Curcumin (diferuloylmethane), a yellow pigment in turmeric, has been shown to inhibit the activation of nuclear factor-κB (NF-κB), a transcription factor closely linked to chemoresistance in multiple myeloma cells. Whether curcumin can overcome chemoresistance and enhance the activity of thalidomide and bortezomib, used to treat patients with multiple myeloma, was investigated in vitro and in xenograft model in nude mice. Our results show that curcumin inhibited the proliferation of human multiple myeloma cells regardless of their sensitivity to dexamethasone, doxorubicin, or melphalan. Curcumin also potentiated the apoptotic effects of thalidomide and bortezomib by down-regulating the constitutive activation of NF-κB and Akt, and this correlated with the suppression of NF-κB-regulated gene products, including cyclin D1, Bcl-xL, Bcl-2, TRAF1, cIAP-1, XIAP, survivin, and vascular endothelial growth factor. Furthermore, in a nude mice model, we found that curcumin potentiated the antitumor effects of bortezomib (P < 0.001, vehicle versus bortezomib + curcumin; P < 0.001, bortezomib versus bortezomib + curcumin), and this correlated with suppression of Ki-67 (P < 0.001 versus control), CD31 (P < 0.001 versus vehicle), and vascular endothelial growth factor (P < 0.001 versus vehicle) expression. Collectively, our results suggest that curcumin overcomes chemoresistance and sensitizes multiple myeloma cells to thalidomide and bortezomib by down-regulating NF-κB and NF-κB-regulated gene products. [Mol Cancer Ther 2009;8(4):959–70]

In the United States, 50,000 patients are affected by multiple myeloma, 16,000 patients are newly diagnosed with multiple myeloma, and 11,000 multiple myeloma patients die of the disease each year (1). Multiple myeloma is a late-stage B-cell malignancy characterized by the infiltration of malignant plasma cells in bone marrow and is associated with high monoclonal protein in the blood. Multiple myeloma can occur de novo or can evolve from benign monoclonal gammapathy of undetermined significance that involves low levels of bone marrow plasmacytosis and monoclonal protein. Approximately 1% of patients with monoclonal gammapathy of undetermined significance develop multiple myeloma each year. The current standard treatments for multiple myeloma are high-dose chemotherapy and stem cell transplantation. Although a wide range of new drugs, including bortezomib (Velcade), thalidomide (Thalomid), and lenalidomide (Revlimid), a thalidomide analogue, have been recently approved, multiple myeloma remains an incurable disease (2). The majority of multiple myeloma patients eventually experience a relapse, their disease becomes chemoresistant, and they die of the disease.

Why multiple myeloma patients develop resistance to chemotherapy is not well understood, but numerous lines of evidence suggest that the transcription factor nuclear factor-κB (NF-κB) may play a major role in pathogenesis of multiple myeloma. First, NF-κB has been shown to be constitutively active in multiple myeloma cell lines (3) and CD138+ multiple myeloma cells obtained from patients (4). Second, the NF-κB-regulated cyclin D1 gene is frequently dysregulated in multiple myeloma patients (5). Third, interleukin-6 (IL-6), which is also regulated by NF-κB, can suppress apoptosis in multiple myeloma cells (6). Fourth, the NF-κB-regulated antiapoptotic proteins, C-reactive protein, and cell adhesion molecules have been linked to chemoresistance in multiple myeloma cells (79). Fifth, two recent reports used a whole genome-based screen approach and showed that cells from multiple myeloma patients have alterations in numerous genes that control the constitutive activation of NF-κB (10, 11). The mechanisms involved are the inactivation of TRAF2, TRAF3, CYLD, cIAP-1, and cIAP-2 and the activation of NF-κB1, NF-κB2, CD40, LTBR, TACI, and NIK. Sixth, NF-κB signaling in stromal cells can lead to production of IL-6, BAFF, or APRIL, which can activate NF-κB and cause proliferation of multiple myeloma cells (12). Based on these lines of evidence, agents that can inhibit NF-κB activation and are pharmacologically safe have the potential to overcome chemoresistance and potentiate the antitumor effects of existing chemotherapeutic agents, such as thalidomide and bortezomib.

Because of its ability to suppress NF-κB activation (13), we hypothesize that curcumin (diferuloylmethane), a pharmacologically safe agent, can reverse chemoresistance and potentiate effects of existing chemotherapy. First, curcumin can suppress NF-κB activation and inhibit the proliferation of multiple myeloma cells (3, 4). Additional support for this hypothesis include the following: curcumin can down-regulate cyclin D1 expression (14); curcumin has been shown to suppress the expression of IL-6 (15); curcumin can inhibit cell survival, proliferation, invasion, and angiogenic gene products (16); curcumin can down-regulate the activation of STAT3 (4, 15), a transcription factor that has also been linked with chemoresistance in multiple myeloma cells (17); and curcumin binds directly to P-glycoprotein and inhibits drug transport (18). In addition, a clinical trial of curcumin showed that it is safe even when consumed at 12 g/d (19). Furthermore, the administration of curcumin at a dose of 2 g/d for 4 weeks to asymptomatic multiple myeloma patients down-regulated NF-κB activation in peripheral blood lymphocytes (20).

We tested our hypothesis by examining whether curcumin overcomes the resistance of multiple myeloma cells to chemotherapy and potentiates the antitumor effects of thalidomide and bortezomib. Our results show that curcumin can overcome the chemoresistance of multiple myeloma cells to conventional chemotherapeutic agents and potentiate the effects of bortezomib and thalidomide by inhibiting the NF-κB activation pathway, which leads to the down-regulation of NF-κB-regulated antiapoptotic gene products.

