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Division of Hematology/Medical Oncology [D. D., Y. X. X., X. G., N. J., R. A. C., S. C. G.], and William T. Gossett Neurology Laboratories [H. J.], Department of Neurology, Henry Ford Health System, Detroit, Michigan 48202
2 To whom requests for reprints should be addressed, at Oncology Research Laboratory, 4D, Henry Ford Health System, One Ford Place, Detroit, MI 48202. Phone: (313) 874-6998; Fax: (313) 874-3770; E-mail: sgotam{at}msn.com
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Abstract |
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15%) in LNCaP cells, as measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium dye reduction assay. On the other hand, cell death was markedly enhanced (2–3-fold) if tumor cells were treated with curcumin and TRAIL together. The combined curcumin and TRAIL treatment increased the number of hypodiploid cells and induced DNA fragmentation in LNCaP cells. The combined treatment induced cleavage of procaspase-3, procaspase-8, and procaspase-9, truncation of Bid, and release of cytochrome c from the mitochondria, indicating that both the extrinsic (receptor-mediated) and intrinsic (chemical-induced) pathways of apoptosis are triggered in prostate cancer cells treated with a combination of curcumin and TRAIL. These results define a potential use of curcumin to sensitize prostate cancer cells for TRAIL-mediated immunotherapy. |
Introduction |
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Failure to undergo programmed cell death (apoptosis) has been implicated in tumor development and resistance to cancer therapy (6, 7). Promotion of apoptosis in prostate cancer cells may lead to the regression and improved prognosis of refractory disease. Indeed, prostate cancer cells have been shown to remain somewhat sensitive to apoptosis induced by chemotherapeutic agents (8, 9) and members of the TNF3-
superfamily of death inducing ligands, including TNF- and FasL (Apo-1L or CD95L; Refs. 10–12). However, the use of these apoptosis-inducing drugs and cytokines is limited by unacceptable systemic toxicity. TRAIL (Apo-2L), the most recently identified member of the TNF family of apoptosis-inducing ligands, has been shown to induce apoptosis in various cancer cell types in vitro with little or no cytotoxicity to the normal cells (13–15). Furthermore, unlike TNF- and FasL (16–18), TRAIL exhibits antitumor activity in vivo without systemic toxicity (19). Thus, induction of cell death by TRAIL might be an attractive new treatment option for advanced prostate cancer.
In contrast to the high incidence of prostate cancer in North America, the incidence of this disease in Japan and China is very low (20, 21). The low incidence of prostate cancer in Asian men has been attributed to the dietary consumption of large amounts of plant-based foods rich in phytochemicals (22–24). Because of these observations, nutritional supplements such as soybean, garlic, green tea, and so on, which are rich in polyphenolic compounds, have been used to augment the anticancer therapies (25, 26). Curcumin, the active component of turmeric, and resveratrol (3,5,4'-trihydroxystilbene), found in grapes, are two such dietary constituents that have received a great deal of attention recently as chemoprotective agents (27–29). We have previously shown that both of these phytochemicals inhibitproliferation and induce apoptosis in human and mouse leukemia cell lines (30, 31). Whether these agents also chemosensitize tumor cells to the apoptosis-inducing effects of cytokines has not been explored. In this report, we demonstrate that curcumin enhances the TRAIL-induced apoptosis in human prostate cancer cell line LNCaP. No such cooperation was observed between resveratrol and TRAIL or curcumin and TNF-. Cell death after combined treatment with curcumin and TRAIL correlated with DNA fragmentation and activation of initiator caspase-8 and caspase-9 and effector caspase-3. The combined curcumin/TRAIL treatment also induced the truncation of proapoptotic Bid and release of cytochrome c from the mitochondria.
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Materials and Methods |
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was purchased from Pepro Tech Inc. (Rocky Hill, NJ). A 100-mM solution of resveratrol or curcumin was prepared in DMSO, and all test concentrations were prepared by diluting the stock solution in tissue culture medium.
Cell Lines.
