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DOI:10.1158/1535-7163.

MCT-06-0545

Curcumin potentiates the apoptotic effects of chemotherapeutic agents and cytokines through down-regulation of nuclear factor-B and nuclear factor-B regulated gene products in IFN-sensitive and IFN- resistant human bladder cancer cells
Ashish M. Kamat, Gautam Sethi and Bharat B. Aggarwal Mol Cancer Ther 2007;6:1022-1030. Published online March 15, 2007.

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Curcumin potentiates the apoptotic effects of chemotherapeutic agents and cytokines through down-regulation of nuclear factor-KB and nuclear factor-KBregulated gene products in IFN-Asensitive and IFN-Aresistant human bladder cancer cells
Ashish M. Kamat,1 Gautam Sethi,2 and Bharat B. Aggarwal2
Departments of 1Urology and Cancer Biology and 2Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Abstract
Bladder cancer mortality varies between the countries; whereas being highest in Western countries, it is lowest in Eastern countries, such as India. Cigarette smoking is one of the major risk factors for bladder cancer in affluent nations, such as United States. Localized early-stage bladder cancer is treated with resection and intravesical cytokine therapy, whereas metastatic cancer is typically treated with various combinations of systemic chemotherapy. Whether curcumin, a yellow curry pigment commonly consumed in countries, such as India, has any role in prevention or treatment of bladder cancer was investigated. We found that curcumin inhibited the proliferation, induced cell cycle arrest, and DNA fragmentation in both IFN-A sensitive (RT4V6) and IFN-A resistant (KU-7) bladder cancer cells. Curcumin also potentiated the apoptotic effects of the chemotherapeutic agents (gemcitabine and paclitaxel) and of cytokines [tumor necrosis factor (TNF) and TNF-related apoptosisinducing ligand]. This effect of curcumin was independent of sensitivity and resistance to IFN-A, commonly used for treatment of bladder cancer. Whether the effects of curcumin are mediated through modulation of the nuclear

factor-KB (NF-KB) pathway known to mediate antiapoptosis was investigated. Both gemcitabine and TNF activated NF-KB in bladder cancer cells and curcumin suppressed this activation. Similarly, cigarette smoke, a major risk factor for bladder cancer, also activated NF-KB and curcumin suppressed it. Cigarette smoke induced expression of the NF-KB regulated gene products cyclooxygenase-2 and vascular endothelial growth factor, linked with proliferation and angiogenesis, respectively, was also down-regulated by curcumin. [Mol Cancer Ther 2007;6(3):1022 30]

Introduction
Bladder cancer is diagnosed in more than 61,000 people annually in the United States, and 14,000 people die of this disease each year (1). The treatment of early-stage bladder cancer involves complete transurethral resection of tumor followed by instillation of intravesical agents, such as Bacillus Calmette-Guerin (believed to act via induction of cytotoxic cytokines and IFN-a; ref. 2). Despite initial responses, these tumors can eventually recur and can progress to more invasive stages requiring surgical removal of the bladder. Most of the deaths from bladder cancer are due to advanced unresectable disease, which is resistant to chemotherapy (3, 4). For more than a decade, the standard treatment for advanced bladder cancer has been the combination of methotrexate, vinblastine, doxorubicin (Adriamycin), and cisplatin. This regimen is consistently reported to produce median survival durations ranging from 13 to 15 months (5). In spite of considerable effort to escalate the doses of the components of methotrexate, vinblastine, doxorubicin (Adriamycin), and cisplatin, there has been no observed improvement in survival with this regimen. Recently, paclitaxel, gemcitabine, and ifosfamide have been recognized to be quite active against invasive transitional cell carcinoma, and many novel combination regimens have been reported (69). Unfortunately, although some of these newer regimens are less toxic than methotrexate, vinblastine, doxorubicin (Adriamycin), and cisplatin, there is still no compelling evidence that survival rates are improving. Indeed, there is a growing conviction that the cytotoxic paradigm will not provide the means to qualitatively change the outcome for patients with metastatic bladder cancer. Bladder cancer mortality rates vary in different countries. The highest rates are noted in European countries, such as Denmark, the United Kingdom, Belgium, and Italy,
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Received 8/31/06; revised 1/4/07; accepted 1/31/07. Grant support: Clayton Foundation for Research (B.B. Aggarwal and A.M. Kamat), NIH P01 grant CA91844 on lung chemoprevention (B.B. Aggarwal), NIH Bladder Specialized Programs in Research Excellence 5 P50 CA091846-03 (Project PI; A.M. Kamat), and grant 5P30 CA01667232 for flow cytometric analysis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: A.M. Kamat and G. Sethi contributed equally to this work. Requests for reprints: Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Unit 143, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3503; Fax: 713-794-1613; E-mail: aggarwal@mdanderson.org Copyright C 2007 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-06-0545