Materials

Curcumin (77.5% curcumin, 18.27% demethoxycurcumin, and 4.21% bisdemethoxycurcumin; also called C3 complex) was kindly supplied by Sabinsa. A 25 mmol/L solution was prepared in DMSO, stored as small aliquots at −20°C, and diluted as needed in cell culture medium. Penicillin, streptomycin, and RPMI 1640 were obtained from Invitrogen. Fetal bovine serum was obtained from Atlanta Biologicals. Bortezomib (PS-341) was obtained from Millennium, and thalidomide was obtained from Tocris Cookson. Dexamethasone, melphalan, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, and anti-β-actin were obtained from Sigma-Aldrich. Antibodies against poly(ADP-ribose) polymerase, cIAP-1, Bcl-2, Bcl-xL, and TRAF1 were obtained from Santa Cruz Biotechnology. Anti-XIAP and survivin antibodies were obtained from BD Biosciences. Anti-vascular endothelial growth factor (VEGF) and Ki-67 antibodies were obtained from NeoMarkers. Polyclonal antibody against p65 was obtained from Abcam. γ32P-ATP was obtained from ICN Pharmaceuticals. Phosphospecific anti-Akt (Ser473) and anti-Akt antibodies were obtained from Cell Signaling.

Cell Lines

Human multiple myeloma cell lines U266, RPMI-8226, RPMI-8226-Dox-6 (a doxorubicin-resistant clone), RPMI-8226-LR-5 (a melphalan-resistant clone), MM.1 (also called MM.1S), and MM.1R (a dexamethasone-resistant variant of MM.1) cell lines were described previously (21). The U266, RPMI-8226, MM.1, and MM.1R cells as well as the Dox-6 and LR-5 variants were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1× antibiotic/antimycotic.

Proliferation Assay

The antiproliferative effects of curcumin on drug-sensitive and drug-resistant cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide uptake method as described previously (21).

Activation of NF-κB in Multiple Myeloma Cells and Tumor Samples

To assess NF-κB activation, we isolated nuclei from multiple myeloma cell lines and tumor samples and carried out electrophoretic mobility shift assays (EMSA) essentially as described previously (22). In brief, nuclear extracts prepared from multiple myeloma cancer cells (1 × 106/mL) and tumor samples were incubated with 32P-end-labeled 45-mer double-stranded NF-κB oligonucleotide from the HIV long terminal repeat (5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′; boldface indicates NF-κB binding sites) for 15 min at 37°C. The resulting DNA-protein complex was separated from free oligonucleotides on 6.6% native polyacrylamide gels. To verify the equal loading of nuclear proteins, the binding of Oct-1 to DNA was determined by incubating 15 μg nuclear extracts with 16 fmol 32P-end-labeled with the octamer-binding protein (Oct-1) consensus oligonucleotide 5′-TGTCGAATGCAAATCACTAGAA-3′ (boldface indicates Oct-1 binding site) for 30 min at 37°C and then analyzed using 5% native polyacrylamide gel. The dried gels were visualized, and radioactive bands were quantitated with a PhosphorImager (Molecular Dynamics) using ImageQuant software.

Western Blot Analysis

To determine the expression levels of the cIAP-1, XIAP, survivin, Bcl-2, Bcl-xL, TRAF1, VEGF, and poly(ADP-ribose) polymerase proteins in the curcumin-treated cells, we subjected the whole-cell extracts to Western blot analysis as described previously (23). In brief, the whole-cell extracts were prepared by lysing curcumin-treated cells with lysis buffer [20 mmol/L Tris (pH 7.4), 250 mmol/L NaCl, 2 mmol/L EDTA (pH 8.0), 0.1% Triton X-100, 0.01 mg/mL aprotinin, 0.005 mg/mL leupeptin, 0.4 mmol/L phenylmethylsulfonyl fluoride, and 4 mmol/L sodium orthovanadate] and loaded. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blocked with 5% nonfat milk, and probed with various antibodies overnight at 4°C. The blots were then washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h, and finally examined using an enhanced chemiluminescence reagent (Amersham).

Apoptosis Assay

To determine whether curcumin potentiates the apoptotic effects of bortezomib and thalidomide in multiple myeloma cells, we used a Live/Dead assay kit (Molecular Probes), which determines intracellular esterase activity and plasma membrane integrity. This assay uses calcein, a polyanionic, green fluorescent dye that is retained within live cells, and a red fluorescent ethidium bromide homodimer dye that can enter cells through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membranes of live cells (23). In brief, cells (1 × 106 per well) were incubated in 12-well plates, pretreated with curcumin for 4 h, and treated with either thalidomide or bortezomib for 24 h. Cells were then stained with the assay reagents for 30 min at ambient temperature. Cell viability was determined under a fluorescence microscope by counting live (green) and dead (red) cells. This experiment was repeated twice, and the statistical analysis was done. The values were initially subjected to one-way ANOVA, which revealed significant differences between groups, and then compared among groups using an unpaired Student’s t test (P < 0.05 considered to be statistically significant), which revealed significant differences between the two sample means.

Annexin V Assay

One of the early indicators of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cell’s cytoplasmic interface to the extracellular surface. This loss of membrane asymmetry can be detected by using the binding properties of Annexin V. This assay was done as described previously (23).

Animals

Male athymic nu/nu mice (4 weeks old) were obtained from the breeding colony of the Department of Experimental Radiation Oncology at The University of Texas M. D. Anderson Cancer Center. The animals were housed (5 mice per cage) in the standard mice Plexiglass cages in a room maintained at constant temperature and humidity under 12 h light and dark cycle and fed with regular autoclaved mouse chow with water ad libitum. None of the mice exhibited any lesions and all were tested pathogen-free. Before initiating the experiment, we acclimatized all mice to a pulverized diet for 3 days. Our experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas M. D. Anderson Cancer Center.