LNCaP cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 (Life Technologies, Inc., Rockville, MD) supplemented with 10% FCS (Hyclone, Logan, UT), 1% penicillin/streptomycin, 1 µg/ml hydrocortisone, and 100 nM testosterone as described previously (32). DU145 and NIH 3T3 cells (American Type Culture Collection) were grown in DMEM (Life Technologies, Inc., Rockville, MD) supplemented with 10% FCS (Hyclone, Logan, UT) and 1% penicillin/streptomycin. The mouse bladder tumor cell line MBT-2 obtained from Dr. James A. Hampton (Medical College of Ohio, Toledo, OH) was grown in RPMI 1640 as described for LNCaP cells but without hydrocortisone and testosterone. All cell lines were cultured at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air.
Measurement of Cell Viability.
Cells were seeded into 96-well plates at a density of 2 x 104 cells/well in 100-µl tissue culture medium in triplicate. After 24-h incubation to allow cells to adhere, cells were treated for 48 h either with curcumin, resveratrol, TRAIL, or TNF- separately or in different combinations, as described in individual experiments. Cell viability was determined by the colorimetric MTS using CellTiter 96 AQueous Assay System from Promega (Madison, WI). In this assay, the quantity of formazan product formed is directly proportional to the number of viable cells in the cultures.
Flow Cytometry.
To study the expression of TRAIL-R, LNCaP cells (106 cells) were pretreated with Fc block (anti-CD16/CD32) and reacted with primary goat anti-TRAIL-R1, TRAIL-R2, TRAIL-R3, or TRAIL-R4 antibodies (50 µg/ml) for 45 min on ice. Cells were washed once with PBS and were then incubated with phycoerythrin-conjugated donkey antigoat secondary antibody (1:50 dilution) for 45 min at 4°C. Cells were washed and analyzed by FACS on a FACScan flow cytometer (Becton Dickinson).
DNA Fragmentation.
To measure DNA fragmentation, 6 x 106 untreated or treated LNCaP cells were washed twice with 1x PBS and then lysed in cold lysis solution [5 mM of Tris (pH 7.4), 20 mM of EDTA, 0.5% Triton X-100) for 20 min. Cell lysates were centrifuged at 27,000 x g for 15 min, and DNA was extracted from the aqueous phase with phenol:chloroform:isoamyl alcohol (25:24:1, by volume) containing 0.1% hydroxyquinoline. DNA was precipitated with 0.3 M sodium acetate and 2 volumes of cold 100% ethanol. DNA samples isolated from equal number of untreated and treated cells were fractionated on 1.2% agarose gel in Tris-acetate (40m M Tris-acetate, 1 mM EDTA) electrophoresis buffer. After fractionation of DNA, gels were treated with RNase (20 µg/ml) for 3 h and stained with ethidium bromide.
Western Blot Analysis.
Total cellular proteins were isolated by detergent lysis (1% Triton X-100, 10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 10% glycerol, 2 mM sodium vanadate, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatinin, and 10 µg/ml 4–2-aminoethyl-benzenesulfinyl fluoride) as described by Keane et al. (33). Lysates were clarified by centrifugation at 14,000 x g for 10 min at 4°C, and protein concentration was determined by Bio-Rad colorimetric assay (Bio-Rad, Hercules, CA). Samples (50 µg) were boiled in an equal volume of sample buffer (20% glycerol, 4% SDS, 0.2% Bromphenol Blue, 125 mM Tris-HCl, and 640 mM ß-mercaptoethanol) and separated on 10% SDS-polyacrylamide gels. Proteins resolved on the gels were transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% milk in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl with 0.05% Tween 20 (TPBS) and probed with antibodies to caspase-3, caspase-8, caspase-9, Bid, cytochrome c, or actin. Immune complexes were visualized with enhanced chemiluminescence detection system (Amersham Corp., Arlington Heights, IL).
Measurement of Caspase-3 Activity.
LNCaP cells were grown to confluence in 100-mm diameter culture dishes and treated with test agents for 20 h. Cell lysates were prepared using the lysis buffer included in the ApoAlert Caspase Assay kit (Clonetech Laboratories, Inc., Palo Alto, CA) and clarified by centrifugation at 14,000 x g for 5 min. Caspase-3 activity in cell lysates (100 µg each lysate) was measured in 96-well microtiter plates using reagents and instructions provided in the assay kit. Plates were read at 405 nm in a microplate reader.
