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CELLULAR AND MOLECULAR

Curcumin [1,7-Bis(4-hydroxy-3-methoxyphenyl)-1–6-heptadine-3,5-dione; C₂₁H₂₀O₆] Sensitizes Human Prostate Cancer Cells to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand/Apo2L-Induced Apoptosis by Suppressing Nuclear Factor-B via Inhibition of the Prosurvival Akt Signaling Pathway

Department of Surgery (D.D., X.G., S.A-H., A.L.D., S.A.D., S.C.G.) and Department of Neurology (H.J.), Henry Ford Health System, Detroit, Michigan

Received November 28, 2006; accepted February 7, 2007.

	Abstract

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Our previous studies have shown that dietary pigment curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1–6-heptadine-3,5-dione; C₂₁H₂₀O₆] sensitizes human prostate cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced apoptosis by inhibiting nuclear factor (NF)-B. In the present study, we demonstrate that activated (phosphorylated) Akt kinase plays a pivotal role in regulation of NF-B and sensitization of LNCaP and PC3 prostate cancer cells to TRAIL by curcumin. Curcumin inhibited the expression of phospho-Akt (p-Akt), which was not due to activation of phosphatase and tensin homolog deleted on chromosome 10 phosphatase activity by curcumin. Because NF-B is a downstream target of Akt, we investigated whether inhibition of NF-B by curcumin is mediated through suppression of p-Akt. Data demonstrate that treatment of PC3 cells with SH-6 (JAm Chem Soc 125:1144–1145, 2003), a specific inhibitor of Akt, or transfection with small inhibitory RNA (siRNA)-Akt not only inhibited p-Akt but also abrogated the expression and transcriptional activity of NF-B. Furthermore, overexpression of constitutively active Akt1 in cancer cells prevented the inhibition of NF-B by curcumin. In addition, treatment with SH-6 or transfection with siRNA-Akt sensitized PC3 cells to TRAIL-induced cytotoxicity. On the other hand, SH-6 does not inhibit NF-B or sensitize DU145 cancer cells to TRAIL because these cells do not express p-Akt. Because expression of antiapoptotic Bcl-2, Bcl-xL, and X-chromosome-linked inhibitor of apoptosis protein (XIAP) is regulated by NF-B, both curcumin and SH-6 decreased the levels of these proteins in PC3 cells through inhibition of NF-B. Furthermore, gene silencing of Bcl-2 with siRNA-Bcl-2 sensitized PC3 cells to TRAIL. Collectively, these data define a pathway whereby curcumin sensitizes prostate cancer cells to TRAIL by inhibiting Akt-regulated NF-B and NF-B-dependent antiapoptotic Bcl-2, Bcl-xL, and XIAP.

Epidemiological studies suggest that in addition to race and age, diet is a prominent risk factor for prostate cancer (Sonn et al., 2005). These and laboratory studies suggest a link between high-fat diet and increased risk of metastatic prostate cancer (Fleshner et al., 2004). On the other hand, consumption of low-fat diet along with high intake of dark green leafy vegetables, fruits, and soy products has been linked to the low rate of prostate cancer. Several essential and nonessential dietary constituents found in plant-derived foods have been recognized for anticarcinogenic properties. The cancer-preventing effects of these foods are attributed to a group of naturally occurring polyphenolic compounds (Kuo, 1997). Indeed, there has been an increasing usage of plant derived flavonoids and phenolic/polyphenolic antioxidants as dietary supplements to prevent and/or treat prostate cancer.

Curcumin is a non-nutritive yellow pigment found in the spice turmeric derived from the rhizome of the plant Curcuma longa. Curcumin has shown strong anti-inflammatory, antioxidative, anticancer, antiangiogenic, and proapoptosis properties (Aggarwal et al., 2003). Because of its ability to scavenge free radicals and induce apoptosis, curcumin has been investigated for prevention and inhibition of tumorigenesis. Curcumin inhibited the development of cancers of skin, forestomach, duodenum, tongue, colon, and mammary glands in models of chemical carcinogenesis in rodents (Huang et al., 1994; Rao et al., 1995). It was also shown to inhibit human prostate cancer xenografts in nude mice (Dorai et al., 2001). In vitro, curcumin inhibited cell proliferation or induced apoptosis in cancer cells (Moragoda et al., 2001; Anto et al., 2002). The chemopreventive and antitumor effects of curcumin were attributed to its ability to inhibit protein kinases (Huang et al., 1991) and cyclooxygenase-2 (Liu et al., 1993) involved in production of tumor-promoting prostaglandin E₂.

