We first measured the effects of curcumin, with or without TRAIL, on cell viability by XTT assay (Fig. 1). Curcumin inhibited cell viability in prostate cancer PC-3 and LNCaP cells in a dose-dependent manner. TRAIL inhibited cell viability in PC-3 cells (Fig. 1A) but had no effect in LNCaP cells (Fig. 1B). Furthermore, curcumin enhanced the inhibitory effects of TRAIL on cell viability in PC-3 cells, and sensitized TRAIL-resistant LNCaP cells. These data suggest that curcumin alone is effective in both PC-3 and LNCaP cells, enhances the anti-proliferative activity of TRAIL in PC-3 cells, and sensitizes TRAIL-resistant LNCaP cells.

Anchorage independent colony growth is one of the characteristics of cancer cells. We next measured the interactive effects of curcumin and TRAIL on colony growth in soft agar (Fig. 1C and 1D). Curcumin inhibited number of PC-3 and LNCaP colonies formed in soft agar. Colonies formed by PC-3 cells were more sensitive to curcumin than those formed by LNCaP cells. By combination, TRAIL inhibited number of PC-3 colonies, but had no effect on LNCaP colonies. Interestingly, curcumin enhanced the inhibitory effects of TRAIL on PC-3 cell colony formation, and sensitized LNCaP cell colonies. These data suggest that curcumin has ability to inhibit anchorage-dependent and – independent growth of prostate cancer cells, and can enhance the apoptosis-inducing potential of TRAIL.

In order to examine the involvement of death receptor pathway on interactive effects of curcumin plus TRAIL, we used dominant negative FADD (DN-FADD). We have previously shown that DN-FADD blocked the interactions of TRAIL with histone deacetylase inhibitors, resveratrol, ionizing radiation or anticancer drugs 233334. As before, curcumin induced apoptosis in both PC-3/neo and LNCaP/neo cells (Fig. 1E and 1F). Curcumin enhanced the apoptosis-inducing potential of TRAIL in PC-3/neo cells, and sensitized TRAIL-resistant LNCaP/neo cells to undergo apoptosis. Transfection of DN-FADD had no effect on curcumin-induced apoptosis in both PC-3 and LNCaP cells, whereas it inhibited TRAIL-induced apoptosis in PC-3 cells. The ability of curcumin to enhance TRAIL-induced apoptosis in PC-3 cells, and its sensitization of TRAIL-resistant LNCaP cells was blocked by DN-FADD. These data suggest that death receptor pathway is involved in transducing effects of curcumin and TRAIL.

Activation of death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) by TRAIL transmits caspase-dependent apoptosis signal 35. We have recently shown that chemotherapeutic drugs, histone deacetylase inhibitors and γ-irradiation upregulate death receptors DR4 and/or DR5, and thus enhance the effectiveness of TRAIL 222333343637. Since curcumin enhances the apoptosis-inducing potential of TRAIL, we measured the expression of cell surface death receptors by flowcytometry (Fig. 2 and 3). Curcumin had no significant effect on the expression of DcR1 and DcR2 in PC-3 (Fig. 2) and LNCaP (Fig. 3) cells. By comparison, treatment of these cells with curcumin resulted in an increased expression of DR4 and DR5 in both cell lines. These data suggest that upregulation of death receptor DR4 and DR5 by curcumin may enhance the apoptosis-inducing potential of TRAIL in PC-3 and LNCaP cells.

Bcl-2 family members regulate apoptosis induced by stress stimuli primarily at the level of mitochondria 343839. We, therefore, examined the effects of curcumin on the expression of Bcl-2 family members. The Western blot analysis showed that curcumin induced expression of pro-apoptotic proteins Bak, Bax, PUMA, Bim and Noxa, and inhibited expression of anti-apoptotic protein Bcl-X_L and Bcl-2 in PC-3 cells (Fig. 4A). It is important to note that the expression of BimL isoform was significantly higher with all the doses of curcumin treatment at 48 h compared to control. The cleavage of Bcl-2 family member Bid to tBid (truncated Bid) is essential for linking death receptor pathway to mitochondrial pathway. We therefore measured whether curcumin treatment causes cleavage of Bid. Treatment of PC-3 cells with curcumin resulted in cleavage of Bid to tBid. Similarly, curcumin induced expression of Bak, Bax, PUMA, Bim and Noxa, and inhibited expression of Bcl-X_L and Bcl-2 in LNCaP cells (Fig. 4B). Furthermore, Bid was cleaved by curcumin in LNCaP cells. These data suggest that Bcl-2 family members may regulate sensitivity of prostate cancer cells to curcumin and/or TRAIL.