Implantation of U266 Cells

U266 cells were injected s.c. into the mice as described previously (24). In brief, U266 cells were harvested from subconfluent cultures, washed once in serum-free medium, and resuspended in PBS. Only suspensions consisting of single cells, with >90% viability, were used for the injections. Mice were anesthetized with a ketamine-xylazine solution, and U266 cells [2 × 106 per 100 μL PBS/Matrigel (1:1)] were injected s.c. into the left flank of the mice using a 25-gauge needle and a calibrated push button-controlled dispensing device (Hamilton Syringe). To prevent leakage, a cotton swab was held cautiously for 1 min over the site of injection.

Experimental Protocol

After 1 week of implantation, tumor diameters were measured using Vernier calipers. The mice were then randomized into treatment groups (n = 5) based on the tumor volume. Group I (control) was treated with corn oil (100 μL, orally, daily) and saline (100 μL, orally, weekly), group II was treated with curcumin alone (1 g/kg, orally, daily), group III was treated with bortezomib alone (0.25 mg/kg, 100 μL, i.p., weekly), and group IV was treated with a combination of curcumin (1 g/kg, orally, daily) and bortezomib (0.25 mg/kg, 100 μL, weekly). Treatment was continued for up to 20 days from the date of randomization (day 0). The tumor volume was measured at 5-day intervals. The mice were killed 25 days after randomization. The tumors were carefully excised and measured to calculate tumor volume. The tumor volume was derived using the formula: V = 2/3πr3, where r is the mean of the three dimensions (length, width, and depth). The tumor volumes were initially subjected to one-way ANOVA and then later compared among groups using an unpaired Student’s t test, with P < 0.05 considered to be significant.

Preparation of Nuclear Extract from Tumor Samples

Multiple myeloma tumor tissues (75-100 mg/mouse) from control and treated mice were minced and incubated on ice for 30 min in 0.5 mL ice-cold buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L KCl, 10 mmol/L MgCl2, 0.5 mmol/L DTT, 0.1% Igepal CA-630, and 0.5 mmol/L phenylmethylsulfonyl fluoride]. The minced tissue was homogenized using a Dounce homogenizer and centrifuged at 16,000 × g at 4°C for 10 min. The resulting nuclear pellet was suspended in 0.2 mL buffer B [20 mmol/L HEPES (pH 7.9), 25% glycerol, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.5 mmol/L DTT, 0.2 mmol/L EDTA, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 2 μg/mL leupeptin] and incubated on ice for 2 h with intermittent mixing. The suspension was then centrifuged at 16,000 × g at 4°C for 30 min. The supernatant (nuclear extract) was collected and stored at −70°C until use (25).

Immunolocalization of NF-κB p65 and VEGF in Tumor Samples

The nuclear localization of p65 and the expression of VEGF was examined using an immunohistochemical method described previously (25). In brief, multiple myeloma tumor samples were embedded in paraffin and fixed with paraformaldehyde. After being washed in PBS, the slides were blocked with protein block solution (DakoCytomation) for 20 min and then incubated overnight with rabbit polyclonal anti-human p65 and mouse monoclonal anti-human VEGF antibodies (1:400 and 1:100, respectively). Slides were washed and then incubated with biotinylated link universal antiserum followed by horseradish peroxidase-streptavidin conjugate (LSAB+ kit). The slides were rinsed, and color was developed using 3,3′-diaminobenzidine hydrochloride as a chromogen. Finally, sections were rinsed in distilled water, counterstained with Mayer’s hematoxylin, and mounted with DPX mounting medium for evaluation. Pictures were captured with a Photometrics CoolSnap CF color camera (Nikon) using MetaMorph software (version 4.6.5; Universal Imaging).

Ki-67 Immunohistochemistry

Frozen sections (5 μm) were stained with anti-Ki-67 antibody as described previously (25). Results were expressed as percentage ± SE of Ki-67+ cells per ×40 magnification. A total of ten ×40 fields were examined and counted from three tumors of each treatment group. The values were initially subjected to one-way ANOVA and then later compared among groups using an unpaired Student’s t test, with P < 0.05 considered to be significant.

Microvessel Density

Frozen sections (5 μm) were stained with rat anti-mouse CD31 monoclonal antibody (Pharmingen) as described previously (25). Areas of greatest vessel density were then examined under higher magnification (×100) and counted. Any distinct area of positive staining for CD31 was counted as a single vessel. Results were expressed as the mean ± SE number of vessels per high-power field (×100). A total of 20 high-power fields were examined and counted from three tumors of each treatment group. The values were initially subjected to one-way ANOVA and then later compared among groups using an unpaired Student’s t test, with P < 0.05 considered to be significant.

The goals of this study were (a) to determine whether curcumin (Fig. 1A) sensitizes chemoresistant multiple myeloma cells that have developed resistance to conventional chemotherapeutic agents, (b) to determine whether curcumin potentiates the antitumor effects of thalidomide and bortezomib, two chemotherapeutic agents used extensively to treat multiple myeloma patients, (c) to determine whether curcumin potentiates the effects of these chemotherapeutic agents in vivo, and (d) to determine the mechanism by which curcumin sensitizes multiple myeloma cells to these drugs. We used multiple myeloma cell lines that are sensitive and resistant to dexamethasone, doxorubicin, and melphalan in our studies.

Curcumin Suppresses the Proliferation of Drug-Sensitive and Drug-Resistant Multiple Myeloma Cells

To determine whether curcumin suppresses the proliferation of drug-sensitive and drug-resistant human multiple myeloma cells, we tested the cell proliferation using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Curcumin suppressed the proliferation of all multiple myeloma cell types tested, including U266 cells, MM.1R cells (resistant to dexamethasone), RPMI-8226-Dox-6 cells (resistant to doxorubicin), and RPMI-8226-LR-5 cells (resistant to melphalan) in a dose- and time-dependent manner (Fig. 1B). Whether a cell line is a sensitive or resistant to a conventional chemotherapeutic agent, no difference in the sensitivity to curcumin was observed.