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whereas the lowest rates are noted in Asian countries, such as India, Japan, China, and Singapore (10). Populations in Southeast Asia have 4- to 10-fold lower incidences of and fewer deaths of bladder cancer than do populations in the United States (11). However, Asian individuals who migrate to the United States have an increased risk of bladder cancer equal to that in Americans in one generation (12). Cigarette smoking is the major risk factor for bladder cancer in affluent nations; exposure to chemical carcinogens in the environment, particularly in the workplace, is also a contributing factor (10, 13). However, a definitive etiology of bladder cancer is still unknown. Considering the unknown etiologic factors for bladder cancer carcinogenesis and the fact that patients with bladder cancer face the threat of both metastasis and uncontrolled local recurrence after treatment, additional efforts to define alternative interventions for the prevention of bladder cancer progression and metastasis are urgently needed. Nuclear factor-nB (NF-nB) is a transcription factor present in the cytoplasm as an inactive heterotrimer consisting of p50, p65, and InBa subunits. On activation, InBa undergoes phosphorylation- and ubiquitinationdependent degradation leading to nuclear translocation and binding to a specific consensus sequence in the DNA, which results in gene transcription. The kinase that phosphorylates InBa is termed InB kinase, which is composed of InB kinase a, InB kinase h, and InB kinase g (also called NF-nB essential modulator). NF-nB regulates the expression of genes involved in antiapoptosis (e.g., bcl-2 and bcl-xl), proliferation [e.g., cyclooxygenase-2 (COX-2) and cyclin D1], and metastasis [e.g., vascular endothelial growth factor (VEGF); refs. 14, 15]. Curcumin (diferuloylmethane), a yellow curry pigment derived from turmeric (Curcuma longa), is a pharmacologically safe agent that has been shown to suppress NFnB activation and NF-nB gene products (16, 17). We did the study described herein to determine whether treatment with curcumin alone or in combination with chemotherapeutic agents or cytokines has a role in modulating the proliferation and apoptosis of bladder cancer cells by regulating the NF-nB pathway. Our results showed that curcumin alone can suppress the proliferation of these cells and further enhance the apoptotic effect of chemotherapeutic agents and cytokines through the down-regulation of NF-nB and NF-nB regulated gene products.

