSO (Fig. 3C). We observed a marked increase in JNK2 phosphorylation as early as 4 hours after TRAIL/5-FU treatment in presence of BSO when compared with JNK2 phosphorylation observed after a similar treatment but in absence of BSO (Fig. 3C). This enhanced JNK2 phosphorylation persisted at 8 and 24 hours after treatment with TRAIL/5-FU in presence of BSO (Fig. 3C). Taken together, our data suggest that depending on the anticancer drug used in combination with TRAIL, GSH depletion triggered different cell signaling pathways that enhance the apoptotic effect.

Combined treatment of tumor necrosis factor {alpha}–related apoptosis-inducing ligand with 5-fluorouracil is not cytotoxic to human primary hepatocytes even after reduced glutathione depletion. We then studied the sensitivity of human primary hepatocytes to compare normal cells versus tumor cells. We showed that TRAIL alone was not cytotoxic to human primary hepatocytes for concentrations ranging from 12.5 to 100 ng/mL (Fig. 4A). On contrary, combined treatment with TRAIL and CDDP induced cytotoxicity that was significantly enhanced by a 4-hour BSO treatment (Fig. 4A). Interestingly, combined treatment with TRAIL and 5-FU was not cytotoxic to human primary hepatocytes and was not enhanced by BSO treatment (Fig. 4A), even at higher concentrations of TRAIL (200 ng/mL) or 5-FU (200 and 400 µg/mL; data not shown). These results suggest that TRAIL sensitization of human primary hepatocytes is dependent on the anticancer agent used. Furthermore, we showed that BSO treatment induced a 40% decrease in intracellular GSH level (Fig. 4B), which is less than what we have observed in HT29 or HepG2 cells (Fig. 4A), thus showing that GSH depletion induced by BSO could be more efficient in cancer cells.



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  		Fig. 4. Combination of TRAIL/5-FU is not cytotoxic towards human primary hepatocytes even after GSH depletion. A, cell viability was estimated by a methylene blue colorimetric assay after treatment with increased concentrations of TRAIL (12.5, 25, 50, and 100 ng/mL cross-linked with 2 µg/mL anti-Flag M2) in combination with CDDP (5 µg/mL) or 5-FU (100 µg/mL) following a 4-hour BSO treatment (BSO) or not. Points, mean (n = 6); bars, ±SD. *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.01. B, GSH level was measured by HPLC in hepatocytes from six donors following a 4-hour BSO (0.2 mmol/L) treatment (BSO) or not. Columns, mean (n = 6); bars, ±SD. *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.01.

 
Bcl-2 expression inhibits both apoptosis and superoxide anion production induced by combined treatment with tumor necrosis factor {alpha}–related apoptosis-inducing ligand and CDDP or 5-fluorouracil. In the following set of experiments, we analyzed Bcl-2 expression in stable Bcl-2 transfected HT29 and HepG2 cells by Western blotting. As shown in Fig. 4A, Bcl-2 protein was not constitutively expressed in HT29 or HepG2 cells transfected with an empty vector (neo) whereas detected in both HT29 and HepG2 cells transfected with a human Bcl-2 coding vector (Bcl-2). Inhibition of both apoptosis and superoxide anion production following a 24-hour treatment with TRAIL and CDDP or 5-FU was observed in Bcl-2–expressing HT29 and HepG2 cells (Fig. 5B and C). All these data show that Bcl-2 expression is a resistance factor to TRAIL/anticancer drug combinations in both tumor cell types.



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  		Fig. 5. Bcl-2 expression inhibits both apoptosis and superoxide anion production induced by combined treatment with TRAIL and 5-FU in HT29 and HepG2 cells. A, Western blot analysis of Bcl-2 expression was done in HT29 neo/Bcl-2 and HepG2 neo/Bcl-2 clones. An antihuman HSC70 antibody was used as a control of protein loading. B, cells are treated as in Fig. 1 and percentage of apoptotic cells was estimated as in Fig. 1. Columns, mean (n = 3); bars, ±SD. C, superoxide anion production was analyzed as in Fig. 1 in HT29 neo/Bcl-2 and in HepG2 neo/Bcl-2 clones after treatments as described above. Numbers indicate the percentage of cells producing superoxide anion. One representative of three independent experiments.

 
Intracellular reduced glutathione level is increased in Bcl-2–expressing HT29 cells but not in HepG2 cells. To further test a link between Bcl-2 expression and GSH level, we analyzed the effect of Bcl-2 expression on intracellular GSH level in both Bcl-2–transfected HT29 and HepG2 cell lines. A 2-fold increase in GSH level was observed in Bcl-2–expressing HT29 cells compared with HT29 cells transfected with an empty vector (neo; Fig. 6). Surprisingly, no change was observed in Bcl-2–expressing HepG2 cells (Fig. 6), although these cells expressed the highest Bcl-2 protein level (Fig. 5A), suggesting that the effect of Bcl-2 expression on intracellular GSH level is cell type dependent.