Curcumin Potentiates the Apoptotic Effects of Bortezomib and Thalidomide against Multiple Myeloma Cells

Bortezomib, a proteasome inhibitor, and thalidomide, a tumor necrosis factor inhibitor, have been approved for the conventional treatment of multiple myeloma patients. To determine whether curcumin potentiates the apoptotic effect of these drugs, we treated U266 cells with curcumin combined with either bortezomib or thalidomide and then examined these cells with a Live/Dead assay. Curcumin potentiated the apoptotic effect of bortezomib from 25% to 85% (Fig. 2A, middle) and thalidomide from 10% to 75% (Fig. 2A, right).

To further confirm the potentiating effect of curcumin on bortezomib and thalidomide, we used the Annexin V method, which detects an early stage of apoptosis. Again, curcumin enhanced the apoptotic effects of bortezomib from 16% to 60% and thalidomide from 18% to 57% (Fig. 2B).

When we examined the poly(ADP-ribose) polymerase cleavage, which indicates caspase-3 activation, a well-known characteristic of apoptosis, we found that curcumin potentiated the effect of bortezomib (Fig. 2C, top) and thalidomide (Fig. 2C, bottom). The potentiating effect of curcumin was more pronounced on thalidomide than on bortezomib. This may be because bortezomib induces apoptosis by activating caspase-9, whereas thalidomide induces apoptosis by activating caspase-8 (26).

Curcumin Potentiates the Inhibitory Effect of Bortezomib and Thalidomide on Constitutive NF-κB Activation in Multiple Myeloma Cells

Our results indicate that curcumin can potentiate the apoptotic effects of bortezomib and thalidomide. How the effects were potentiated was investigated. The constitutive activation of NF-κB is associated with growth and survival of cancer cells, including multiple myeloma cells (27, 28). In addition, studies have shown that bortezomib and thalidomide have been shown to suppress NF-κB activation (29, 30). To determine whether the apoptotic effects of curcumin alone and the combination of curcumin with these drugs correlates with down-regulation of NF-κB activation was examined. To investigate this, we incubated U266 cells with suboptimal concentrations of curcumin, bortezomib, and thalidomide alone or a combination with curcumin and these drugs and then examined NF-κB activation by EMSA. Curcumin potentiated the inhibitory effect of bortezomib and thalidomide on NF-κB activation in multiple myeloma cells (Fig. 2D).

Curcumin Suppresses Akt Activation in Multiple Myeloma Cells

The phosphatidylinositol 3-kinase/Akt pathway is one of the signaling pathways whose dysregulation has been linked with chemoresistance in multiple myeloma cells (31). We investigated whether curcumin suppressed the activation of Akt in multiple myeloma cells. We found that Akt was constitutively active in U266 cells and that curcumin suppressed the levels of phosphorylated Akt in a time-dependent manner (Fig. 3A), indicating that these reduced levels of activated Akt may contribute toward increasing the apoptosis in multiple myeloma cells.

Curcumin Inhibits the Expression of NF-κB-Regulated Gene Products in Multiple Myeloma Cells

Numerous proteins, including cyclin D1, survivin, Bcl-xL, cIAP-1, XIAP, Bcl-2, TRAF1, and VEGF, have been linked with chemoresistance; all regulated by NF-κB. To determine whether curcumin mediates its effects by down-regulating the expression of NF-κB-regulated proteins, we examined the effect of curcumin on the expression of NF-κB-regulated gene products implicated in cell proliferation (cyclin D1), antiapoptosis (cIAP-1, XIAP, survivin, Bcl-2, Bcl-xL, and TRAF1), and angiogenesis (VEGF). Our results showed that curcumin down-regulated the constitutive expression of cyclin D1, cIAP-1, XIAP, survivin, Bcl-2, Bcl-xL, and TRAF1 and VEGF in U266 cells (Fig. 3B). The effect of curcumin on apoptosis showed the consistent to our previous report (3).

Curcumin Potentiates the Antitumor Effects of Bortezomib in Human Multiple Myeloma Xenograft in Nude Mice

To determine whether curcumin enhances the antitumor effects of bortezomib against multiple myeloma, we developed a human multiple myeloma xenograft in nude mice using U266 cells. A week after implantation, the animals were randomized into four treatment groups based on tumor volume. Treatment was started 1 week after tumor cell implantation and was continued up to 20 days, in accordance with the experimental protocol (Fig. 4A). The tumor diameters were measured at 5-day intervals. Animals were killed 32 days after tumor cell injection and 25 days after the treatment start date (Fig. 4A), and the tumors were excised and the tumor diameters were measured. We found that the tumor volume increased rapidly in the control group compared with the other treatment groups (Fig. 4B). Both curcumin alone and bortezomib alone significantly decreased the tumor volume; the tumor volume in the curcumin + bortezomib group was significantly lower than that in the bortezomib alone group and in the control group at day 25 after treatment (P < 0.001, vehicle versus curcumin + bortezomib; P < 0.001, bortezomib versus curcumin + bortezomib; Fig. 4C). When examined for tumor volume on different days, we found that curcumin + bortezomib combination was much more effective in reducing the tumor volume compared with either agent alone (P < 0.001 versus vehicle; Fig. 4D).

Curcumin Inhibits NF-κB Activation in Human Multiple Myeloma Xenograft in Nude Mice

We evaluated whether the effects of curcumin on multiple myeloma tumor growth were associated with the inhibition of NF-κB activation. The DNA-binding assay for NF-κB in nuclear extracts from tumor samples showed that curcumin alone significantly suppressed NF-κB activation. The effect of bortezomib alone was less pronounced than curcumin in inhibiting NF-κB activation (Fig. 5A). Interestingly, the curcumin + bortezomib combination was not more effective than curcumin alone (P < 0.001 versus vehicle).