Inhibitors of apoptosis proteins (IAPs) play a major role in inhibiting caspase activation and apoptosis 40. Since curcumin augments TRAIL-induced apoptosis by activation of caspases, we sought to examine the effects of curcumin on the expression of IAPs (XIAP, survivin, cIAP1 and cIAP2) in PC-3 and LNCaP cells (Fig. 4C and 4D). These cells were treated with curcumin for 48 h, and expressions of IAPs were measured by the Western blot analysis. Curcumin inhibited expression of survivin and XIAP, and had no effect on levels of cIAP1 and cIAP2 in both PC-3 and LNCaP cells. These data suggest that inhibition/cleavage of IAPs by curcumin may be one of the mechanisms regulating apoptosis.

During apoptosis, engagement of the mitochondrial pathway involves the permeabilization of the outer mitochondrial membrane (OMM), which leads to the release of proteins such as cytochrome c and Smac/DIABLO 41. OMM permeabilization depends on activation, translocation and oligomerization of multidomain Bcl-2 family proteins such as Bax or Bak. We have shown that curcumin causes drop in mitochondrial membrane potential (ΔΨ_m), and release of Smac/DIABLO, cytochrome c and Omi/HtrA2 from mitochondria to cytosol in prostate cancer cells 12. Curcumin treatment of PC-3 and LNCaP cells caused a drop in ΔΨ_m in a time-dependent manner (Fig. 5A and 5B). Treatment of these cells with curcumin, TRAIL or curcumin plus TRAIL had no effect on mitochondrial membrane potential at 0 h (Fig. 5C and 5D). In PC-3 cells, curcumin and TRAIL alone caused a ΔΨ_m at both 8 and 16 h (Fig. 5C). The combination of curcumin and TRAIL had enhanced loss of ΔΨ_m when compared with either agent alone. In LNCaP cells, although TRAIL alone was ineffective in reducing ΔΨ_m, in combination with curcumin it caused significant loss of ΔΨ_m(Fig. 5D). These data suggest that curcumin exerts antiproliferative effects at the level of mitochondria.

The apoptotic death of cells requires proteolytic activation of caspases which are synthesized as latent proenzymes. Once activated, caspases cleave a wide range of molecules (e.g. PARP) that eventually result in the dismantlement of cells. Active caspases can be specifically inhibited by inhibitors of apoptosis (IAP). IAP antagonists such as Smac/DIABLO and Omi/HtrA2 compete with caspases for IAP-binding and consequently relieve caspase inhibition by IAPs and promote cell death. Since curcumin augments TRAIL-induced apoptosis, we sought to examine the activation/cleavage of caspase-9, -3 and -8, and PARP (Fig. 6). Curcumin induced caspase-3 activity in PC-3 and LNCaP cells (Fig. 6A and 6B). TRAIL induced caspase-3 activity in PC-3 cells, but had no effect in LNCaP cells. Curcumin has no effect on caspase-8 activity in PC-3 and LNCaP cells (Fig. 6C and 6D). However, TRAIL induced caspase-8 activity in PC-3 cells but not in LNCaP cells. Interestingly, the combination of curcumin and TRAIL resulted in more caspase-3 and caspase-8 activities in both cells lines (Fig. 6A–D).

PC-3 and LNCaP cells were treated with curcumin for 24 h followed by treatment with TRAIL for another 24 h, and the cleavage of caspase-3, caspase-9, caspase-8, and PARP was determined. Treatment of PC-3 cells with curcumin slightly increased caspase-3, caspase-9, caspase-8, and PARP cleavage (Fig. 6E). The combination of curcumin and TRAIL enhanced the cleavage of caspase-3, caspase-9, caspase-8 and PARP in PC-3 cells. In LNCaP cells, curcumin induced cleavage of capase-3, caspase-9, caspase-8 and PARP, whereas TRAIL had no effect on the cleavage of these proteins (Fig. 6F). Interestingly, pretreatment of curcumin with TRAIL resulted in cleavage of capase-3, caspase-9, caspase-8 and PARP. These data suggest that curcumin induces apoptosis through caspase activation. It is important to not that the combined treatments were more robust in the case of caspase-8 and PARP cleavage.

