Curcumin Induces Apoptosis in Human Neuroblastoma Cells via Inhibition of NF Î B

Background: Metastasised neuroblastoma is a largely incurable neoplasia in children over one year of age using current treatment protocols. After dissemination to the bone, the survival rate is <7%, indicating an urgent need for novel therapeutic regimes. As curcumin (diferuloylmethane) had been shown to exert strong anticancer effects against diverse human malignancies different from neurblastoma, the antiproliferative effect of curcumin on the growth of human neuroblastoma cell lines was tested. Materials and Methods: Proliferation of neuroblastoma cell lines Lan-5, SK-N-SH and Kelly under the treatment of curcumin over a broad concentration range (1x10–5 to 1x102 ÌM) was assessed using XTT cell proliferation assays. Possible induction of apoptosis through curcumin treatment was assessed by detection of DNA fragmentation. To investigate the effect of curcumin on NFÎB activation, the protein levels of the NFÎB subunit p65 of curcumin-treated cells were compared to untreated cells using Western blots. Results: Curcumin showed a significant dosedependent antiproliferative effect on all three neuroblastoma cell lines starting at a concentration of 1x10–3 ÌM. The highest concentration of 1x102 ÌM significantly reduced the viable cell count to 8-48% depending on the cell line. This antiproliferative effect was mediated through an increased induction of apoptosis by inhibition of NFÎB, corroborating earlier findings indicating an antiapoptotic effect of NFÎB. Conclusion: Our results suggest that curcumin might hold promise in the treatment of patients suffering from neuroblastoma. Neuroblastoma represents the third most common malignancy in children and accounts for at least 15% of all childhood cancer deaths (1). Despite advances in existing therapeutic modalities, including surgery, radiotherapy and dose-intensive chemotherapy, the long-term survival for stage 4 disease has remained over the years at less than 15% (2). Hence, improved therapy for neuroblastoma is imperatively needed. In several studies, the antiproliferative effects of curcumin have been demonstrated in a broad range of human malignancies, including breast, prostate and colon cancer, and hepatocellular carcinoma (3). Curcumin (diferuloylmethane) is a major active component of tumeric (Curcuma longa) being responsible for the specific flavour and yellow colour of the common spice, curry. The compound has been found to be pharmacologically safe: clinical trials indicated no doselimiting toxicity when administered at doses up to 8 g/day for 3 months (4). From a molecular point of view, curcumin has been shown to suppress cellular transformation, proliferation, invasion, angiogenesis and metastasis of several tumour cell types through mechanisms not fully understood (5). However, several reports indicate that curcumin provides these antitumour effects through inhibition of NFÎB activation. Under normal conditions, NFÎB, a heterodimer consisting of a p50 and a p65 subunit, is kept from translocating into the nucleus through binding to its inhibitor IÎB·. In response to diverse stimuli, NFÎB activation proceeds sequentially through phosphorylation of IÎB·, ubiquitination of IÎB· and finally degradation of IÎB· in the 26S proteasome, leading to translocation from NFÎB into the nucleus (6), where NFÎB activates transcription of several genes involved in apoptosis, tumorigenesis and inflammation (7). In this study, the antiproliferative effect of curcumin on three different human neuroblastoma cell lines was investigated. Furthermore, whether curcumin inhibits NFÎB expression and might therefore induce apoptosis in neuroblastoma cells was investigated. Materials and Methods Reagents. Curcumin used in this study was kindly provided by Professor Dr. J. Greten (Deutsche Gesellschaft für Traditionelle 209 Correspondence to: Dr. Christian Freudlsperger, Institut für Anatomie II: Experimentelle Morphologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. Tel: + 49 40428032589, Fax: + 49 40428035427, e-mail: c.freudlsperger@uke.uni-hamburg.de