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  		Fig. 6. Bcl-2 expression increases intracellular GSH level in HT29 cells but not in HepG2 cells. GSH level was measured by HPLC. Percentage of GSH level measured in control neo cells. Columns, mean (n = 3); bars, ±SD.

 
Depletion of reduced glutathione level does not overcome resistance of Bcl-2–expressing cells. We tested the effect of GSH depletion in Bcl-2–expressing cells. Whereas GSH level was initially higher in Bcl-2–expressing HT29 cells, BSO treatment induced GSH depletion to the same extent in Bcl-2–expressing cells as in cells transfected with an empty vector (neo; Fig. 7A). In HepG2 cells, BSO treatment induced similar GSH depletion in both Bcl-2– and neo-transfected cells (Fig. 7A). In Bcl-2–expressing cells, BSO-induced GSH depletion did not reverse resistance of both HT29 or HepG2 cells to combined treatment with TRAIL and CDDP or 5-FU (Fig. 7B). These data further imply Bcl-2 as exerting its antiapoptotic effect on combined treatment with TRAIL and anticancer agents independently of intracellular GSH level.



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  		Fig. 7. GSH depletion cannot overcome resistance of Bcl-2 expressing cells. A, intracellular GSH level was measured by HPLC in HT29 neo/Bcl-2 and HepG2 neo/Bcl-2 clones after a 4-hour BSO (0.2 mmol/L) treatment (BSO) or not. Percentage of GSH level of untreated neo cells. Columns, mean (n = 3); bars, ±SD. B, cells are treated as in Fig. 1 after GSH depletion (BSO) or not and percentage of apoptotic cells was estimated as in Fig. 1. Columns, mean (n = 3); bars, ±SD. *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.01.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Reactive oxygen species are now thought to be involved in several cellular mechanisms including apoptosis (14). A production of hydrogen peroxide has been observed in HeLa cells during TRAIL-induced apoptosis (15). We described here a production of superoxide anion during apoptosis induced by combined treatment with TRAIL and cisplatin or 5-FU in HT29 colon cancer and HepG2 hepatocarcinoma cells. This ROS production was completely inhibited by Bcl-2 expression that also blocked apoptosis induction in both cellular systems, thus suggesting a contribution of ROS in the mitochondrial apoptotic pathway amplification induced by TRAIL/anticancer drug combinations (6).

GSH has for long been known a chemoresistance factor in cancer cells (42). To test whether GSH was a resistance factor towards combined treatment with TRAIL and cisplatin or 5-FU, we used GSH monoethyl ester. A 4- to 5-fold increase of GSH level completely inhibited apoptosis induced by combined treatment with TRAIL and cisplatin but not with TRAIL and 5-FU in HT29 and HepG2 cells. Whereas a role for GSH in resistance to platinum compounds has been well described (43), such a role in resistance to 5-FU is still a matter of debate (44, 45). In fact, the detoxification of cisplatin by GSH via nonenzymatic or enzymatic reaction with GSH S-transferases has been shown (46). This could explain why in both HT29 colon cancer and HepG2 hepatocarcinoma cells apoptosis induced by combined treatment with TRAIL and CDDP was completely inhibited upon GSH repletion, in contrast to that induced by combined treatment with TRAIL and 5-FU. In the same order, the fact that an overall decrease in detoxication mechanisms of CDDP is likely to occur upon GSH depletion could explain the higher sensitivity of cancer cells to TRAIL/CDDP combination. In this context, combination of TRAIL with 5-FU might therefore be more suitable to treat tumors with high level of intracellular GSH. Regarding this latter point, tumors often exhibit a higher GSH level than normal tissues, and GSH depletion could consequently help overcoming chemoresistance. In line with this, several evidences exist to show that GSH depletion by BSO is efficient for increasing sensitivity of resistant cells to platinum-containing compound, alkylating agents, anthracyclins, and arsenic trioxide (29, 47). To our knowledge, GSH depletion had never been tested on combined treatment of TRAIL with chemotherapeutic agents. We showed here that BSO treatment increases apoptosis induced by combined treatment with TRAIL and cisplatin or 5-FU and might thus be of interest in clinical cancer treatment.

