Curcumin Suppresses the Paclitaxel-Induced Nuclear Factor-KB Pathway in Breast Cancer Cells and Inhibits LungMetastasis of Human Breast Cancer in NudeMice (2005)

Currently, there is no effective therapy for metastatic breast cancer after surgery, radiation, and chemotherapy have been used against the primary tumor. Because curcumin suppresses nuclear factor-nB (NF-nB) activation and most chemotherapeutic agents activate NF-nB that mediates cell survival, proliferation, invasion, and metastasis, we hypothesized that curcumin would potentiate the effect of chemotherapy in advanced breast cancer and inhibit lung metastasis. We tested this hypothesis using paclitaxel (Taxol)-resistant breast cancer cells and a human breast cancer xenograft model. As examined by electrophoretic mobility gel shift assay, paclitaxel activated NF-nB in breast cancer cells and curcumin inhibited it; this inhibition was mediated through inhibition of InBa kinase activation and InBa phosphorylation and degradation. Curcumin also suppressed the paclitaxel-induced expression of antiapoptotic (XIAP, IAP-1, IAP-2, Bcl-2, and Bcl-xL), proliferative (cyclooxygenase 2, c-Myc, and cyclin D1), and metastatic proteins (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1). It also enhanced apoptosis. In a human breast cancer xenograft model, dietary administration of curcumin significantly decreased the incidence of breast cancer metastasis to the lung and suppressed the expression of NF-nB, cyclooxygenase 2, and matrix metalloproteinase-9. Overall, our results indicate that curcumin, which is a pharmacologically safe compound, has a therapeutic potential in preventing breast cancer metastasis possibly through suppression of NF-nB and NF-nB ^ regulated gene products. Although early-stage breast cancer is highly treatable, no effective treatment is available for metastatic breast cancer that follows surgery, radiation, and chemotherapy for the primary tumor (1). Paclitaxel (Taxol) is currently used as the front-line chemotherapeutic agent in breast cancers (1); however, because the drug frequently induces drug resistance (2–4), probably through the activation of nuclear factor-nB (NF-nB; ref. 5), it is not useful in treating advanced breast cancer. Curcumin (diferuloylmethane), a polyphenol (see Fig. 1A) derived from turmeric, Curcuma longa, is a pharmacologically safe and effective agent that can block NF-nB activation. Curcumin has been shown by us and others to suppress NFnB activation induced by various inflammatory stimuli (6) through inhibition of the activation of InBa kinase (IKK) activity needed for NF-nB activation (7, 8). Based on the paclitaxel and curcumin data, we hypothesized that curcumin would improve the therapeutic outcome of paclitaxel treatment for breast cancer. We tested this hypothesis using breast cancer cells and a nude mouse xenograft model. Our goal was to determine whether curcumin can suppress paclitaxel-induced NF-nB activation and NF-nB–regulated gene products and prevent breast cancer metastasis to the lung. We found that curcumin did block paclitaxel-induced NF-nB activation and NF-nB–regulated gene expression in breast cancer cells and inhibited breast cancer metastasis to the lung in nude mice. Materials andMethods Materials. Paclitaxel was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). It was dissolved in ethanol as a 10 mmol/L stock solution and stored at 4jC. Penicillin, streptomycin, Iscove’s modified Dulbecco’s medium, RPMI 1640, and fetal bovine serum were obtained from Life Technologies, Inc. (Grand Island, NY). Tris, glycine, NaCl, SDS, bovine serum albumin, and phorbol 12-myristate 13-acetate were obtained from Sigma Chemical Co. (St. Louis, MO) The following polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): anti-p65, against the epitope corresponding to amino acids mapping within the amino terminal domain of human Cancer Therapy: Preclinical Authors’ Affiliations: Cytokine Research Laboratory and Departments of Experimental Therapeutics, Pathology, and Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas Received 6/1/05; accepted 7/14/05. Grant support: Odyssey Program and theTheodore N. LawAward for Scientific Achievement at University of Texas M.D. Anderson Cancer Center (S. Shishodia); Clayton Foundation for Research (B.B. Aggarwal); Department of Defense U.S. Army Breast Cancer Research Program grant BC010610 (B.B. Aggarwal), and Cancer Center support grant CA16672. 