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Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0037 Research Article Cancer Prevention Research Thiocolchicoside Exhibits Anticancer Effects through Downregulation of NF-κB Pathway and Its Regulated Gene Products Linked to Inflammation and Cancer Simone Reuter, Sahdeo Prasad, Kanokkarn Phromnoi, Jayaraj Ravindran, Bokyung Sung, Vivek R. Yadav, Ramaswamy Kannappan, Madan M. Chaturvedi, and Bharat B. Aggarwal Abstract The discovery of new uses for older, clinically approved drugs is one way to expedite drug development for cancer. Thiocolchicoside, a semisynthetic colchicoside from the plant Gloriosa superba, is a muscle relaxant and used to treat rheumatologic and orthopedic disorders because of its analgesic and anti- inflammatory mechanisms. Given that activation of the transcription factor NF-κB plays a major role in inflammation and tumorigenesis, we postulated that thiocolchicoside would inhibit NF-κB and exhibit anticancer effects through the modulation of NF-κB–regulated proteins. We show that thiocolchicoside inhibited proliferation of leukemia, myeloma, squamous cell carcinoma, breast, colon, and kidney cancer cells. Formation of tumor colonies was also suppressed by thiocolchicoside. The colchicoside induced apoptosis, as indicated by caspase-3 and poly(ADP-ribose) polymerase cleavage, and suppressed the expression of cell survival [e.g., Bcl-2, X-linked inhibitor of apoptosis (XIAP), MCL-1, bcl-xL, cIAP-1, cIAP-2, and cFLIP] proteins. Cell proliferation biomarkers such as c-MYC and phosphorylation of phosphoinosi- tide 3-kinase and glycogen synthase kinase 3β were also blocked by thiocolchicoside. Because most cell survival and proliferation gene products are regulated by NF-κB, we studied the effect of thiocolchicoside on this transcription factor and found that thiocolchicoside inhibited NF-κB activation, degradation of inhibitory κBα (IκBα), IκBα ubiquitination, and phosphorylation, abolished the activation of IκBα kinase, and suppressed p65 nuclear translocation. This effect of thiocolchicoside on the NF-κB pathway led to inhibition of NF-κB reporter activity and cyclooxygenase-2 promoter activity. Our results indicate that thiocolchicoside exhibits anticancer activity through inhibition of NF-κB and NF-κB–regulated gene products, which provides novel insight into a half-century old drug. Cancer Prev Res; 3(11); 1462–72. ©2010 AACR. Introduction low back pain accompanied by muscle spasm (2) and is at least as effective as tizanidine, the standard drug used Thiocolchicoside is a semisynthetic drug derived from to treat low back pain (3, 4). Biochemical studies indicated colchicoside, a natural glucoside present in the plant that thiocolchicoside can interact with γ-aminobutyric acid Gloriosa superba. Thiocolchicoside has been used clinically receptors, given that it was able to inhibit the binding of for >35 years as a muscle relaxant as well as an anti- both [3H]γ-aminobutyric acid and [3H]strychnine to rat inflammatory and analgesic drug (1). Given these proper- cerebrocortical and spinal cord membranes, respectively, ties, thiocolchicoside has long been used to treat a number in vitro and in vivo (5–7). Whereas these findings suggest of orthopedic, traumatic, and rheumatologic conditions. that thiocolchicoside, a γ-aminobutyric acid agonist, in- Furthermore, clinical trials showed that thiocolchicoside duces depression of the central nervous system and, in turn, is an efficient and safe treatment for patients with acute myorelaxation (8), the mechanism of its anti-inflammatory effect is still unknown. Authors' Affiliation: Cytokine Research Laboratory, Department of One possible mechanism by which thiocolchicoside Experimental Therapeutics, University of Texas M.D. Anderson Cancer may exert its anti-inflammatory effect is by modulating Center, Houston, Texas the NF-κB pathway, which is commonly involved in in- Note: Current address for M.M. Chaturvedi: Department of Zoology, flammation and tumorigenesis. NF-κB is a transcription University of Delhi, Delhi, India. factor that resides in the cytoplasm in its resting stage Corresponding Author: Bharat B. Aggarwal, Cytokine Research Labora- and then, when activated, translocates to the nucleus and tory, Department of Experimental Therapeutics, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 143, Houston, mediates the transcription of >400 different genes (9). Var- TX 77030. Phone: 713-794-1817; Fax: 713-745-6339; E-mail: aggarwal@ ious inflammatory agents can activate NF-κB, including cy- mdanderson.org. tokines [e.g., tumor necrosis factor (TNF)], carcinogens, doi: 10.1158/1940-6207.CAPR-10-0037 tumor promoters, cigarette smoke, environmental pollu- ©2010 