Measurement of Cytochrome c.
To determine whether combination treatment induces release of cytochrome c from mitochondria, LNCaP cells were treated with TRAIL (20 ng/ml), curcumin (10 µM), or a combination of TRAIL and curcumin for 20 h. Using ApoAlert Cell Fractionation Kit (Clonetech Laboratories, Inc.), mitochondria and cytosol fractions were prepared from treated cells after instructions and reagents included in the kit. Ten µg of cytosolic or mitochondrial protein were separated on a 12% SDS-PAGE gel and after protein transfer membrane was probed with cytochrome c antibody.
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Results |
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or TRAIL-mediated cytotoxicity, we first examined the effect of these phytochemicals individually on the viability of LNCaP cells in MTS assay. As shown in Fig. 1A, curcumin at 20–40 µM concentration reduced cell viability by 40–45%. The effect of curcumin on cell viability was minimal at 5–10 µM concentration (10–15% reduction). In contrast, LNCaP cells were less sensitive to the cytotoxic effect of resveratrol. A small reduction in cell viability (15–20%) was observed at 20–40 µM resveratrol (Fig. 1B). Next, we tested the effect of TNF- and TRAIL separately and in combination with curcumin or resveratrol on the viability of LNCaP cells. Treatment with TNF- or TRAIL at concentrations from 6.25 to 50 ng/ml for 48 h produced very little loss in cell viability (<10%; Fig. 1, C and D, respectively). In the combined treatment, resveratrol (10 µM) in combination with TNF- or TRAIL over a concentration range of 6.25- 50 ng/ml produced cytotoxicity, which was no more than that observed by treating cells with each of these agents individually (Fig. 1E). Cytotoxic effect of treatment with curcumin (10 µM) and TNF- together was also not different from that of each agent alone (Fig. 1F). In contrast, treatment with a combination of curcumin and TRAIL produced greater cytotoxicity compared with the sum of toxicity produced by each as a single agent (Fig. 1F). Overall, the reduction in cell viability with combined treatment was 2.5–3-fold higher than the sum of the loss of cell viability by either agent alone.
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and TRAIL at concentrations ranging from 5 to 50 ng/ml. In contrast, curcumin, but not resveratrol, in combination with TRAIL, but not TNF-, caused 2–3-fold enhanced cell death in LNCaP cells at concentrations that each agent alone is ineffective or only poorly effective. Cooperation between curcumin and TRAIL was also observed in inhibition of the clonal growth of these cells.
Several recent reports have shown the ability of nontoxic concentrations of chemotherapeutic agents to sensitize tumor cells for apoptosis by death-inducing ligands such as FasL, TRAIL, or TNF- (8–11). The enhanced cytotoxicity of combined curcumin and TRAIL treatment may result from augmentation of TRAIL-induced apoptosis by curcumin. Indeed, treatment of LNCaP cells with curcumin and TRAIL together caused a decrease in the diploid cells with a corresponding increase in the hypodiploid (sub-G1) population, suggesting that cytotoxicity induced by curcumin and TRAIL is mediated by induction of apoptosis. This observation was also supported by the induction of internucleosomal DNA fragmentation and its inhibition in the presence of caspase-3 inhibitor z-DEVD-fmk. These results are consistent with the previously reported data by other investigators that anticancer agents such as doxorubicin, 5-fluorouracil, cisplatin, paclitaxel, synthetic retinoids, etoposide, and so on enhance TRAIL-mediated apoptosis in tumor cells at subtoxic concentrations (8, 33, 36, 37). In the present studies, curcumin lowered the signaling threshold required for TRAIL-induced cell death in LNCaP cells. As low as 6.25 ng/ml TRAIL induced significant cell death in combination with subtoxic concentration of curcumin, whereas treatment with TRAIL alone, up to a concentration of 50 ng/ml, was inconsequential.