Materials and Methods


Reagents Curcumin (purity, >98%) was obtained from Sabinsa Corp. (Piscataway, NJ). Bacteria-derived human recombinant human tumor necrosis factor (TNF) that was purified to homogeneity with a specific activity of 5 107 units/mg was provided by Genentech (South San Francisco, CA). TNF-related apoptosis-inducing ligand (TRAIL) was purchased from R&D Systems (Minneapolis, MN). Cigarette
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smoke condensate prepared as described previously (18) was supplied by C. Gary Gairola (University of Kentucky, Lexington, KY). Penicillin, streptomycin, RPMI 1640, and fetal bovine serum were obtained from Invitrogen (Grand Island, NY). Paclitaxel and an anti h-actin antibody were obtained from Sigma-Aldrich (St. Louis, MO). Gemcitabine was purchased from MD Anderson Pharmacy (manufactured by Eli Lilly, Indianapolis, IN). An antibody against poly(ADP-ribose) polymerase (PARP) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody against VEGF was obtained from NeoMarkers (Fremont, CA). An anti COX-2 antibody was obtained from BD Biosciences (San Diego, CA). Cell Lines and Culture Conditions The human bladder carcinoma cell line KU-7 was obtained from the American Type Culture Collection (Manassas, VA). The cell line RT4V6 was generated in Dr. Kamats laboratory by in vivo recycling of RT4 (ATCC, Manassas, VA). The cells were grown as monolayers in modified Eagles MEM supplemented with 10% fetal bovine serum, vitamins, sodium pyruvate, L-glutamine, nonessential amino acids, and penicillin-streptomycin. Electrophoretic Mobility Shift Assay To assess NF-nB activation, nuclear extracts were prepared, and electrophoretic mobility shift assay (EMSA) was done as described previously (19). The dried gels were visualized, and the radioactive bands were quantitated using the Storm820 and ImageQuant software (Amersham, Piscataway, NJ). Western Blot Analysis To determine the levels of protein expression, whole-cell extracts were prepared (20) and fractionated using SDSPAGE. After electrophoresis, the proteins were electrotransferred onto nitrocellulose membranes, blotted with each antibody, and detected using an enhanced chemiluminescence regent (Amersham). The bands obtained were quantitated using the NIH Image software program (NIH, Bethesda, MD). Live/Dead Assay To measure apoptosis, the Live/Dead assay (Molecular Probes, Carlsbad, CA), which measures intracellular esterase activity and plasma membrane integrity, was used. This assay uses calcein, a polyanionic dye that is retained within the live cells and provides green fluorescence. It also uses the ethidium monomer dye (red fluorescence), which can enter the cells only through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membranes of live cells. Briefly, cells were incubated with curcumin and/or chemotherapeutic agents (paclitaxel and gemcitabine) and cytokines (TNF and TRAIL) at 37jC for 24 h. Thereafter, cells were stained with the Live/Dead reagent (5 Amol/L ethidium homodimer and 5 Amol/L calcein-AM) and then incubated at 37jC for 30 min. Cells were then analyzed under a fluorescence microscope (Labophot-2). Annexin V Assay An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid

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Curcumin suppresses proliferation of bladder cancer cells. A, RT4V6 and KU-7 cells (5,000 cells/0.1 mL) were incubated with the indicated concentrations of curcumin at 37jC for 24 h, and cell proliferation was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent. Results are representative of three independent experiments. Columns, cell proliferation; bars, SD (between the triplicates). B, curcumin induces cell cycle arrest in bladder cancer cells. RT4V6 and KU-7 cells (1 106) were incubated with the indicated concentrations of curcumin for 24 h, and cell cycle analysis was done as described in Materials and Methods. Results are representative of three independent experiments. C, curcumin induces DNA fragmentation in bladder cancer cells. RT4V6 and KU-7 cells (1 106) were incubated with the indicated concentrations of curcumin for 48 h, and DNA fragmentation was analyzed as described in Materials and Methods. Columns, DNA fragmentation; bars, SD (between the triplicates).

Figure 1.

phosphatidylserine from the cytoplasmic interface of membrane to the extracellular surface. This loss of membrane asymmetry can be detected by using the binding properties of Annexin V. To identify apoptosis, we used an Annexin V antibody, which was conjugated with the FITC fluorescence dye. Briefly, cells were incubated with curcumin and/or chemotherapeutic agents (paclitaxel and gemcitabine) and cytokines (TNF

and TRAIL) at 37jC for 24 h and subjected to Annexin V staining. The cells were washed in PBS, resuspended in 100 AL of binding buffer containing a FITC-conjugated anti Annexin V antibody, and then analyzed with a flow cytometer (FACSCalibur, BD Biosciences). Immunoblotting Analysis for PARP Cleavage Apoptosis was assessed by determining the proteolytic cleavage of PARP (21). Briefly, cells (2 106/mL) were
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treated with indicated concentrations of curcumin and/or gemcitabine for indicated periods. The cells were then washed and extracted by incubation for 30 min on ice in 0.05 mL of a buffer consisting of 20 mmol/L HEPES (pH 7.4), 2 mmol/L EDTA, 250 mmol/L NaCl, 0.1% NP40, 2 Ag/mL leupeptin, 2 Ag/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 Ag/mL benzamidine, 1 mmol/L DTT, and 1 mmol/L sodium vanadate. The lysate was centrifuged, and the supernatant was collected. Cell extract proteins (30 Ag) were resolved on 7.5% SDS-PAGE gels, electrotransferred onto a nitrocellulose membrane, blotted with a mouse anti-PARP antibody, and then visualized using an enhanced chemiluminescence reagent. Apoptosis was represented by cleavage of 116-kDa PARP into an 85-kDa product.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay The antiproliferative effect of curcumin on bladder cancer cells was determined by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye uptake in bladder cancer cells (22). Briefly, cells were incubated in triplicate in 96-well plates in the presence or absence of indicated concentrations of curcumin in a final volume of 0.1 mL at 37jC for 24 h. Afterward, 0.025 mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg/mL in PBS) was added to each well. After 2 h of incubation at 37jC, 0.1 mL of lysis buffer (20% SDS, 50% dimethylformamide) was added, incubation was continued overnight at 37jC, and the absorbance at 590 nm was read using a 96-well multiscanner

