Published OnlineFirst November 14, 2016; DOI: 10.1158/1541-7786.MCR-16-0101 Cell Death and Survival Molecular Cancer Research Combined Parthenolide and Balsalazide Have Enhanced Antitumor Efficacy Through Blockade of NF-kB Activation Se-Lim Kim1, Seong Hun Kim1, Young Ran Park1, Yu-Chuan Liu1, Eun-Mi Kim2, Hwan-Jeong Jeong2, Yo Na Kim3, Seung Young Seo1, In Hee Kim1, Seung Ok Lee1, Soo Teik Lee1, and Sang-Wook Kim1 Abstract Balsalazide is a colon-specific prodrug of 5-aminosalicylate balsalazide together resulted in significant recovery of body that is associated with a reduced risk of colon cancer in patients weight and improvement in histologic severity. Administration with ulcerative colitis. Parthenolide, a strong NF-kB inhibitor, of parthenolide and balsalazide to CAC mice also suppressed has recently been demonstrated to be a promising therapeutic carcinogenesis as demonstrated by uptake of 18F-fluoro-2- agent, promoting apoptosis of cancer cells. In the current study, deoxy-D-glucose (FDG) using micro-PET/CT scans. These the antitumor effect of balsalazide combined with parthenolide results demonstrate that parthenolide potentiates the efficacy in human colorectal cancer cells and colitis-associated colon of balsalazide through synergistic inhibition of NF-kB activa- cancers (CAC) was investigated. The results demonstrate that tion and the combination of dual agents prevents colon car- the combination of balsalazide and parthenolide markedly cinogenesis from chronic inflammation. suppress proliferation, nuclear translocation of NF-kB, IkB-a phosphorylation, NF-kB DNA binding, and expression of Implications: This study represents the first evidence that com- NF-kB targets. Apoptosis via NF-kB signaling was confirmed bination therapy with balsalazide and parthenolide could be a by detecting expression of caspases, p53 and PARP. Moreover, new regimen for colorectal cancer treatment. Mol Cancer Res; 15(2); treatment of a CAC murine model with parthenolide and 1–11. Ó2016 AACR. Introduction intestine and does not reach the colon (2). Sulfasalazine, the first 5-ASA–containing drug, functions by releasing an active compo- Colorectal cancer is a serious complication of ulcerative colitis nent in the colon through the activity of azo reductase expressed and responsible for up to 15% of all deaths in patients with by colonic bacterial. Sulfasalazine has been approved for thera- inflammatory bowel disease (1). Early detection and prevention peutic usage because of its ability to improve intestinal mucosal strategies (such as colonoscopy, mucosal biopsies, and procto- permeability (3, 4). However, the high rate of adverse effects colectomy) for colorectal cancer patients with ulcerative colitis related to sulfapyridine limits its use for patients. Balsalazide is a have limitations, and therefore, there is increased interest in novel orally administered prodrug of 5-ASA in which an inert identifying chemopreventive agents to reduce the overall risk of carrier molecule, 4-aminobenzoilb-alanine, is bonded to 5-ASA. colitis-associated colorectal cancer (CAC). Balsalazide has been shown to be more effective than sulfasala- 5-Aminosalicylate (5-ASA) is used to treat ulcerative colitis zine in the treatment of active ulcerative colitis (5, 6) and to be as because of its ability to control and relieve inflammation. In most effective and well tolerated as delayed release 5-ASA in the chronic cases, 5-ASA is absorbed rapidly and extensively in the upper treatment of ulcerative colitis (7). A recent study has shown that 5- ASA use is associated with reduced risk of colorectal cancer in 1Department of Internal Medicine, Research Institute of Clinical Medicine of patients with ulcerative colitis (8). However, the molecular Chonbuk National University-Biomedical Research Institute of Chonbuk Nation- mechanisms of balsalazide in the ulcerative colitis to colorectal al University Hospital, Jeonju, Korea. 2Department of Nuclear Medicine, cancer development have not been fully elucidated and the Research Institute of Clinical Medicine of Chonbuk National University-Biomed- antitumor effect of balsalazide remains controversial. ical Research Institute of Chonbuk National University Hospital, Jeonju, Korea. NF-kB is one of the key regulators in inflammation and cancer 3 Department of Pathology, Research Institute of Clinical Medicine of Chonbuk progression (9). NF-kB activation is markedly enhanced in National University-Biomedical Research Institute of Chonbuk National Univer- fl sity Hospital, Jeonju, Korea. patients with in ammatory bowel disease and the level of acti- vated NF-kB is significantly correlated with the severity of intes- S.