We also examined whether the EMSA results were consistent with the immunohistochemical analyses. We found that the nuclear translocation of p65 was significantly inhibited by curcumin alone but not by bortezomib alone, which confirmed the EMSA results (Fig. 5B, left and right).

Curcumin Down-Regulates the Expression of the Cell Proliferation Marker Ki-67 in Human Multiple Myeloma Xenograft in Nude Mice

To determine whether curcumin decreases multiple myeloma tumor growth by inhibiting proliferation, we examined the expression of Ki-67-positive cells in multiple myeloma tumors from mice. Our results showed that curcumin alone and bortezomib alone significantly decreased the expression of Ki-67 in tumor tissue (Fig. 5C, left and right). The curcumin + bortezomib combination was more effective in reducing Ki-67 expression than either agent alone (P < 0.001 versus vehicle).

Curcumin Inhibits Angiogenesis and Down-Regulates the Expression of VEGF in Human Multiple Myeloma Xenograft in Nude Mice

To determine whether curcumin decreases multiple myeloma tumor growth by inhibiting angiogenesis, we examined the expression of the microvessel density marker CD31+ in multiple myeloma tumors from nude mice. Our results showed that curcumin alone and bortezomib alone significantly reduced microvessel density. The curcumin + bortezomib combination was more effective in reducing microvessel density than either agent alone (Fig. 6A, left and right; P < 0.001 versus vehicle).

Because VEGF plays an important role in angiogenesis, we also examined its expression in multiple myeloma tumors. We found that both curcumin alone and bortezomib alone effectively suppressed VEGF expression and that the curcumin + bortezomib combination had the highest inhibitory effect on VEGF expression in multiple myeloma tumors (Fig. 6B, left and right).

Despite the fact that several treatments for multiple myeloma are currently available, multiple myeloma remains an incurable disease, with a median survival time of 3 to 5 years (3234). The disease relapses in the majority of multiple myeloma patients regardless of the treatment regimen or their initial response to a given treatment. Recently, three new agents (bortezomib, thalidomide, and lenalidomide) were approved for the treatment of multiple myeloma patients. Bortezomib (Velcade) was approved for the treatment of multiple myeloma patients who have received at least one prior therapy, thalidomide (Thalomid) in combination with dexamethasone was approved for the treatment of newly diagnosed multiple myeloma patients, and the thalidomide analogue lenalidomide (Revlimid) in combination with dexamethasone was recently approved for the treatment of multiple myeloma patients who have received one prior therapy (35). All these three drugs mediate their antimyeloma activities through modulation of intracellular signaling pathways within tumor cells and microenvironments (36, 37). Preclinical studies have shown that these drugs induce apoptosis in myeloma cells resistant to melphalan, doxorubicin, and dexamethasone and potentiate the antimyeloma activity of conventional therapies (38, 39). However, these drugs exhibit several side effects, such as fatigue, anemia, and peripheral neuropathy. Furthermore, studies have shown that patients eventually develop resistance to these drugs. Thus, new treatment approaches that are more effective than conventional therapies against multiple myeloma are required to improve the outcome and extend the survival of multiple myeloma patients.

Because the transcription factor NF-κB has been closely linked with chemoresistance, cell survival, and proliferation, we investigated whether curcumin suppresses the proliferation of multiple myeloma cells that have developed resistance to chemotherapeutic agents. We found that curcumin suppressed human multiple myeloma cell proliferation regardless of the cells’ sensitivity or resistance to melphalan, doxorubicin, or dexamethasone. Similar to our results, these drug-resistant cells have been shown to be sensitive to bortezomib and thalidomide (38, 40). We also found that curcumin potentiated the antitumor effects of bortezomib and thalidomide. What is the mechanism of resistance of multiple myeloma to melphalan, doxorubicin, or dexamethasone? First, cells resistant to chemotherapeutic agents have been shown to express increased activation of NF-κB and suppression of this NF-κB can sensitize the cells to the drug (41). Second, multiple myeloma cells and mantel cell lymphoma are known to express constitutive active NF-κB that is resistant to bortezomib (42, 43). Our results indicate that the mechanism of potentiation is the suppression of NF-κB activation. Why NF-κB is constitutively active in multiple myeloma cells is not fully understood. Recently, two studies found mutations on the genes encoding positive and negative regulators of NF-κB signaling in many multiple myeloma cell lines and primary tumor cells; these mutations are thought to mediate the constitutive activation of NF-κB in multiple myeloma cells (10, 11). It is likely that the constitutive activation of NF-κB in multiple myeloma cells leads to chemoresistance. For example, C-reactive protein, whose expression is regulated by NF-κB, has been shown to enhance the proliferation of myeloma cells and protect myeloma cells from chemotherapy-induced apoptosis both in vitro and in vivo (8). C-reactive protein has also been shown to bind to Fcγ receptors and activate Akt, extracellular signal-regulated kinase, and NF-κB pathways. C-reactive protein also enhanced the secretion of IL-6 and synergized with IL-6 to protect myeloma cells from chemotherapy-induced apoptosis. In our study, we showed that curcumin down-regulates Akt activation, another possible mechanism that curcumin sensitizes tumor cells to chemotherapeutic agents. Previously, we showed that curcumin also down-regulates the expression of IL-6 (4), a major growth factor for multiple myeloma cells. We also found that curcumin down-regulated various proliferative and antiapoptotic proteins, including cyclin D1, cIAP-1, XIAP, survivin, Bcl-2, Bcl-xL, and TRAF1. As mentioned above, cyclin D1, Bcl-2, Bcl-xL, and survivin have been linked with chemoresistance in multiple myeloma cells (4446). Thus, in addition to the suppression of NF-κB activation, these mechanisms may help explain the effects of curcumin.