Figure 2. Curcumin potentiates apoptosis induced by chemotherapeutic agents and cytokines. RT4V6 cells (A) and KU-7 cells (B), 1 106, were incubated with 5 Amol/L curcumin alone or in combination with 5 nmol/L gemcitabine, 300 nmol/L paclitaxel, 1 nmol/L TNF, and 1 ng/mL TRAIL for 24 h. Cells were stained with the Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope. RT4V6 cells (C) and KU-7 cells (D), 1 106, were incubated with 5 Amol/L curcumin alone or in combination with 5 nmol/L gemcitabine, 300 nmol/L paclitaxel, 1 nmol/L TNF, and 1 ng/mL TRAIL for 24 h. Cells were incubated with anti Annexin V antibody conjugated with FITC and then analyzed with a flow cytometer for early apoptotic effects. Results are representative of three independent experiments. Mol Cancer Ther 2007;6(3). March 2007

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1026 Curcumin Induces Apoptosis and Suppresses NF-kB

ethanol. After incubation overnight at 20jC, cells were washed with PBS and then suspended in a staining buffer (10 Ag/mL propidium iodide, 0.5% Tween 20, 0.1% RNase in PBS). The cells were analyzed using a FACSVantage flow cytometer with the CellQuest acquisition and analysis software program (Becton Dickinson and Co., San Jose, CA). Gating was set to exclude cell debris, doublets, and clumps.

Results
The focus of this study was to determine whether curcumin has a role in prevention or treatment of bladder cancer. Apoptosis, NF-nB, COX-2, and VEGF, which we used as targets, are relevant to both prevention and treatment of cancer (25 28). Throughout, we used two different bladder cancer cell lines: RT4V6, which is highly sensitive to IFN-a and TRAIL, and KU-7, which is resistant to IFN-a and TRAIL. Curcumin Suppresses the Proliferation of Bladder Cancer Cells Because NF-nB has been implicated in cell survival and proliferation (29, 30), we examined the effect of treatment with curcumin on the proliferation of RT4V6 and KU-7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Treatment with curcumin at a concentration as low as 5 Amol/L inhibited the growth of both cell types (Fig. 1A); at 25 Amol/L, the treatment significantly suppressed the growth of both cell types. We also examined whether curcumin has any effect on cell cycle by using flow cytometric analysis. The results showed a significant, dose-dependent increase in the percentage of cells in the S phase of cell cycle (Fig. 1B). These results clearly showed that curcumin induces arrest of these bladder cancer cells. In addition, we examined whether curcumin can induce DNA fragmentation in bladder cancer cells. We found that treatment with curcumin induced substantial DNA fragmentation in both RT4V6 and KU-7 cells in a dose-dependent manner (Fig. 1C). The results of Fig. 1C, which measures DNA fragmentation, correlates well with Fig. 1A, which measures mitochondrial activity. For instance, 10 Amol/L curcumin induced f40% DNA fragmentation and 50% reduction in mitochondrial activity in RT4V6 cell line. Curcumin Potentiates Apoptosis Induced by Chemotherapeutic Agents and Cytokines Because we found that curcumin inhibited the proliferation of KU-7 and RT4V6 cells, we next sought to determine whether curcumin potentiates the apoptotic effects of chemotherapeutic agents and cytokines used against bladder cancer. The Live/Dead assay, which measures membrane stability, indicated that treatment with combination of curcumin and gemcitabine increased the percentage apoptosis in RT4V6 cells from 10% to 90% compared with cells treated with gemcitabine alone. Curcumin also potentiated the cytotoxic effects of paclitaxel, TNF, and TRAIL, to the same extent (Fig. 2A). We obtained similar results with KU-7 cells (Fig. 2B). To
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Figure 3. Curcumin activates caspase-3 and potentiates the apoptotic effect of gemcitabine in bladder cancer cells. A, RT4V6 cells (2 106) were treated with 25 Amol/L curcumin for 24 h. Whole-cell extracts were prepared and subjected to Western blot analysis using anti-PARP antibody. Results are representative of three independent experiments. B, RT4V6 cells (2 106) were treated with 5 Amol/L curcumin and 5 nmol/L gemcitabine for 24 h. C, KU-7 cells (2 106) were treated with 25 Amol/L curcumin for 24 h. D, KU-7 cells (2 106) were treated with 5 Amol/L curcumin and 5 nmol/L gemcitabine for 24 h. Whole-cell extracts were prepared and subjected to Western blot analysis using an anti-PARP antibody. Results are representative of three independent experiments.