TRAIL or Apo2L is a member of the TNF- superfamily of death-inducing ligands that also includes TNF- and FasL (CD95). Like TNF- and FasL, TRAIL also induces apoptosis in a variety of cancer cell lines, but unlike TNF- and FasL, TRAIL shows little cytotoxicity to normal cells (Pitti et al., 1996). TRAIL binds to five receptors, i.e., TRAIL-R1, -R2, -R3, -R4, and osteoprotegerin (Ashkenzi and Dixit, 1999). The binding of TRAIL to TRAIL-R1 and -R2 induces death signals, whereas binding to TRAIL-R3 and -R4 fails to transduce death signaling (Ashkenzi and Dixit, 1999). Furthermore, in contrast to the severe inflammatory response syndrome induced by TNF- and the hepatotoxicity of FasL, treatment of mice and nonhuman primates with TRAIL lacks systemic toxicity (Ashkenazi et al., 1999; Walczak et al., 1999). TRAIL has shown potent antitumor activity against human tumor xenografts in nude mice without systemic toxicity (Ashkenazi et al., 1999; Walczak et al., 1999). Thus, TRAIL is an attractive cytokine for cytokine therapy of advanced cancers including prostate cancer.

We have shown previously that although prostate cancer cells LNCaP, DU145, and PC3 are mostly resistant to TRAIL, they can be sensitized with curcumin to TRAIL-induced apoptosis (Deeb et al., 2003, 2004, 2006). Chemosensitization of LNCaP cells to TRAIL by curcumin involved the inhibition of constitutively active NF-B through suppression of IB phosphorylation (Deeb et al., 2004). Because Akt can signal activation of NF-B through phosphorylation of IB kinase (IKK) or RelA (Kane et al., 1999; Madrid et al., 2000), in the present study, we investigated whether modulation of Akt activity by curcumin is part of the mechanism by which it inhibits NF-B and sensitizes prostate cancer cells to TRAIL. Our data demonstrate that down-regulation of Akt by curcumin, Akt-specific inhibitor SH-6 (Kozikowski et al., 2003), or siRNA-Akt inhibits NF-B and NF-B-dependent antiapoptotic proteins, leading to the sensitization of prostate cancer cells to TRAIL-induced apoptosis.

	Materials and Methods

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Reagents and Antibodies. Curcumin was purchased from Sigma Chemical Co. (St. Louis, MO). Anti-NF-B (p65), anti-Bcl-2, and anti-Bcl-xL antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-XIAP antibody was from Trevigen (Gaithersburg, MD). TRAIL was purchased from R&D Systems (Minneapolis, MN). CellTiter 96 AQueous One Solution Proliferation Assay System was from Promega (Madison, WI). SignalSilence Akt and Bcl-2 siRNA kits were purchased from Cell Signaling Technology, Inc. (Beverly, MA). A 100 mM solution of curcumin was prepared in dimethyl sulfoxide, and all test concentrations were prepared by diluting the stock solution in tissue culture medium.

Cell Lines. LNCaP, DU145, and PC3 human prostate cancer cell lines were obtained from American Type Culture Collection (Rockville, MD). LNCaP cells were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 1 µg/ml hydrocortisone, and 100 nM testosterone as described previously (Deeb et al., 2003). PC3 cells were grown in F-12K nutrient mixture (Invitrogen) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 25 mM HEPES buffer. DU145 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 25 mM HEPES buffer. All cell lines were cultured at 37°C in a humidified atmosphere consisting of 5% CO₂ and 95% air and maintained by subculturing cells twice a week.

Measurement of Cell Viability (MTS Assay). Cells (2 x 10⁴) were seeded into each well of a 96-well plate in 100 µl of tissue culture medium. After 24-h incubation to allow cells to adhere, cells were treated either with curcumin or TRAIL or a combination of the two agents. Cultures were incubated for an additional 48 h. Cell viability was then determined by the colorimetric MTS assay using CellTiter 96 AQueous One Solution Proliferation Assay System from Promega. This assay measures the bioreduction by intracellular dehydrogenases of the tetrazolium compound MTS in the presence of the electron-coupling reagent phenazine methosulfate. MTS and phenazine methosulfate were added to the culture wells, and the mixture was incubated for 2 h at 37°C. The absorbance was measured at 490 nm using a microplate reader and is directly proportional to the number of viable cells in the cultures. Percent cytotoxicity was calculated from the loss of cell viability in cultures.