Like curcumin, bortezomib inhibits NF-κB activation but through different mechanisms. The pathway by which bortezomib prevents degradation of IκB differs from that of curcumin. Bortezomib inhibits the proteasome, resulting in the accumulation of IκBα, whereas curcumin prevents IκB phosphorylation, thus blocking its subsequent ubiquitination and degradation through suppression of upstream kinase IKK. Thus, these different mechanisms of NF-κB suppression provide the rationale for combining these agents to effectively inhibit NF-κB activation. Furthermore, other mechanisms of bortezomib and curcumin may support their combination to maximize their cytotoxicity against multiple myeloma cells. For instance, curcumin is a potent blocker of STAT3 activation (15), a transcription factor that has been linked with chemoresistance (47). Moreover, bortezomib has been shown to overcome chemoresistance in multiple myeloma cells both in the laboratory (41, 48) and in the clinic (49). Indeed, bortezomib has been shown to exhibit considerable clinical efficacy against multiple myeloma. Whether this activity is due to its anti-NF-κB activity is not clear (35), as its ability to suppress NF-κB in patients has not been shown.

We next determined whether curcumin potentiates the effects of bortezomib on human multiple myeloma xenografts in nude mice. Our results showed that curcumin alone and bortezomib alone inhibited the multiple myeloma xenograft in nude mice. We found that the curcumin + bortezomib combination had a higher antimyeloma effect than either agent alone. When we investigated the mechanism, we found that curcumin down-regulated the expression of NF-κB, the cell proliferation marker Ki-67, and the microvessel density marker CD31, all have been linked with chemoresistance. Because curcumin alone inhibited most of NF-κB activity with partial antitumor effect, the down-regulation of NF-κB by curcumin alone may not be sufficient to explain the effect of this agent in vivo. Moreover, when combined with bortezomib in vivo, there is slight increase in NF-κB DNA-binding activity, although this combination leads to the greatest effect on myeloma growth in vivo. Thus, it is possible that the in vivo effect of curcumin is not entirely mediated by its ability to suppress NF-κB. Curcumin has been shown to modulate multiple targets (50) that may be involved in its in vivo antitumor action.

Multiple myeloma is usually diagnosed in elderly patients who are unable to tolerate highly toxic drugs, so curcumin, a pharmacologically safe compound, is a viable therapeutic agent for these multiple myeloma patients. A recent phase I study of curcumin showed that this agent can be administered safely at oral doses of up to 12 g/d (19). There was no dose-limiting toxicity; dosing was limited by the number of pills that patients could or would swallow daily. In addition, a recent phase II clinical trial with curcumin in pancreatic cancer patients showed that tumor progression was suppressed by 73% in some patients (51). Clinical trials with asymptomatic multiple myeloma patients have shown that curcumin down-regulates NF-κB activation in peripheral blood mononuclear cells even at dose of 2 g/d (20). Thus, curcumin can be used (a) to enhance the effects of current chemotherapeutic agents and (b) to overcome chemoresistance in multiple myeloma to conventional therapy. Fatigue and peripheral neuropathy are two symptoms commonly observed in multiple myeloma patients. Because these symptoms are mediated through the expression of proinflammatory cytokines, curcumin may be used to alleviate these symptoms. In fact, curcumin has been shown to reverse these symptoms (5254), which further supports its use in multiple myeloma patients.

In conclusion, the chemoresistance remains a major challenge in the treatment of patients with multiple myeloma as well as other cancers. Multiple myeloma patients who have relapsed after conventional dose chemotherapy or stem cell transplantation are typically treated with high-dose corticosteroids, thalidomide, or bortezomib. However, a large number of these patients do not respond to treatment with these agents. Moreover, prolonged exposure leads to the development of resistance and toxicity, and progression-free and overall survival times for multiple myeloma patients are short. The ability of curcumin to suppress NF-κB activation, down-regulate the expression of cyclin D1 and Bcl-xL, inhibit cell proliferation, potentiate the effects of bortezomib and thalidomide, and overcome chemoresistance provides a sound basis for conducting clinical trials with curcumin, alone or in combination with other agents, to enhance treatment efficacy, reduce toxicity, and overcome chemoresistance of relapsed or refractory multiple myeloma.

Curcumin is commonly available from numerous health food stores and various companies, including Indogen which was set up by spouse of one of the authors (B.B. Aggarwal). No potential conflicts of interest were disclosed.

We thank Lionel Santibañez for carefully editing this article.