Because curcumin treatment significantly decreased HUVEC viability, we sought to examine whether curcumin inhibited in vitro angiogenesis. We explored this possibility by determining the effect of curcumin treatment on capillary tube formation by HUVEC on growth factor-reduced matrigel, which is well-accepted technique to measure in vitro angiogenesis 42. The data revealed that curcumin inhibited capillary tube formation in a dose-dependent manner (Fig. 7A). We next examined the involvement of ERK on capillary tube formation by using a specific inhibitor of ERK pathway. As shown in Fig. 7B and 7C, curcumin and ERK inhibitor alone inhibited capillary tube formation, and this effect of curcumin was further enhanced in the presence of ERK inhibitor. These results indicated that curcumin inhibition of capillary tube formation involved ERK MAP kinase.

Next we determined the effect of curcumin treatment on invasion potential (migration) of HUVEC cells using a modified Boyden Chamber assay. In DMSO-treated controls, a large fraction of HUVEC migrated to the bottom face of the membrane (Fig. 7D and 7E). Treatment of chambers with curcumin resulted in inhibition of migration of HUVECs in a dose-dependent manner (Fig. 7D). Inhibition of ERK blocked migration of HUVEC cells, and the inhibitory effects of curcumin on cell migration were further enhanced in the presence of ERK inhibitor (Fig. 7E). These results indicated that the migration by HUVEC cells was inhibited by curcumin, and ERK is an important regulator of this process.

Antibodies against cIAP1, cIAP2, survivin, XIAP, Bcl-2, Bcl-X_L, Bax, Bak, Bid, PUMA, Noxa, Bim, and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-caspae-3, anti-caspase-8, anti-caspase-9, and anti-PARP antibodies were purchased from BD Biosciences/Pharmingen (San Diego, CA). JC-1 was purchased from Invitrogen/Molecular Probes, Inc. (Eugene, OR). Enhanced chemiluminescence (ECL) Western blot detection reagents were from Amersham Life Sciences Inc. (Arlington Heights, IL). MEK1/2 inhibitor PD98059, and kits for Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL), caspase-3 and caspase-8 assays were purchased from EMD Biosciences/Calbiochem (San Diego, CA). Antibodies against TRAIL-R1/DR4, TRAIL-R2/DR5, DcR1, and DcR2 for flowcytometry were purchased from R&D Systems, Inc. (Minneapolis, MN). TRAIL was purified as described elsewhere 60. Curcumin was purchased from LKT Laboratories, Inc. (St. Paul, MN).

Androgen-sensitive LNCaP and androgen-insensitive PC-3 cell lines from human prostate cancer were obtained from the American Type Culture Collection (Manassas, VA). Cultures were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Invitrogen) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (Walkersville, MD) and maintained in endothelial cell growth factor medium-2 (EGM2 MV SingleQuots, Clonetics) supplemented with 5% FBS. The effect of curcumin on HUVEC viability was determined by trypan blue dye exclusion assay.

Cell growth inhibition was analyzed by the spectrophotometric measurement of the mitochondrial dehydrogenase activity using 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazo-lium hydroxide (XTT). XTT is converted to a colored formazan in the presence of metabolic activity (the primary mechanisms of XTT-to-formazan conversion are the mitochondrial succinoxidase and cytochrome P450 systems, as well as flavoprotein oxidases). Since the formazan product is water soluble, it is easily measured in cellular supernatants. Prostate cancer cells (1 × 10⁴ in 200 μl culture medium per well) were seeded in 96-well plate (flat bottom), treated with or without drugs and incubated for various time points at 37°C and 5% CO₂. Before the end of the experiment, 50 μl XTT labeling mixture (final concentration, 125 μM sodium XTT and 25 μM PMS) per well was added and plates were incubated for further 4 h at 37°C and 5% CO₂. The spectrophotometric absorbance of the sample was measured using a microtitre plate (ELISA) reader. The wavelength to measure absorbance of the formazon product was 450 nm, and the reference wavelength was 650 nm.