Isolation of Nuclear Proteins. Nuclear extracts were prepared as described previously (Deeb et al., 2004). After treatment with curcumin for 24 h, cells were washed three times with phosphate-buffered saline and incubated on ice for 15 min in hypotonic buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.6% Nonidet P-40). Cells were vortexed gently for lysis, and nuclei were separated from the cytosol by centrifugation at 12,000g for 1 min. Nuclei were resuspended in buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and shaken for 30 min at 4°C. Nuclear extracts were obtained by centrifugation at 12,000g, and protein concentration was measured by Bradford assay (Bio-Rad, Richmond, CA). NF-B in nuclear extracts was detected by Western blotting as described below.

Western Blotting. Total cellular proteins were isolated by detergent lysis [1% (v/v) Triton X-100, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 10% glycerol, 2 mM sodium vanadate, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, and 10 µg/ml 4–2-aminoethyl-benzenesulfinyl fluoride]. Lysates were clarified by centrifugation at 14,000g for 10 min at 4°C, and protein concentrations were determined by the Bradford assay. Samples (50 µg) were boiled in an equal volume of sample buffer (20% glycerol, 4% SDS, 0.2% bromphenol blue, 125 mM Tris-HCl, pH 7.5, and 640 mM 2-mercaptoethanol) and separated on 10 to 14% SDS-polyacrylamide gels. Proteins resolved on the gels were transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in 10 mM Tris-HCl, pH 8.0, and 150 mM NaCl with 0.05% Tween 20 and probed with protein-specific antibodies to NF-B (1:500), Akt (1:500), phospho-Akt (p-Akt) (1:500), PTEN (1:1000), p-IB (1:1000), Bcl-2 (1: 500), Bcl-xL (1:1000), XIAP (1:1000), or -actin (1:500) followed by horseradish peroxidase-conjugated secondary antibody. Immune complexes were visualized with an enhanced chemiluminescence detection system from Amersham Corp. (Arlington Heights, IL).

DNA Transfection. The transcriptional activity of NF-B was measured in NF-B-dependent luciferase reporter gene expression assay. In brief, 0.5 x 10⁶ cancer cells were plated in each well of a six-well plate for 24 h and then transfected using the Lipofectamine reagent (Invitrogen) with p3B-Luc expression plasmid (5 µg of DNA) containing three human immunodeficiency virus B sites upstream of the thymidine kinase minimal reporter and the luciferase cDNA. Cells were incubated at 37°C for 24 h, and culture medium was replaced with fresh medium. After further incubation for 24 h, cell extracts were prepared, and 50 µl of cell extract was used to measure luciferase activity in a luminometer using reagents and instructions provided with the luciferase assay system from Promega.

For overexpression of Akt, semiconfluent cultures of PC3 cells in 100-mm² Petri dish were transfected with 10 µg of empty or expression vector (pUSEamp) DNA containing Myc-His tagged mouse Akt1 (activated) under the control of the cytomegalovirus promoter (Upstate Cell Signaling, Lake Placid, NY) using the Lipofectamine Plus reagent. After incubation for 24 h, cells were analyzed for the expression of exogenous Akt1/PKB by immunoblotting using anti-Myc Tag antibody.

For gene silencing of Akt and Bcl-2, PC3 cells were transfected with double-stranded siRNA of Akt or Bcl-2 using SignalSilence siRNA kits from Cell Signaling Technology. In brief, 10⁶ cancer cells were plated in each 60-mm Petri dish for 24 h and treated with 3 ml of transfection medium containing 20 µg of Lipofectamine and 100 nM siRNA for 24 h. Cell extracts were prepared, and gene silencing was confirmed by protein inhibition by Western blotting.

Statistical Analysis. Data are presented as means ± S.D. Interaction between TRAIL and curcumin was tested by two-way analysis of variance. The degree of interaction was expressed as the percentage difference between the combined TRAIL and curcumin response and the sum of the responses to TRAIL and curcumin alone.