American Association for Cancer Research. tants, ionizing radiation, and stress (9). NF-κB activation 1462 Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on September 27, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0037 Thiocolchicoside Exhibits Anticancer Effects Through NF-κB Inhibition has been shown to control the expression of genes linked to in DMEM containing 10% FBS, nonessential amino acids, inflammation, apoptosis, survival, proliferation, invasion, pyruvate, glutamine, and vitamins. All media were also angiogenesis, metastasis, chemoresistance, tumor cell supplemented with 100 units/mL penicillin and 100 μg/mL transformation, and radioresistance (9). streptomycin. Because of the critical role of NF-κB in inflammation and tumorigenesis, we postulated that thiocolchicoside Cytotoxicity assay mediates its anti-inflammatory effect through modulation Cell proliferation and cell viability experiments were as- of the NF-κB pathway. Indeed, our results show that thio- sayed by the modified tetrazolium salt MTT assay as de- colchicoside inhibits NF-κB activated by inflammatory cy- scribed previously (10). For the cell proliferation assay, tokines (TNF), okadaic acid (OA), tumor promoter [phorbol 2,000 cells were incubated with various concentrations 12-myristate 13-acetate (PMA)], and lipopolysaccharide of thiocolchicoside, in triplicate, for 1, 3, and 5 days in (LPS) through inhibition of phosphorylation, ubiquitina- 96-well plates at 37°C. For the cell viability assay, 5,000 tion, and degradation of inhibitory κBα (IκBα), the inhibitor cells were incubated with various concentrations of thio- of NF-κB. Thiocolchicoside also inhibited the phosphoryla- colchicoside, in triplicate, for 24 hours in 96-well plates tion and nuclear translocation of p65, the major isoform of at 37°C. Thereafter, an MTT solution was added to each NF-κB. Thiocolchicoside inhibition of NF-κB leads to sup- well. After 2 hours of incubation at 37°C, lysis buffer pression of NF-κB–regulated proteins, which are responsible (20% SDS and 50% dimethylformamide) was added, for the anticancer effect of thiocolchicoside on various cancer the cells were incubated overnight at 37°C, and then ab- cell lines, characterized by induction of apoptosis and inhi- sorbance was measured at 570 nm using a 96-well multi- bition of cell proliferation as well as colony formation. To- scanner (MRX Revelation, Dynex Technologies). gether, our results provide a new role for thiocolchicoside as an anticancer agent. Clonogenic assay for HCT-116 The clonogenic assay or colony formation assay is an Materials and Methods in vitro cell survival assay based on the ability of a single cell to grow into a colony (11). HCT-116 cells have been Reagents used for the clonogenic assay, because these are adherent A 100 mmol/L solution of thiocolchicoside, kindly pro- cells and give a good response for this assay. To test the vided by Sarv Bio Labs, was prepared in water, stored at ability of thiocolchicoside to inhibit single cells to grow +4°C, and then diluted as needed in cell culture medium. into colonies, 500 cells were seeded in six-well plates Bacteria-derived recombinant human TNF-α was kindly and incubated overnight to allow attachment. The follow- provided by Genentech. ing day, the cells were treated with various concentrations Penicillin, streptomycin, Iscove's modified Dulbecco's of thiocolchicoside, in triplicate, for 24 hours. The next medium, DMEM, RPMI 1640, and fetal bovine serum day, the medium was changed, and the cells were incubated (FBS) were purchased from Invitrogen. The proteasome for 9 days to form colonies. Medium was replaced after inhibitor N-acetyl-leucylleucyl-norleucinal (ALLN) was 4 days. At the end of the ninth day, medium was removed, purchased from Calbiochem. and 0.3 mL of clonogenic acid reagent was added. Cells PMA,OA,LPS,andanti-β-actin antibody were pur- were incubated for 30 minutes and washed twice, and blue chased from Sigma-Aldrich. Antibodies against p65, poly colonies were counted (12). (ADP-ribose) polymerase (PARP), inhibitor of apoptosis protein 1, BCL-2, BCL-xL, c-MYC, and caspase-3 were pur- Electrophoretic mobility shift assay chased from Santa Cruz Biotechnology. The antibody To assess NF-κB activation, we did electrophoretic mo- against the inhibitory subunit of NF-κB(IκBα)waspur- bility shift assay (EMSA) as described previously (13). chased from Imgenex. Anti-XIAP antibodies were In brief, nuclear extracts prepared from TNF-treated cells purchased from BD Biosciences. Anti-phosphorylated IκBα (1.5 × 106/mL) were incubated with 32P end-labeled 45-mer (Ser32/36) and anti-phosphorylated p65 (Ser536)were double-stranded NF-κB

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