Prostate cancer cells were seeded on soft agar at a density of 500 per well in a six-well plate containing 1 ml of RPMI/10% fetal bovine serum. Cells were then incubated at 37°C in a humidified atmosphere containing 5% CO₂. Colony growth was assessed by the size and number of colonies after three weeks. Colonies exceeding the minimum diameter of 80 μm were counted.

Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated nick end-labeling method, which examines DNA strand breaks during apoptosis. Briefly, 1 × 10⁵ cells were treated with curcumin and/or TRAIL at the indicated doses for various time points at 37°C. Thereafter, cells were washed with PBS, air-dried, fixed with 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. After washing, cells were incubated with reaction mixture for 60 minutes at 37°C. Stained cells were mounted and analyzed under a microscope. In some cases, the data were confirmed by staining cells with Hoechst 33248.

Capillary tube formation assays were performed as we described earlier 61. In brief, matrigel (100 μl) was added to wells of a 96-well culture plate and allowed to polymerize for 1 h at 37°C. To examine the effects of curcumin on in vitro angiogenesis, subconfluent HUVECs were resuspended in complete medium and added to matrigel containing wells (1 × 10⁴ cells/well), and exposed to various concentrations of curcumin, MEK1/2 inhibitor (PD098059) or DMSO (control). The plates were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂ 62. Capillary tube formations (tubular structure formations) on 3-D Matrigel were visualized after 24 h under an inverted phase-contrast microscope (×200), and photomicrographs were documented by a SPOT digital camera (Sterling Heights, MI). Tube formation was defined as straight cellular extensions joining two cell masses or at branch points.

Cell migration assays were performed as we described earlier 61. In brief, migration of HUVEC cells was assessed using Transwell Boyden chamber (Corning, Acton, MA) containing a polycarbonated filter with a pore size of 8-μM. HUVECs (4 × 10⁴ cells in 0.2 ml) cells in complete medium was mixed with desired concentration of curcumin, MEK1/2 inhibitor (PD098059) or DMSO (control), and the cell suspension was added to the upper chamber. The lower chamber contained 0.6 ml of complete medium with the same concentration of curcumin, ERK inhibitor or DMSO. Migration through the membrane was determined after 24 h of incubation at 37°C. Cells remaining on the topside of the transwell membrane were removed using a cotton swab. The membrane was washed with ice-cold PBS. Cells that had migrated to the underside were fixed with 90% ethanol and stained with hematoxylin and eosin. Cell migration was quantified by counting the number of cells per field.

Cells (4 × 10⁴ per well) were seeded in a 96-well plate with 200 μl culture medium. Approximately 16 h later, cells were treated with various doses of curcumin and/or TRAIL to induce apoptosis. Casapse-3 and caspase-8 activities were measured as per manufacturer's instructions (EMD Biosciences) with a fluorometer.

Cell pellets were lysed in RIPA buffer containing 1 × protease inhibitor cocktail, and protein concentrations were determined using the Bradford assay (Bio-Rad, Philadelphia, PA). Cell lysates were electrophoresed in 12.5% SDS polyacrylamide gels and then transferred onto nitrocellulose membranes. After blotting in 5% nonfat dry milk in TBS, the membranes were incubated with primary antibodies at 1:1,000 dilution in TBS-Tween 20 overnight at 4°C, and then secondary antibodies conjugated with horseradish peroxidase at 1:5,000 dilution in TBS-Tween 20 for 1 hour at room temperature. Protein bands were visualized on X-ray film using an enhanced chemiluminescence system.

Mitochondrial energization was determined by retention of JC-1 dye (Molecular Probes Inc., Eugene) as we described earlier 2263. Briefly, drug treated cells (5 × 10⁵) were loaded with JC-1 dye (1 μg/ml) during the last 30 min of incubation at 37°C in a 5% CO₂ incubator. Cells were washed in PBS twice. Fluorescence was monitored in a fluorometer using 570-nm excitation/595-nm emission for the J-aggregate of JC1 64. ΔΨ_m was calculated as a ratio of the fluorescence of J-aggregate (aqueous phase) and monomer (membrane-bound) forms of JC1.

The mean and SD were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA using PRISM statistical analysis software (GrafPad Software, Inc., San Diego, CA). Significant differences among groups were calculated at P < 0.05.