	Results

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Curcumin Sensitizes Prostate Cancer Cells to TRAIL-Induced Cell Death. Dose-response studies have identified 10, 20, and 30 µM as nontoxic concentrations of curcumin for LNCaP, DU145, and PC3 cells, respectively, having little or no effect on the viability of these cells in 48-h MTS assay (supplemental Fig. 1). Likewise, 20 ng/ml TRAIL was found to have minimal affect on the viability of each of these cell lines (Deeb et al., 2003, 2006). In contrast, the viability of cultures is significantly decreased when cells were treated with combination of curcumin and TRAIL together at these nontoxic concentrations. As shown in Fig. 1A, whereas treatment with 10 µM curcumin and 20 ng/ml TRAIL alone for 48 h reduced the viability of LNCaP cells by 5 and 8%, viability was dramatically decreased (66% reduction, p < 0.001) when cells were treated with combination of curcumin and TRAIL together. The same kinds of effects were observed when DU145 cells were treated with 20 µM curcumin and 20 ng/ml TRAIL (Fig. 1A, curcumin or TRAIL alone, 8 and 16% reduction; combined treatment, 68% reduction, p < 0.001). Treatment of PC3 cells at 30 µM curcumin and 20 ng/ml TRAIL as single agents and in combination reduced the viability 4, 10, and 62%, respectively (Fig. 1A). These data indicate that curcumin or TRAIL as single agents have minimal effect on the viability of prostate cancer cell lines; however, the viability of each cell line is significantly reduced following treatment with curcumin and TRAIL together at nontoxic concentration of each agent. Furthermore, in each case, sensitization of cancer cells by curcumin was associated with a decrease in expression level of NF-Bin cells treated with curcumin (insets, Fig. 1A) corroborating the results of our earlier studies with LNCaP cells (Deeb et al., 2004, 2006).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Effect of curcumin, TRAIL, or curcumin/TRAIL combination on viability of prostate cancer cells. A, short-term treatment with curcumin. Tumor cells (2 x 104) were seeded in each well of a microtiter plate in 0.1 ml of culture medium. Plates were incubated for 20 h before treating cells with curcumin at 10 (LNCaP), 20 (DU145), or 30 (PC3) µM curcumin or TRAIL at 20 ng/ml (all cell lines) or a combination of curcumin and TRAIL together. Plates were incubated at 37°C, 95% relative humidity, and 5% CO2 for 48 h. Cell viability was measured by MTS assay. B, long-term treatment with curcumin. DU145 and PC3 cells (5 x 105/2.5 ml of culture medium) plated in 60-mm2 culture dishes were pretreated with curcumin at 10 µM for 96 h with a medium change at 48 h. After pretreatment with curcumin, cells were harvested, and 2 x 104 tumor cells were seeded in each well of a microtiter plate in 0.1 ml of culture medium and treated with TRAIL at 20 ng/ml in triplicates. Viability was measured by MTS assay after 24 h. Bar graphs show percent reduction in viability (mean ± S.D.) in three independent experiments. Insets (A and B) show effect of curcumin on NF-B expression.

Curcumin Inhibits p-Akt in Prostate Cancer Cells. Recent evidence suggests that Akt, a serine-threonine kinase, regulates NF-B activation through phosphorylation of IKK or RelA directly (Kane et al., 1999; Madrid et al., 2000), raising the possibility that suppression of NF-B by curcumin in prostate cancer cells might also involve suppression of p-Akt. To examine this, PC3, LNCaP, and DU145 cells were treated with curcumin for 24 h, and p-Akt and basal Akt levels were analyzed by Western blotting. Untreated PC3 and LNCaP cells showed p-Akt and treatment with curcumin inhibited it in a dose-related manner (Fig. 2A). In contrast, DU145 cells lacked p-Akt and therefore could not be evaluated for the effect of curcumin. There was little effect of curcumin on basal Akt in three tumor cell lines.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Effects of curcumin on p-Akt, PTEN phosphatase and p-IB. A, PC3, DU145, and LNCaP cells were treated with curcumin at concentrations as shown for 24 h, and p-Akt was analyzed by Western blotting. B, immunoblot showing expression of PTEN in LNCaP, DU145, and PC3 cells or PC3 cells treated with curcumin for 24 h. C, immunoblot showing inhibition of p-IB in DU145 cells by curcumin. Values above blots represent the change in protein expression compared with untreated control represented as 1.0. Data presented are representative of two to three experiments.

To determine the mechanism by which curcumin inhibits NF-B and sensitizes DU145 cells to TRAIL because these cells lack p-Akt, we investigated the effect of curcumin on phosphorylation of IB, an inhibitor of NF-B, which binds to and prevent translocation of NF-B to the nucleus. As shown in Fig. 2C, treatment of DU145 cells with curcumin almost completely inhibited phosphorylation of IB at a dose of 20 µM. This result suggests that curcumin most probably inhibits NF-B in DU145 cells by inhibiting phosphorylation and degradation of IB through inactivation of IKK independent of p-Akt.