autoreader (Dynatech MR 5000; Dynatech, Chantilly, VA) with the lysis buffer used as the blank. Cell viability was expressed as a percentage: (absorbance of the experiment samples / absorbance of the control) 100. DNA Fragmentation Assay Bladder cancer cells were grown in six-well plates in the presence of 10% MEM. After reaching 70% confluence, the cells were exposed to various concentrations of curcumin for 48 h. Cells were harvested using trypsinization and pelleted using centrifugation. The pellets were then resuspended in PBS containing 50 Ag/mL propidium iodide, 0.1% Triton X-100, and 0.1% sodium citrate. Propidium iodide incorporation, as measured using fluorescence-activated cell sorting analysis [FACScan (FL-3 channel), Becton Dickinson and Co., Mountain View, CA] was used to measure DNA fragment sizes and plotted on a histogram (23). Cells in the sub-G1 population were assumed to be apoptotic (24). Cell Cycle Analysis To determine the effect of curcumin on the cell cycle, bladder cancer cells were treated with various concentrations of curcumin for 24 h, washed, and fixed with 70%

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further confirm the potentiation effect of curcumin, we used Annexin V staining, which detects an early-stage of apoptosis. These results also suggested the enhancement of apoptotic effects of TNF, TRAIL, paclitaxel, and gemcitabine in RT4V6 (Fig. 2C) and KU-7 cells (Fig. 2D). Thus, all these results together indicated that curcumin could potentiate the apoptotic effects of chemotherapeutic agents and cytokines against different bladder cancer cell lines. We also investigated whether curcumin mediates its apoptotic effects through activation of caspases. As shown in Fig. 3A, treatment of RT4V6 cells with curcumin alone induced caspase-3 mediated PARP cleavage in a timedependent manner. At a dose at which neither curcumin nor gemcitabine alone had much effect on PARP cleavage, treatment with the two together was found to be very effective in inducing PARP cleavage (Fig. 3B). We obtained similar results on treatment with curcumin alone (Fig. 3C) and in combination with gemcitabine (Fig. 3D) in KU-7 cells. Gemcitabine Induces NF-KB Activation, but Curcumin Inhibits this Activation How curcumin potentiates the apoptotic effects of gemcitabine was investigated. Because chemotherapeutic agents have been shown to activate NF-nB (31, 32), and NF-nB is known to suppress apoptosis (33), we investigated the role of NF-nB pathway as a potential mechanism. We first investigated whether gemcitabine can activate NF-nB in bladder cancer cells. We found that gemcitabine induced NF-nB activation in both RT4V6 (Fig. 4A) and KU-7 (Fig. 4B) cells in a time-dependent manner. We also examined whether curcumin suppresses gemcitabineinduced NF-nB activation in RT4V6 and KU-7 cells. We found that curcumin inhibited gemcitabine-induced NF-nB activation in both cell types (Fig. 4C and D). Because treatment with curcumin potentiates TNFinduced apoptosis of bladder cancer cells, we also sought to determine whether curcumin suppresses TNF-induced NF-nB activation in bladder cancer cells. We found that TNF activated NF-nB and curcumin inhibited TNF-induced NF-nB activation in both RT4V6 (Fig. 4E) and KU-7 cells (Fig. 4F). Cigarette Smoke Induces NF-KB Activation in Bladder Cancer Cells, but Curcumin Suppresses this Activation Cigarette smoking is a potential risk factor for bladder cancer (13). Therefore, we investigated whether cigarette smoke induces NF-nB activation in RT4V6 and KU-7 cells. Our results showed that exposure of bladder cancer cells to cigarette smoke activated NF-nB in both cell types in a time-dependent manner (Fig. 5A). Cigarette smoke induced NF-nB activation in these cells was persistent. We also investigated whether curcumin can suppress cigarette smoke induced NF-nB activation in bladder cancer cells. We found that treatment with curcumin inhibited cigarette smoke induced NF-nB activation in both RT4V6 and KU-7 cells in a dose-dependent manner (Fig. 5B).
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Curcumin Represses Cigarette Smoke ^ Induced Expression of NF-KB ^ Dependent Gene Products Involved in the Proliferation and Metastasis of Tumor Cells Additionally, we investigated whether curcumin can modulate NF-nB regulated gene products involved in the proliferation and metastasis of tumor cells. NF-nB activation has been shown to regulate the expression of COX-2 and VEGF (14). Western blot analysis using specific antibodies showed that cigarette smoke induced the expression of both COX-2 and VEGF in a time-dependent manner, whereas treatment with curcumin suppressed their expression in both RT4V6