FACS analysis revealed that LNCaP cells express death signal transducing receptors TRAIL-R1 and TRAIL-R2, suggesting that these cells are capable of responding to TRAIL. However, LNCaP cells are not killed when treated with TRAIL alone. This is most likely because these cells also express decoy (inhibitory) receptors TRAIL-R3 and TRAIL-R4. Unlike signaling by FasL, little is known about the events associated with binding of TRAIL to its receptors. Because combination of curcumin and TRAIL induced the cleavage of the most apical initiator caspase-8, this suggests that apoptosis in LNCaP cells is, at least in part, mediated via the death receptor-signaling pathway. Whether increased cell death in cultures treated with curcumin and TRAIL together results from the enhancement of the expression or binding affinity of death receptors TRAIL-R1 and TRAIL-R2 by curcumin has not been established. Similarly, the argument can also be made that curcumin may increase the sensitivity of LNCaP cells to TRAIL by reducing the expression of inhibitory TRAIL-R3 and TRAIL-R4.
Two major modes of apoptotic cell death program have been identified, namely receptor-mediated and chemical-induced apoptosis. In both cases, caspases, a family of cysteine proteases, play an important role (38). Binding of the death-inducing ligands (e.g., TNF-, FasL, and TRAIL) with their cognate receptors activates caspase-8 and caspase-3 leading to apoptosis (39). In chemical-induced (chemotherapeutic agents) apoptosis, undefined signals induce release of cytochrome c from mitochondria, which in conjunction with Apaf-1 causes activation of caspase-9. Activated caspase-9, in turn, can activate effector caspase-3, caspase-6, and caspase-7 (38). Recent evidence also suggests existence of cross-talk between caspase-8- and caspase-9-mediated apoptotic pathways through Bid, a proapoptotic Bcl-2 family member (40, 41). Our studies have demonstrated enhanced cleavage of procaspase-3, procaspase-8, and procaspase-9 in LNCaP cells treated with curcumin and TRAIL together. Furthermore, the results also demonstrated that combined curcumin/TRAIL treatment induces cleavage of Bid and release of cytochrome c from the mitochondria. These finding are consistent with the previously reported effect of curcumin in which it was shown to induce cleavage of Bid and release of cytochrome c from mitochondria in HL-60 leukemia cells (42). The release of cytochrome c from mitochondria and activation of caspase-9 indicate that the intrinsic pathway is also activated in cells treated with combination of curcumin and TRAIL. The activation of initiator/effector caspases (caspase-8, caspase-9, caspase-3) and proapoptotic Bid seen in LNCaP cells treated with combined curcumin and TRAIL treatment may result from the enhancement by curcumin of TRAIL-initiated cleavage processes. Alternatively, curcumin and TRAIL may promote apoptosis by inducing two independent but complementary activating signals.
Chemotherapy agents can augment TRAIL-induced apoptosis in cancer cells by inducing p53 or lowering NF-
B activity. Expression of NF-B transcription factor in tumor cells has been shown to inhibit apoptosis by TNF- (43). We have previously shown that curcumin inhibits the activation and translocation of NF-B to the nucleus (44). This suggests that up-regulation of TRAIL-induced apoptosis by curcumin may result from the suppression of NF-B activity in LNCaP cells. Further, suppression of the endogenous inhibitors of apoptosis such as cFLIP or XIAP is another mechanism by which curcumin could augment TRAIL-mediated apoptosis (45). It remains undetermined at present whether curcumin abrogates the activity of these intracellular negative regulators of apoptosis.
In summary, this study demonstrates that curcumin augments TRAIL-mediated apoptosis in androgen-sensitive prostate cancer cells. The induction of apoptosis by combined curcumin and TRAIL treatment involves the activation of initiator/effector caspases (caspase-8, caspase-9, caspase-3), cleavage of proapoptotic Bid, and the release of cytochrome c from the mitochondria. Thus, combination of TRAIL with curcumin, a pharmacologically safe compound, may provide a more effective adjuvant treatment for prostate cancer.
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Acknowledgments |
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Footnotes |
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1 Supported by USPHS Award RO1 CA85976 (to S. C. G.) and a grant from the American Institute for Cancer Research. 
3 The abbreviations used are: TNF, tumor necrosis factor; FasL, Fas ligand; TRAIL, TNF-related apoptosis-inducing ligand; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium; FACS, fluorescent-activated cell sorting; CHX, cycloheximide; NF-B, nuclear factor B.
Received 5/ 6/02; revised 8/30/02; accepted 7/11/02.
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