Inhibition of p-Akt Suppresses the Expression and Transcriptional Activity of NF-B and Sensitizes PC3 Cell to TRAIL. To explore Akt regulation of NF-B and resistance of prostate cancer cells to TRAIL, we investigated whether inhibition of p-Akt would modulate the levels and the transcriptional activity of NF-B. For this purpose, PC3 cells were treated with SH-6, a potent inhibitor of Akt for 24 h. Cellular and nuclear proteins were isolated and analyzed for p-Akt and NF-B, respectively, by Western blotting. SH-6 reduced the levels of p-Akt in a dose-dependent manner without affecting basal Akt (Fig. 3A). More significantly, the inhibition of p-Akt by SH-6 led to the inhibition of NF-B, also in a dose-related manner.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Inhibition of p-Akt inhibits the expression and transcriptional activity of NF-B and sensitizes PC3 cells to TRAIL. A, tumor cells were treated with SH-6 at concentrations of 5, 10, and 20 µM for 24 h after, which the expression of p-Akt, NF-B, and Akt (loading control) was analyzed by Western blotting. B, to test whether overexpression of Akt will prevent the inhibition of NF-B by curcumin, PC3 cells were transfected with empty or Akt1 expression plasmid (myr-Akt1-pUSE) using LipofectAMINE Plus reagent, and 24 h later, cells were treated with curcumin (30 µM) for 24 h and analyzed for p-Akt and NF-B expression. C, to test whether Akt inhibition affects transcriptional activity of NF-B, PC3 cells were transfected with control vector (pGL3-Luc) or p3xB-Luc vector or p3xB-Luc vector and siRNA-Akt together. In separate cultures, PC3 cells transfected with p3xB-Luc vector were treated with SH-6 at 10 or 20 µM. After incubation for 48 h, cell lysates were prepared, and luciferase activity was determined in a luminometer using the luciferase assay system from Promega. D, to test the sensitivity of PC3 cells treated with SH-6 to TRAIL, control and SH-6 treated cells were treated with TRAIL (20 ng/ml) for 24 h, and cell viability was measured by MTS assay. Data shown in A and B are representative of three experiments. Values above blots represent the change in protein expression compared with untreated control represented as 1.0. Bar graphs (C and D) represent mean ± S.D. of three separate experiments.

We next analyzed the effect of Akt inhibition on the transcriptional activity of NF-B. For this, PC3 cells were double transfected with p3B-Luc expression vector, and siRNA-Akt or PC3 cells transfected with p3B-Luc expression vector were treated with Akt inhibitor SH-6 for 24 h. Cells were then washed and reincubated in fresh medium for an additional 24 h. After incubation, cells were lysed, and 50 µl of cell extract was used to measure luciferase activity in a luminometer. As shown in Fig. 3C, the relative luciferase activity of PC3 cells transfected with p3B-Luc reporter vector was severalfold higher compared with cells transfected with the control plasmid pGL3-Luc without the B sites. The luciferase activity was markedly suppressed in cells that were transfected with siRNA-Akt (80%) or treated with SH-6 (80–88%). The inhibition of p3B-Luc expression vector by siRNA or p-Akt inhibitor SH-6 demonstrated that Akt regulates the transcriptional activity of NF-B in PC3 cells.

Whether inhibition of p-Akt sensitizes prostate cancer cells to TRAIL through down-regulation of NF-B was examined next. For this purpose, PC3 cells were treated with SH-6 for 24 h and then tested for susceptibility to TRAIL in MTS assay. Treatment with SH-6 resulted in sensitization of PC3 to TRAIL-induced cytotoxicity (Fig. 3D). At 5 µM SH-6, there was little change in p-Akt or NF-B, and it correlated with the lack of sensitization to TRAIL. On the other hand, both p-Akt and NF-B were significantly reduced at 10 and 20 µM SH-6, which also resulted in an increase in sensitivity to TRAIL (55% cytotoxicity). These data demonstrate that Akt regulates NF-B in PC3 cells, and inhibition of p-Akt sensitizes them to TRAIL through the inhibition of NF-B.