Curcumin suppresses gemcitabine- and TNF-induced NF-nB activation in bladder cancer cells. RT4V6 cells (A), 2 106, were treated with 100 Amol/L gemcitabine and KU-7 cells (B), 2 106, were treated with 10 Amol/L gemcitabine for indicated time intervals. The cells were then analyzed for NF-nB activation using EMSA. RT4V6 cells (C) and KU-7 cells (D), 2 106, were pretreated with 10, 25, and 50 Amol/L curcumin for 4 h. RT4V6 cells were stimulated with 100 Amol/L gemcitabine for 8 h, and KU-7 cells were stimulated with 10 Amol/L gemcitabine for same period. The cells were then analyzed for NF-nB activation using EMSA. RT4V6 cells (E) and KU-7 cells (F), 2 106 each, were pretreated with 10 and 50 Amol/L curcumin for 4 h. Thereafter, cells were stimulated with 0.1 nmol/L TNF for 30 min. The cells were then analyzed for NF-nB activation using EMSA. Results are representative of three independent experiments.

Figure 4.

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Cigarette smoke (CS) induces NF-nB activation and curcumin suppresses cigarette smoke-induced NF-nB activation in bladder cancer cells. A, RT4V6 and KU-7 cells (2 106 each) were treated with 10 Ag/mL cigarette smoke for the indicated periods. The cells were then analyzed for NF-nB activation using EMSA. B, RT4V6 and KU-7 cells (2 106 each) were pretreated with 10 and 50 Amol/L curcumin for 4 h. Thereafter, cells were stimulated with 10 Ag/mL cigarette smoke for 1 h. The cells were then analyzed for NF-nB activation using EMSA. Results are representative of three independent experiments.

Figure 5.

This is the first study to show that curcumin alone can suppress the proliferation of both TRAIL/IFN-a sensitive and TRAIL/IFN-a resistant bladder cancer cells. Specifically, curcumin seems to induce cell cycle arrest and DNA fragmentation in these cells. Considering that TRAIL and IFN-a are implicated mechanistically in response of bladder tumors to Bacillus Calmette-Guerin, our results suggest that combination therapy with curcumin would be worth exploring. How curcumin overcomes resistance to IFN-a is unclear, but a previous study showed that this cytokine mediates its apoptotic effects through the induction of TRAIL (34). Similar to curcumin, genistein (derived from soy) and analogues of indole-3-carbinol (derived from cruciferous vegetables) have been shown to suppress the proliferation of bladder cancer cells (35, 36). Curcumin has been shown to have no effect on normal cells. For instance, treatment of normal lymphocytes, hepatocytes, human foreskin fibroblast cells, and rat skin fibroblasts with 50 Amol/L curcumin for 24 h did not affect cell proliferation (37, 38). We also found that treatment with curcumin potentiated the apoptotic effects of chemotherapeutic agents (gemcitabine and paclitaxel) and cytokines (TNF and TRAIL) against bladder cancer cells. Histone deacetylase inhibitors have been found to enhance the TRAIL-induced apoptosis of bladder cancer cells (39). We have shown previously that treatment with a proteasome inhibitor bortezomib enhanced gemcitabine-induced apoptosis of bladder cancer cells (24). When investigated for the mechanism by which curcumin mediates its effects, we found that both cytokines and chemotherapeutic agents activated NF-nB and that curcumin inhibited this activation. The role of NF-nB in

(Fig. 6A) and KU-7 cells (Fig. 6B). These results support our hypothesis that treatment with curcumin blocks the expression of cigarette smoke induced NF-nB regulated gene products.