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 with18 U.S.C. Section1734 solely to indicate this fact. Note: B.B. Aggarwal is a Ransom HorneJr. Professor of Cancer Research. Requests for reprints: Bharat B. Aggarwal, Department of Experimental Therapeutics, M.D. Anderson Cancer Center, Box 143, 1515 Holcombe Boulevard, Houston,TX 77030. Phone: 713-792-3503, 713-792-6459; Fax: 713-794-1613; E-mail: aggarwal@mdanderson.org. F2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1192 www.aacrjournals.org Clin Cancer Res 2005;11(20) October15, 2005 7490 Fig. 1. A, structure andmass spectrophotometric analysis of curcumin. B, curcumin inhibited paclitaxel-dependent NF-nB activation. MDA-MB-435 cells (2 10/mL) were preincubated with different concentrations of curcumin for 2 hours at 37jC and then treated with 50 Amol/L paclitaxel for16 hours. Nuclear extracts were prepared and tested for NF-nB activation as described in Materials and Methods. Curcumin Suppresses NF-kBand Breast CancerMetastasis www.aacrjournals.org Clin Cancer Res 2005;11(20) October15, 2005 7491 NF-nB p65; anti-p50, against a 15-amino-acid-long peptide mapping at the nuclear localization sequence region of NF-nB p50; anti-InBa, against amino acids 297 to 317 mapping at the carboxy terminus of InBa/MAD-3; and anti–c-Rel and anti–cyclin D1, against amino acids 1 to 295, which represents full-length cyclin D1 of human origin. Phospho-InBa (Ser) antibody was purchased from New England BioLabs (Beverly, MA). Anti-IKKa and anti-IKKh antibodies were kindly provided by Imgenex (San Diego, CA). Anti–cyclooxygenase 2 (COX-2) antibody was purchased from Transduction Labs (now Invitrogen, Carlsbad, CA) and anti–matrix metalloproteinase (MMP-9) antibody was purchased from Cell Sciences, Inc. (Norwood, MA). Curcumin. Curcumin with a purity of >98% was obtained from either LKT Laboratories (St. Paul, MN) or Sabinsa Corp. (Piscataway, NJ). To ensure identity and quality of the curcumin used in this study, curcumin was dissolved in 50:50 methanol/0.2% formic acid and maintained at 5jC in a refrigerated autosampler before analysis by electrospray ionization liquid chromatography tandem mass spectrometry. Curcuminoids (curcumin, demethoxycurcumin, and bisdesmethoxycurcumin) were separated on a Phenomenex Gemini 5 Am C18 2 100 mm analytic column using a linear acetonitrile/0.1% formic acid gradient. Detection was accomplished using a Waters Quattro Micro tandem mass spectrometer equipped with electrospray positive ionization capability. Each of these compounds was quantified by using a standard calibration curve prepared from a curcumin reference standard obtained from Sigma-Aldrich Chemical. The calibration curve was prepared by making a 1 mg/mL stock solution of the authentic material in methanol and then serially diluting the stock solution to 1,000, 500, 250, 100, 50, 10, 5, and 1 ng/mL with methanol. A calibration curve was then prepared using the mass spectrometry quantification software. The ratios of the three curcuminoids in the curcumin used in this study were as follows: curcumin, 87.2% (detected at m/z 367); demethoxycurcumin, 10.5% (detected at m/z 337); and bisdemethoxycurcumin, 2.3% (detected at m/z 307; see Fig. 1A). Percentages were calculated based on the peak areas for each of the curcuminoids detected. Cell lines. We used the human breast cancer cell line MDA-MB435. We have previously shown that this cell line is tumorigenic and metastatic in nude mice (9). Nuclear factor-kB activation. To determine NF-nB activation, we carried out electrophoretic mobility gel shift assay on nuclear extracts of paclitaxel-treated cells essentially as previously described (6). IkBa degradation. To determine the effect of curcumin on paclitaxeldependent InBa degradation, cytoplasmic extracts were prepared as previously described (6) from MDA-MB-435 cells (2 10/mL) pretreated with curcumin for 2 hours and then exposed to 50 Amol/L paclitaxel for various times. The extracts were then resolved on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were electrotransferred to nitrocellulose filters, probed with rabbit polyclonal antibodies against InBa, and detected by chemiluminescence (ECL, Amersham, Piscataway, NJ). The bands obtained were quantitated using Personal Densitometer Scan v1.30 using Imagequant software version 3.3 (Amersham). IkBa phosphorylation. To determine the effect of curcumin on paclitaxel-dependent InBa phosphorylation, cytoplasmic extracts were prepared fromMDA-MB-435 cells (2 10/mL) treated with 50 Amol/L curcumin for 2 hours and then treated with 50 Amol/L paclitaxel for various times. The extracts were then resolved on 10% SDS polyacrylamide gels and analyzed by Western blotting using antibody against phosphorylated InBa (Amersham). IkBa kinase assay. To determine the effect of curcumin on paclitaxel-induced IKK activation, we did IKK by a method described