-L. Kim and S.H. Kim contributed equally to this article. tinal inflammation (10, 11). Activated NF-kB has the ability to Corresponding Author: Sang-Wook Kim, Department of Internal Medicine, Chon- promote the expression of various proinflammatory genes, thus buk National University Hospital, 20 Geonji-ro, Deokjin-gu, Jeonju 561-712, Korea. strongly influencing the process of ulcerative colitis (12). Phone: 826-3250-2302; Fax: 826-3254-1609; E-mail: [email protected] Parthenolide, a natural product, has been used for the treat- doi: 10.1158/1541-7786.MCR-16-0101 ment of fever and inflammatory disease. It is well known to inhibit Ó2016 American Association for Cancer Research. IL-1 and TNFa-mediated NF-kB activation (13, 14). Recent www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst November 14, 2016; DOI: 10.1158/1541-7786.MCR-16-0101 Kim et al. studies have demonstrated that parthenolide induces apoptotic a 60-mm culture dish were incubated with the designated doses of cell death through inhibition of NF-kB activation in a number of parthenolide and/or balsalazide for 24 hours. Cells were washed human cancers (13, 15). Several studies have also shown that twice with cold PBS and then resuspended in 500 mL of a binding parthenolide is a potent inhibitor of NF-kB activation and sup- buffer (10 mmol/L HEPES/NaOH pH 7.4, 140 mmol/L NaCl, and 6 presses the expression of proinflammatory cytokines in experi- 2.5 mmol/L CaCl2) at a concentration of 1 Â 10 cells/mL. mental murine models (13, 16). We have also demonstrated that Annexin V-FITC (5 mL) and PI (1 mg/mL) were then added and parthenolide inhibits phosphorylation of IkBa and NF-kB acti- the cells were analyzed with a FACStar flow cytometer. vation, resulting in initiation of apoptosis, suppression of colo- Cell cycle and sub-G1 cell analysis were determined by PI (Ex/ rectal cancer tumor growth, and CAC development (17, 18). On Em ¼ 488/617 nm) staining. In brief, 1 Â 106 cells were incubated the basis of these observations, it can be assumed that the in a 60-mm culture dish with the designated doses of parthenolide combination of parthenolide and balsalazide can be a promising and/or balsalazide for 24 hours. Total cells including floating cells strategy to prevent the development of ulcerative colitis to colo- were then washed with PBS and fixed in 70% (v/v) ethanol. Cells rectal cancer, inhibiting NF-kB activation pathway. were washed again with PBS, then incubated with PI (10 mg/mL) The aim of our study was to evaluate the effects of combination with simultaneous RNase treatment at 37C for 30 minutes. therapy with parthenolide and balsalazide on the inhibition of Cellular DNA content was measured using a FACStar flow cyt- NF-kB activation in human colorectal cancer cells and to inves- ometer (Becton-Dickinson) and analyzed using lysis II and CellFit tigate whether parthenolide and balsalazide are effective in pre- software (Becton-Dickinson). venting carcinogenesis from chronic colitis. Parthenolide-induced apoptosis in colon cancer cells was assessed using Hoechst 33258. The cells were treated with various concentrations of parthenolide for 24 hours, and then stained Materials and Methods with Hoechst 33258 (1 mg/mL) at 37 C for 10 minutes. Nuclear Chemicals and reagents morphology was examined under a Confocal Laser Scanning Parthenolide and z-VAD-FMK were obtained from Calbio- Microscope (Carl Zeiss) to identify cells undergoing apoptosis. chem. Balsalazide was provided by Chong Kun Dang Pharm. Parthenolide was dissolved in dimethylsulfoxide (DMSO; Sigma) Cytoplasmic and nuclear extract preparation to a concentration of 100 mmol/L and stored at À20 C in the dark. Cells were harvested and resolved in a lysis buffer [20 mmol/L Balsalazide was dissolved in FBS-free media to a concentration of Tris-HCl (pH 7.5), 137 mmol/L NaCl, 10% glycerol (v/v), 1% fl 100 mmol/L at 4 C. Annexin V- uorescein isothiocyanate Triton X-100 (v/v), 1 mmol/L Na3VO4, 1 mmol/L phenylmethyl- (Annexin-V FITC) and propidium iodide (PI) were purchased sulphonylfluoride, and protease inhibitor cocktail]. After centri- from Invitrogen. Hoechst 33258 was from Sigma. fugation at 16,000 Â g for 15 minutes, the supernatants were used as cytoplasmic extracts. To extract the nuclear fraction, cells were Cell culture and cell line authentication resuspended in 150 mL of buffer A [10 mmol/L HEPES (pH 7.9), HCT 116, SW480 and HT-29 (ATCC) were employed as repre- 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiotreitol, sentative human colorectal cancer cells. The cells were cultured in 0.5 mmol/L phenylmethylsulphonylfluoride, 0.4% Nonidet P-40 RPMI1640 medium supplemented with 10% FBS, 100 U penicil- (v/v) and protease inhibitor cocktail] for 20 minutes on ice and lin, and 100 U streptomycin. The three cell lines listed above were then centrifuged at 2,300 Â g for 5 minutes. The resulting pellets validated by short-tandem repeat (STR) DNA fingerprinting using were resolved in 100 mL of buffer C [20 mmol/L HEPES (pH 7.9), the Promega PowerPlex 18D System and analyzed by GeneMapper 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 Software 5 at Cosmo Genetech Korea in January 2016.