Discussion
The goal of this study was to determine whether curcumin has a role in the prevention and/or treatment of bladder cancer. We found that curcumin inhibited the proliferation and induced apoptosis of both IFN-a sensitive and IFN-a resistant bladder cancer cells. Curcumin also potentiated the apoptotic effects of gemcitabine, paclitaxel, TNF, and TRAIL. This effect of curcumin was found to be independent of sensitivity or resistance to IFN-a or TRAIL, both of which are believed to mediate the effects of Bacillus CalmetteGuerin, which is the current gold standard treatment for localized, noninvasive bladder cancer. Furthermore, we found that both gemcitabine and TNF activated NF-nB in bladder cancer cells and that treatment with curcumin suppressed this activation. In addition, curcumin suppressed cigarette smoke induced NF-nB activation. Finally, treatment with curcumin down-regulated the cigarette smoke induced expression of the NF-nB regulated gene products COX-2 and VEGF in bladder cancer cells.

Figure 6. Curcumin suppresses cigarette smoke induced COX-2 and VEGF expression in bladder cancer cells. RT4V6 cells (A) and KU-7 cells (B), 2 106 each, were pretreated with 10 Amol/L curcumin for 4 h. Thereafter, cells were stimulated with 10 Ag/mL cigarette smoke for the indicated periods. Whole-cell extracts were prepared and subjected to Western blot analysis using the relevant antibodies. Results are representative of three independent experiments. Mol Cancer Ther 2007;6(3). March 2007

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suppression of apoptosis induced by cytokines and chemotherapeutic agents is well established (40). Thus, suppression of NF-nB by curcumin may sensitize bladder cancer cells to chemotherapeutic agents and cytokines. Cigarette smoking is one of the major risk factors for bladder cancer. We found that cigarette smoke can activate NF-nB in bladder cancer cells and that curcumin can suppress this activation. Because NF-nB activation has been shown to mediate cellular transformation, proliferation, invasion, angiogenesis, and metastasis (14), it is possible that suppression of cigarette smoke induced NF-nB activation by curcumin may have implications in suppression of tumorigenesis. Previously, NF-nB has been targeted in bladder cancer using genistein (35). We also found that cigarette smoke induced expression of NF-nB regulated proteins, COX-2 and VEGF, which are required for cell proliferation and angiogenesis, respectively, is downregulated by curcumin. Whether all of these effects of curcumin are mediated by targeting NF-nB is not clear. Previously, we showed that the indole-3-carbinol analogue DIM can mediate its effects against bladder cancer by targeting peroxisome proliferator-activated receptor-g (36). Curcumin is agonist of peroxisome proliferator-activated receptor-g as well (41). Similarly, a previous study found that curcumin mediates its effects by modulating histone acetylation (42); others have shown that histone deacetylase inhibitors enhance TRAIL-induced apoptosis of bladder cancer cells as described above (39). Thus, curcumin may mediate its effects by targeting more than one cell signaling pathway (26). This is consistent with recent reports that multitargeted therapy has a better chance of success against cancer than monotargeted therapies (25, 28). Phase I clinical trials in patients with resected bladder cancer have indicated that curcumin is pharmacologically safe even when consumed up to 12 g/d for 3 months and there is an indication of a histologic response to it (43). The present study suggests that curcumin can be used either alone for prevention of bladder cancer or in combination with chemotherapeutic agents and cytokines for treatment of the disease. This may account for the lower incidence of bladder cancer in countries where curcumin is consumed on a regular basis. Future animal studies are required to further validate these findings. Overall, our results support the use of curcumin either alone or in combination with existing therapy.
Acknowledgments
We thank Walter Pagel for carefully proofreading the manuscript and providing valuable comments.

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