Silencing of Akt with siRNA-Akt Sensitizes PC3 Cells to TRAIL. To more directly test regulation of NF-BbyAkt and sensitization of PC3 cells to TRAIL, we inhibited Akt expression by transfecting cells with double-stranded siRNA-Akt and tested them for sensitivity to TRAIL. Transfection with siRNA-Akt for 24 h not only inhibited Akt expression but also the expression of NF-B (Fig. 4A) and sensitized PC3 cells to TRAIL-induced cytotoxicity (Fig. 4B, 59%), demonstrating that inhibition of NF-B through targeted disruption of Akt with siRNA-Akt sensitizes PC3 cells to TRAIL.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Transfection with siRNA-Akt abolishes NF-B expression and sensitizes prostate cancer cells to TRAIL. A, PC3 cells were transfected with siRNA-Akt (100 nM) for 24 h, after which Akt and NF-B levels were analyzed by Western blotting. Similar results were obtained in three separate experiments. B, sensitivity of transfected cells to TRAIL (20 ng/ml) was measured by MTS assay as described. Bar graph represents mean ± S.D. of three separate experiments.

Treatment with p-Akt Inhibitor SH-6 Does Not Sensitize DU145 Cells to TRAIL. If indeed sensitization of PC3 cells to TRAIL by SH-6 or with siRNA-Akt is mediated through the inhibition of Akt-regulated NF-B, then cells lacking p-Akt expression should not be sensitized to TRAIL after treatment with SH-6. Because DU145 cells express active NF-B but not p-Akt, we tested the response of DU145 to TRAIL after treatment with SH-6. Treatment with combination of curcumin and TRAIL together induced significant cytotoxicity (68%) compared with curcumin (20 µM) or TRAIL (20 ng/ml) alone (Fig. 5). In contrast, treatment with SH-6 (5–20 µM) neither inhibits nuclear NF-B expression nor sensitizes DU145 cells to TRAIL-mediated cytotoxicity (Fig. 5, A and B). This result demonstrates that sensitization of PC3 cells to TRAIL by SH-6 and siRNA-Akt is indeed through the inhibition of Akt-regulated NF-B.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Treatment with SH-6 does not inhibit NF-B or sensitize DU145 cells to TRAIL. A, DU145 cells were treated with SH-6 at concentrations of 5, 10, and 20 µM for 24 h. Nuclear proteins were isolated from control and treated cells and analyzed by Western blotting. Similar results were obtained in two independent experiments. B, sensitivity of control and SH-6-treated cells to TRAIL (20 ng/ml) was measured by MTS assay as described. Bar graph represents mean ± S.D. of three separate experiments.

View larger version (40K): [in this window] [in a new window]   Fig. 6. Curcumin or SH-6 reduces the expression of NF-B-dependent antiapoptotic proteins, and disruption of Bcl-2 sensitizes PC3 cells to TRAIL. A and B, PC3 cells were treated with curcumin (20, 30, or 40 µM) or SH-6 (5, 10, or 20 µM) for 24 h, after which nuclear (NF-B) and cellular (Bcl-2, Bcl-xL, and XIAP) proteins were analyzed by Western blotting. Similar results were obtained in two independent experiments. C, silencing of Bcl-2 gene in PC3 cells transfected with siRNA-Bcl-2 (100 nM) for 24 h and effect on expression of Bcl-2. D, sensitivity of PC3 cells transfected with siRNA-Bcl-2 to TRAIL (20 ng/ml) measured by MTS assay. Bar graph represents mean ± S.D. of three separate experiments. Values above blots represent the change in protein expression compared with untreated control represented as 1.0.

Since treatment with both curcumin and SH-6 inhibited the expression of NF-B-regulated Bcl-2, we next tested the significance of Bcl-2 inhibition in sensitization of PC3 cells to TRAIL-induced cytotoxicity. PC3 cells were transfected with siRNA-Bcl-2 for 24 h, after which transfected cells were analyzed for the expression of Bcl-2 and sensitivity to TRAIL. As shown in Fig. 6C, transfection with siRNA-Bcl-2 nearly completely abolished the expression of Bcl-2. Furthermore, percent cytotoxicity induced by TRAIL in transfected cells (71%) was higher than that induced by combined curcumin/TRAIL treatment in untransfected PC-3 cells (64%, Fig. 6D). This result demonstrated that abrogation of Bcl-2 whether by siRNA gene silencing or by curcumin through Akt/NF-B pathway increases the sensitivity of PC3 cells to TRAIL-induced cytotoxicity.