Furthermore, the molecular mechanisms involved in apoptosis induced by TRAIL/ anticancer drug treatment upon GSH depletion seem to depend on the anticancer agent used. Indeed, we showed here that JNK2 activation was necessary for the increase in apoptosis induced by TRAIL/5-FU treatment upon GSH depletion in HT29 cells, whereas no such implication was observed when using TRAIL/CDDP/BSO treatment. It is worth noting that TRAIL alone induced JNK2 phosphorylation that was not modified by cotreatment with CDDP or 5-FU. Despite this phosphorylation, apoptosis induced by TRAIL/anticancer drug combination remained unaffected by JNK inhibitor, thus suggesting that JNK2 activation did not play a major role in this cell death pathway. However, JNK inhibitor was effective to reverse the effect of BSO only on apoptosis induced by TRAIL/5-FU combination, showing that the enhanced apoptosis induced by GSH depletion had different molecular mechanisms depending on the anticancer agent used in combination with TRAIL. As stated above, regarding CDDP, we assume that an overall decrease in detoxification mechanisms could be responsible for increased apoptosis induced by TRAIL/CDDP upon GSH depletion. On the whole, our data suggest that such a therapeutic approach might be a useful tool to overcome resistance because BSO treatment could have pleiotropic effects depending on the anticancer drug used.

However, the possible use of TRAIL in clinical cancer therapy raises the problem of toxicity in normal tissues which is not clear today. In this way, we tested the sensitivity of human primary hepatocytes to combined treatment with TRAIL and CDDP or 5-FU with or without BSO treatment. We showed that TRAIL alone is not cytotoxic towards human primary hepatocytes. It has recently been shown that normal hepatocytes could respond to TRAIL-R1 and TRAIL-R2 signal (48). Besides, different types of recombinant TRAIL protein have been previously found to induce different toxicities to human primary hepatocytes (49). We show here that the recombinant protein constituted of the extracellular domain of human TRAIL fused at the NH2 terminus to a FLAG-tag and an 8-amino-acid linker peptide from Alexis Biochemicals is not hepatotoxic. Nevertheless, combined treatment with TRAIL and cisplatin became highly cytotoxic towards human primary hepatocytes, whereas combined treatment with TRAIL and 5-FU was not, showing that TRAIL sensitization is dependent on the anticancer agent used. It would be of great interest to understand this discrepancy and characterize the underlying molecular mechanisms. Furthermore, GSH depletion induced by BSO was found to be less efficient in human primary hepatocytes, sensitizing hepatocytes only to combined treatment with TRAIL and cisplatin. This latter fact is consistent with the observation that BSO treatment would be more efficient to induce GSH depletion in tumor than in normal tissues (36, 37). BSO has also been used to overcome Bcl-2–conferred resistance to different stimuli such as cisplatin (28) or radiation (25). We showed here that GSH depletion in Bcl-2–expressing cells did not restore cell sensitivity to TRAIL/CDDP or TRAIL/5-FU confirming that Bcl-2 has a pleiotropic antiapoptotic effect.

Together, the data provide new and interesting information on the role of intracellular GSH in cell sensitivity to the apoptosis induced by TRAIL/anticancer drug combinations. First, GSH depletion could be useful to increase the therapeutic efficacy of cancer treatment combining TRAIL with anticancer agents. Second, TRAIL/5-FU combination seems insensitive to high intracellular GSH level and not cytotoxic towards human primary hepatocytes, even after GSH depletion. Third, the treatment combining TRAIL with CDDP kills normal primary hepatocytes suggesting that depending on the anticancer used, TRAIL-based therapy could become cytotoxic towards normal cells. Preclinical studies done in rodents and primates predict safe and efficient use of TRAIL in vivo (3, 4). At present, humanized monoclonal antibodies directed against TRAIL-R1 or TRAIL-R2 are tested in phase I of clinical assays by Human Genome Sciences, Inc. (Rockville, MD). However, according to our present data, in human tumors overexpressing Bcl-2, the anticancer therapeutic strategy using TRAIL death pathway may be less promising than expected.


   Acknowledgments
 
We thank the Biological Resource Centre of Rennes for the supply of isolated human primary hepatocytes; Dr. P. Despres for the kind gift of HepG2 neo/Bcl-2 cells; G. Le Moigne for technical assistance; Dr. S. Langouët for helpful advice on intracellular GSH measurement by high-performance liquid chromatography; and Prof. A. Guillouzo, Dr. O. Sergent, and Dr. D. Gilot for helpful discussions on the article.


   Footnotes
 
Grant support: Ligue Nationale Contre le Cancer (Morbihan, Côte d'Armor, and Ille et Vilaine Committees), Rennes Métropole, and Région Bretagne.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/31/04; revised 1/ 6/05; accepted 1/24/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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