1
Ries
LG
,
Alves
MC
,
Berzin
F
. 
Asymmetric activation of temporalis, masseter, and sternocleidomastoid muscles in temporomandibular disorder patients
.
Cranio
2008
;
26
:
59
64
.
2
Hideshima
T
,
Anderson
KC
. 
Preclinical studies of novel targeted therapies
.
Hematol Oncol Clin North Am
2007
;
21
:
1071
91
, viii-ix.
3
Bharti
AC
,
Donato
N
,
Singh
S
,
Aggarwal
BB
. 
Curcumin (diferuloylmethane) down-regulates the constitutive activation of nuclear factor-κB and IκBα kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosis
.
Blood
2003
;
101
:
1053
62
.
4
Bharti
AC
,
Shishodia
S
,
Reuben
JM
, et al
. 
Nuclear factor-κB and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis
.
Blood
2004
;
103
:
3175
84
.
5
Bergsagel
PL
,
Kuehl
WM
,
Zhan
F
,
Sawyer
J
,
Barlogie
B
,
Shaughnessy
J
 Jr
. 
Cyclin D dysregulation: an early and unifying pathogenic event in multiple myeloma
.
Blood
2005
;
106
:
296
303
.
6
Lichtenstein
A
,
Tu
Y
,
Fady
C
,
Vescio
R
,
Berenson
J
. 
Interleukin-6 inhibits apoptosis of malignant plasma cells
.
Cell Immunol
1995
;
162
:
248
55
.
7
Schwarze
MM
,
Hawley
RG
. 
Prevention of myeloma cell apoptosis by ectopic bcl-2 expression or interleukin 6-mediated up-regulation of bcl-xL
.
Cancer Res
1995
;
55
:
2262
5
.
8
Yang
J
,
Wezeman
M
,
Zhang
X
, et al
. 
Human C-reactive protein binds activating Fcγ receptors and protects myeloma tumor cells from apoptosis
.
Cancer Cell
2007
;
12
:
252
65
.
9
Landowski
TH
,
Gleason-Guzman
MC
,
Dalton
WS
. 
Selection for drug resistance results in resistance to Fas-mediated apoptosis
.
Blood
1997
;
89
:
1854
61
.
10
Keats
JJ
,
Fonseca
R
,
Chesi
M
, et al
. 
Promiscuous mutations activate the noncanonical NF-κB pathway in multiple myeloma
.
Cancer Cell
2007
;
12
:
131
44
.
11
Annunziata
CM
,
Davis
RE
,
Demchenko
Y
, et al
. 
Frequent engagement of the classical and alternative NF-κB pathways by diverse genetic abnormalities in multiple myeloma
.
Cancer Cell
2007
;
12
:
115
30
.
12
Moreaux
J
,
Legouffe
E
,
Jourdan
E
, et al
. 
BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone
.
Blood
2004
;
103
:
3148
57
.
13
Singh
S
,
Aggarwal
BB
. 
Activation of transcription factor NF-κB is suppressed by curcumin (diferuloylmethane) [corrected]
.
J Biol Chem
1995
;
270
:
24995
5000
.
14
Mukhopadhyay
A
,
Bueso-Ramos
C
,
Chatterjee
D
,
Pantazis
P
,
Aggarwal
BB
. 
Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines
.
Oncogene
2001
;
20
:
7597
609
.
15
Bharti
AC
,
Donato
N
,
Aggarwal
BB
. 
Curcumin (diferuloylmethane) inhibits constitutive and IL-6-inducible STAT3 phosphorylation in human multiple myeloma cells
.
J Immunol
2003
;
171
:
3863
71
.
16
Aggarwal
S
,
Ichikawa
H
,
Takada
Y
,
Sandur
SK
,
Shishodia
S
,
Aggarwal
BB
. 
Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IκBα kinase and Akt activation
.
Mol Pharmacol
2006
;
69
:
195
206
.
17
Catlett-Falcone
R
,
Landowski
TH
,
Oshiro
MM
, et al
. 
Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells
.
Immunity
1999
;
10
:
105
15
.
18
Limtrakul
P
,
Chearwae
W
,
Shukla
S
,
Phisalphong
C
,
Ambudkar
SV
. 
Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin
.
Mol Cell Biochem
2007
;
296
:
85
95
.
19
Lao
CD
,
Normolle
D
, et al
. 
Dose escalation of a curcuminoid formulation
.
BMC Complement Altern Med
2006
;
6
:
10
.
20
Vadhan-Raj
S
,
Weber
D
,
Giralt
S
, et al
. 
Curcumin downregulates NF-κB and related gene in patients with multiple myeloma: results in phase 1/2 study
.
Am Soc Hematol
2007
(abstract).
21
Bhardwaj
A
,
Sethi
G
,
Vadhan-Raj
S
, et al
. 
Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-κB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells
.
Blood
2007
;
109
:
2293
302
.
22
Chaturvedi
MM
,
Mukhopadhyay
A
,
Aggarwal
BB
. 
Assay for redox-sensitive transcription factor
.
Methods Enzymol
2000
;
319
:
585
602
.
23
Sung
B
,
Pandey
MK
,
Ahn
KS
, et al
. 
Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-κB-regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-κBα kinase, leading to potentiation of apoptosis
.
Blood
2008
;
111
:
4880
91
.
24
Bhutani
M
,
Pathak
AK
,
Nair
AS
, et al
. 
Capsaicin is a novel blocker of constitutive and interleukin-6-inducible STAT3 activation
.
Clin Cancer Res
2007
;
13
:
3024
32
.
25
Kunnumakkara
AB
,
Diagaradjane
P
,
Guha
S
, et al
. 
Curcumin sensitizes human colorectal cancer xenografts in nude mice to γ-radiation by targeting nuclear factor-κB-regulated gene products
.
Clin Cancer Res
2008
;
14
:
2128
36
.
26
Mitsiades
N
,
Mitsiades
CS
,
Poulaki
V
, et al
. 
Apoptotic signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma cells: therapeutic implications
.
Blood
2002
;
99
:
4525
30
.
27
Ni
H
,
Ergin
M
,
Huang
Q
, et al
. 
Analysis of expression of nuclear factor κB (NF-κB) in multiple myeloma: downregulation of NF-κB induces apoptosis
.
Br J Haematol
2001
;
115
:
279
86
.
28