	Discussion

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Apoptosis or programmed cell death plays an essential role in normal development of multicellular organisms and in maintaining tissue homeostasis. Aberration of apoptosis has been implicated in tumor development and resistance to cancer therapies (Wang et al., 1999). Thus, promotion of apoptosis in prostate cancer cells by anticancer agents could potentially lead to the regression and improved prognosis of refractory disease. Human prostate cancer cell lines have shown weak sensitivity to TRAIL in some studies (Yu et al., 2000) but not in others (Nimmanapalli et al., 2001). Our previous studies have shown that at nontoxic concentrations, curcumin sensitizes prostate cancer cell lines LNCaP, DU145, and PC3 to TRAIL-mediated apoptosis. Treatment of prostate cancer cells with curcumin and TRAIL together induced DNA fragmentation, cleavage of procaspases-3, -8, and -9, and release of cytochrome c from mitochondria (Deeb et al., 2003, 2006). These results are consistent with reports by other investigators that anticancer agents such as doxorubicin, cisplatin, paclitaxel, retinoids, etoposide, etc. enhance TRAIL-mediated apoptosis in tumor cells (Nimmanapalli et al., 2001; Zisman et al., 2001). Curcumin sensitized prostate cancer cells to TRAIL independent of their hormonal status. Hormone-sensitive LNCaP cells were sensitized at a lower concentration of curcumin (10 µM), whereas a higher concentration of curcumin (20–30 µM) was needed to sensitize hormone-resistant DU145 and PC3 cells (Deeb et al., 2006). The present study demonstrated that even hormone-resistant DU145 and PC3 cells can be sensitized to TRAIL at a lower concentration of curcumin (10 µM), provided cells are pretreated with curcumin for longer periods of time. Thus, curcumin can sensitize both hormone-sensitive and -resistant prostate cancer cells to TRAIL by short exposure to high concentration or by longer exposure to lower concentration of curcumin.

Sensitization of prostate cancer cells to TRAIL by curcumin is linked to the inhibition of constitutively active NF-B (Deeb et al., 2004, 2006). Inhibition of NF-B by curcumin is especially noteworthy because constitutively active NF-B renders tumor cells resistant to apoptosis by death ligands and chemotherapy (Beg and Baltimore, 1996; Wang et al., 1999). Because activated NF-B promotes proliferation and growth of tumor cells and therefore curtails apoptosis, this might explain why prostate cancer cells are resistant to TRAIL-induced apoptosis. Under normal conditions, NF-B is sequestered in the cytoplasm as a heterodimer composed of Rel proteins p50 and p65 by the inhibitory protein IB (Karin, 1999). In response to cell stimulation, IB is rapidly phosphorylated and degraded allowing NF-B to translocate to the nucleus, where it regulates the expression of target genes. Thus, signal-induced phosphorylation of IB catalyzed by a stimulus-responsive IKK complex is the key event that triggers the activation of NF-B. Indeed, inhibition of NF-B activation by curcumin in LNCaP cells involved the repression of phosphorylation of IB, an event that primes IB for proteasomal degradation (Deeb et al., 2004). Others have also shown inhibition of NF-B activation by curcumin through suppression of IB in a number of tumor cell lines (Singh and Aggarwal, 1995).

Phosphatidylinositol-3-OH kinase is a major signaling pathway that mediates cell survival signals in response to growth factors, cytokines, and oncogenic Ras (Marte and Downward, 1997). Activated phosphatidylinositol-3-OH kinase phosphorylates Akt, a 57-kDa serine/threonine kinase that regulates the generation of cell survival signals. Activated Akt promotes cell survival by stimulating cell proliferation and cell cycle progression and inhibiting apoptosis by phosphorylating and inactivating proapoptotic Bad, procaspase-9, and members of the Forkhead transcription family (Datta et al., 1997; Cardone et al., 1998; Brunet et al., 1999). In addition to blocking apoptosis directly, Akt also signals activation of NF-B through phosphorylation and activation of IKK or by phosphorylating RelA (Kane et al., 1999; Madrid et al., 2000).

Our finding that PC3 and LNCaP cells express p-Akt, which is suppressed by curcumin, suggested that suppression of NF-B by curcumin might in fact be through suppression of the Akt/IKK/IB/NF-B signaling pathway. Indeed, treatment of PC3 cells with the selective Akt inhibitor SH-6 not only inhibited p-Akt but also markedly reduced the levels of constitutive NF-B. Furthermore, overexpression of constitutively active Akt also inhibited curcumin-induced decrease in NF-B. The inhibition of p-Akt by curcumin was not due to the activation of PTEN phosphatase activity because PC3 cells lack PTEN, and it could not be induced after treatment with curcumin. Our results also showed that p-Akt regulates the transcriptional activity of NF-B because B-driven reporter activity of p3xB-Luc expression vector was almost completely abolished in PC3 cells transfected with siRNA-Akt or treated with the p-Akt inhibitor SH-6. In addition, treatment with SH-6 sensitized PC3 cells to TRAIL-induced cytotoxicity. We also considered the possibility that sensitization of tumor cells to TRAIL by SH-6 could have resulted from changes other than Akt-mediated regulation of NF-B; however, selective silencing of Akt by siRNA-Akt ruled out this possibility. Specific inactivation of Akt with siRNA-Akt inhibited not only Akt but also NF-B, leading to sensitization of PC3 cells to TRAIL-induced cytotoxicity. Further proof that Akt regulates NF-B in PC3 cells came from the finding that treatment of DU145 cells that do not express p-Akt with SH-6 does not reduce NF-B or sensitize them to TRAIL. However, DU145 cells are sensitized by curcumin to TRAIL through the suppression of NF-B via an Akt-independent pathway. Thus, the common denominator among these three prostate cancer cell lines for sensitization to TRAIL is the inhibition of NF-B. In prostate cancer cells that express Akt, sensitization to TRAIL by curcumin proceeds through the suppression of NF-B via Akt. Sensitization of prostate cancer cells that lack p-Akt (DU145) also requires inhibition of constitutively active NF-B; however, inhibition of NF-B in these cells by curcumin is mediated through the suppression of IB phosphorylation independent of Akt.

NF-B plays a critical role not only in the transcription of genes involved in immune and inflammatory responses, but it also regulates genes that inhibit apoptosis and promote cell survival. The transcription of many of the Bcl-2 and IAPs families of gene products that regulate apoptosis is controlled by Rel/NF-B family of transcription factors (Zong et al., 1999; Chen et al., 2000); therefore, as expected, inhibition of NF-B by curcumin reduced the expression of antiapoptotic Bcl-2, Bcl-xL, and XIAP, all NF-B-regulated gene products. In addition, inhibition of p-Akt by SH-6 also markedly reduced the levels of these NF-B-dependent gene products, confirming that down-regulation of activated Akt negatively affects NF-B and NF-B-regulated Bcl-2, Bcl-xL, and XIAP.

Our findings that curcumin inhibits NF-B and NF-B-regulated antiapoptotic gene products through suppression of Akt corroborate the results of studies carried out by other investigators. For instance, inhibition of Akt activation by curcumin in human mantle cell lymphoma cells was shown to lead to the inhibition of NF-B and several of the NF-B-regulated gene products related to cell proliferation and apoptosis (Shishodia et al., 2005). In another report, curcumin inhibition of TNF-induced activation of NF-B in human myeloid leukemia cells U937 was also linked the inhibition of Akt activation and the inhibition of NF-B-regulated gene products involved in cell proliferation, angiogenesis, and tumor metastasis (Aggarwal et al., 2006). Together, published reports and findings of the present study support the view that in a variety of cancer cells, the inhibition of NF-B and NF-B-dependent gene products involved in cell proliferation, apoptosis, tumor metastasis, and sensitization of tumor cells to death ligands proceeds through the inhibition of Akt. Although these data provide an insight into the mechanisms responsible for sensitization of prostate cancer cells to TRAIL, besides Akt and NF-B, Jun N-terminal kinase, -catenin, and peroxisome proliferator-activated receptor- are also targets of curcumin and may contribute to the sensitization of prostate cancer cells to TRAIL by curcumin (Jaiswal et al., 2002; Xu et al., 2003). Further understanding of these mechanisms of action of curcumin could potentially lead to the development of a novel curcumin/TRAIL combination therapy to better manage prostate cancer.

	Footnotes

 
This work was supported by the National Institutes of Health Grant 1R21 CA102616 (to S.C.G.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.117721.

ABBREVIATIONS: CaP, carcinoma of prostate; C₂₁H₂₀O₆, curcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1–6-heptadine-3,5-dione; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TNF, tumor necrosis factor; NF, nuclear factor; IKK, IB kinase; siRNA, small inhibitory RNA; MTS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; p-Akt, phospho-Akt; PTEN, phosphatase and tensin homolog deleted on chromosome 10; XIAP, X-chromosome-linked inhibitor of apoptosis protein.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Subhash C. Gautam, Surgical Research 4D, One Ford Place, Detroit, MI 48202. E-mail: sgautam1{at}hfhs.org

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