Hideshima
T
,
Chauhan
D
,
Richardson
P
, et al
. 
NF-κB as a therapeutic target in multiple myeloma
.
J Biol Chem
2002
;
277
:
16639
47
.
29
Palombella
VJ
,
Conner
EM
,
Fuseler
JW
, et al
. 
Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis
.
Proc Natl Acad Sci U S A
1998
;
95
:
15671
6
.
30
Majumdar
S
,
Lamothe
B
,
Aggarwal
BB
. 
Thalidomide suppresses NF-κB activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester
.
J Immunol
2002
;
168
:
2644
51
.
31
Kim
D
,
Dan
HC
,
Park
S
, et al
. 
AKT/PKB signaling mechanisms in cancer and chemoresistance
.
Front Biosci
2005
;
10
:
975
87
.
32
Greipp
PR
,
San Miguel
J
,
Durie
BG
, et al
. 
International staging system for multiple myeloma
.
J Clin Oncol
2005
;
23
:
3412
20
.
33
Attal
M
,
Harousseau
JL
,
Facon
T
, et al
. 
Single versus double autologous stem-cell transplantation for multiple myeloma
.
N Engl J Med
2003
;
349
:
2495
502
.
34
Child
JA
,
Morgan
GJ
,
Davies
FE
, et al
. 
High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma
.
N Engl J Med
2003
;
348
:
1875
83
.
35
Richardson
PG
,
Mitsiades
C
,
Schlossman
R
,
Munshi
N
,
Anderson
K
. 
New drugs for myeloma
.
Oncologist
2007
;
12
:
664
89
.
36
Rajkumar
SV
,
Richardson
PG
,
Hideshima
T
,
Anderson
KC
. 
Proteasome inhibition as a novel therapeutic target in human cancer
.
J Clin Oncol
2005
;
23
:
630
9
.
37
Bartlett
JB
,
Dredge
K
,
Dalgleish
AG
. 
The evolution of thalidomide and its IMiD derivatives as anticancer agents
.
Nat Rev Cancer
2004
;
4
:
314
22
.
38
Hideshima
T
,
Chauhan
D
,
Shima
Y
, et al
. 
Thalidomide and its analogs overcome drug resistance of human multiple myeloma cells to conventional therapy
.
Blood
2000
;
96
:
2943
50
.
39
Mitsiades
N
,
Mitsiades
CS
,
Richardson
PG
, et al
. 
The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications
.
Blood
2003
;
101
:
2377
80
.
40
Chauhan
D
,
Li
G
,
Podar
K
, et al
. 
Targeting mitochondria to overcome conventional and bortezomib/proteasome inhibitor PS-341 resistance in multiple myeloma (MM) cells
.
Blood
2004
;
104
:
2458
66
.
41
Ma
MH
,
Yang
HH
,
Parker
K
, et al
. 
The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents
.
Clin Cancer Res
2003
;
9
:
1136
44
.
42
Markovina
S
,
Callander
NS
,
O’Connor
SL
, et al
. 
Bortezomib-resistant nuclear factor-κB activity in multiple myeloma cells
.
Mol Cancer Res
2008
;
6
:
1356
64
.
43
Yang
DT
,
Young
KH
,
Kahl
BS
,
Markovina
S
,
Miyamoto
S
. 
Prevalence of bortezomib-resistant constitutive NF-κB activity in mantle cell lymphoma
.
Mol Cancer
2008
;
7
:
40
.
44
Kornmann
M
,
Danenberg
KD
,
Arber
N
,
Beger
HG
,
Danenberg
PV
,
Korc
M
. 
Inhibition of cyclin D1 expression in human pancreatic cancer cells is associated with increased chemosensitivity and decreased expression of multiple chemoresistance genes
.
Cancer Res
1999
;
59
:
3505
11
.
45
Reed
JC
,
Kitada
S
,
Takayama
S
,
Miyashita
T
. 
Regulation of chemoresistance by the bcl-2 oncoprotein in non-Hodgkin’s lymphoma and lymphocytic leukemia cell lines
.
Ann Oncol
1994
;
5
:
61
5
.
46
Tran
J
,
Master
Z
,
Yu
JL
,
Rak
J
,
Dumont
DJ
,
Kerbel
RS
. 
A role for survivin in chemoresistance of endothelial cells mediated by VEGF
.
Proc Natl Acad Sci U S A
2002
;
99
:
4349
54
.
47
Boehm
AL
,
Sen
M
,
Seethala
R
, et al
. 
Combined targeting of epidermal growth factor receptor, signal transducer and activator of transcription-3, and Bcl-X(L) enhances antitumor effects in squamous cell carcinoma of the head and neck
.
Mol Pharmacol
2008
;
73
:
1632
42
.
48
Hideshima
T
,
Richardson
P
,
Chauhan
D
, et al
. 
The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells
.
Cancer Res
2001
;
61
:
3071
6
.
49
Berenson
JR
,
Yang
HH
,
Sadler
K
, et al
. 
Phase I/II trial assessing bortezomib and melphalan combination therapy for the treatment of patients with relapsed or refractory multiple myeloma
.
J Clin Oncol
2006
;
24
:
937
44
.
50
Aggarwal
BB
,
Sung
B
. 
Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets
.
Trend Pharmacol Sci
2009
;
30
:
85
94
.
51
Dhillon
N
,
Aggarwal
BB
,
Newman
RA
, et al
. 
Phase II trial of curcumin in patients with advanced pancreatic cancer
.
Clin Cancer Res
2008
;
14
:
4491
9
.
52
Sharma
RA
,
Euden
SA
,
Platton
SL
, et al
. 
Phase I clinical trial of oral curcumin: biomarkers of systemic activity and compliance
.
Clin Cancer Res
2004
;
10
:
6847
54
.
53
Maes
M
,
Mihaylova
I
,
Bosmans
E
. 
Not in the mind of neurasthenic lazybones but in the cell nucleus: patients with chronic fatigue syndrome have increased production of nuclear factor κB
.
Neuro Endocrinol Lett
2007
;
28
:
456
62
.
54
Davis
JM
,
Murphy
EA
,
Carmichael
MD
, et al
. 
Curcumin effects on inflammation and performance recovery following eccentric exercise-induced muscle damage
.
Am J Physiol Regul Integr Comp Physiol
2007
;
292
:
R2168
73
.

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