Cancer Therapy: Preclinical Novel Dithiolethione-Modified Nonsteroidal Anti-Inflammatory Drugs in Human Hepatoma HepG2 and Colon LS180 Cells Sara E. Bass,1Pawel Sienkiewicz,2 ChristopherJ. MacDonald,2 RobertY.S. Cheng,2 Anna Sparatore,3 Piero Del Soldato,4 David D. Roberts,5 Terry W. Moody,6 David A. Wink,7 and Grace ChaoYeh2 Abstract Purpose: Nonsteroidal anti-inflammatory drugs (NSAID) are promising chemopreventive agents against colon and other cancers. However, the molecular basis mediated by NSAIDs for chemo- prevention has not been fully elucidated. Environmental carcinogens induce DNA mutation and cellular transformation; therefore, we examined the effect of NSAIDs on carcinogenesis mediated by the aryl hydrocarbon receptor signaling pathway. In this study, we investigated the activities of a new class of NSAIDs containingdithiolethione moieties (S-NSAID) on both arms of carcinogenesis. Experimental Design: We investigated the effects of the S-NSAIDs, S-diclofenac and S-sulin- dac, on carcinogen activation and detoxification mechanisms in human hepatoma HepG2 and human colonic adenocarcinoma LS180 cells. Results: We found that S-diclofenac and S-sulindac inhibited the activity and expression of the carcinogen activating enzymes, cytochromes P-450 (CYP) CYP1A1, CYP1B1, and CYP1A2. Inhi- bition was mediated by transcriptional regulation of the aryl hydrocarbon receptor (AhR) path- way. The S-NSAIDs down-regulated carcinogen-induced expression of CYP1A1 heterogeneous nuclear RNA, a measure of transcription rate. Both compounds blocked carcinogen-activated AhR from bindingto the xenobiotic responsive element as shown by chromatin immunoprecipi- tation. S-diclofenac and S-sulindac inhibited carcinogen-induced CYP enzyme activity through direct inhibition as well as through decreased transcriptional activation of the AhR. S-sulindac induced expression of several carcinogen detoxification enzymes of the glutathione cycle includ- ingglutathione S-transferase A2, glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase. Conclusions: These results indicate that S-diclofenac and S-sulindac may serve as effective chemoprevention agents by favorably balancing the equation of carcinogen activation and detoxification mechanisms. The use of nonsteroidal anti-inflammatory drugs (NSAID) has tive NSAIDs, such as aspirin, diclofenac, ibuprofen, and been associated with a reduced incidence of colorectal, breast, naproxen, is frequently associated with gastrointestinal toxicity esophageal, stomach, prostate, bladder, ovary, and lung cancers due to the chronic suppression of cyclooxygenase-1and (1). However, chronic treatment with conventional nonselec- subsequent inhibition of mucin secretion in the gut (2). The new generation of NSAIDs that target cyclooxygenase-2, such as rofecoxib and celecoxib, although effective as chemopreventive Authors’Affiliations: 1Basic Research Program, ScienceApplications International 2 agents, have been recently shown to cause an increased Corporation-Frederick, Inc., and Laboratory of Metabolism, National Cancer incidence of stroke and myocardial infarctions (3). Institute-Frederick, Frederick, Maryland; 3Istituto di Chimica Farmaceutica, University of Milan and 4Sulfidris, Milan, Italy; 5Laboratory of Pathology, 6Office of A new series of novel redox-modified NSAIDs has been the Director, and 7Radiation Biology Branch, Center for Cancer Research, National synthesized that may reduce these side effects. These compounds Cancer Institute, NIH, Bethesda, Maryland are conventional NSAIDs linked to a gaseous transmitter. The Received 7/17/08; revised 11/13/08; accepted 12/2/08; published OnlineFirst most extensively studied of these compounds are the nitric 3/10/09. Grant support: This Research was supported (in part) by the Intramural Research oxide–releasing NSAIDs, which were first described in 1994 (4) Program of the NIH, National Cancer Institute, Center for Cancer Research and and have because been evaluated in several clinical trials in National Cancer Institute, NIH, under contract N01-CO-12400. which their gastric sparing properties have been well-established The costs of publication of this article were defrayed in part by the payment of page (5, 6). Additional studies have shown nitric oxide–NSAIDs to be advertisement charges. This article must therefore be hereby marked in accordance promising chemopreventive agents (7). Although nitric oxide with 18 U.S.C. Section 1734 solely to indicate this fact. Note:The contentof thispublicationdoes notnecessarily reflect the views orpolicies is the best characterized of the gaseous transmitters, carbon of the Department of Health and Human Services, nor does mention of trade names, monoxide and hydrogen sulfide (H2S) have also been recog- commercialproducts, ororganizations imply endorsement by the U.S. Government. nized for their ability to affect key physiologic functions (8). Requests for reprints: Grace C. Yeh, Building31, Room 3A11, 31 Center Drive, In particular, H2S has been shown to exhibit complex but poorly Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892- 2440. Phone: 301-451-8296; Fax: 301-496-0775; E-mail: [email protected]. understood roles in inflammation with evidence for proinflam- F 2009 American Association for Cancer Research. matory as well as anti-inflammatory activities. H2S has also been doi:10.1158/1078-0432.CCR-08-1870 reported to reduce NSAID-induced gastric mucosal injury Clin Cancer Res 2009;15(6) March 15, 2009 1964 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2009 American Association for Cancer Research. Modulation of Phase I and II Enzymes by S-NSAIDs There is additional evidence that S-NSAIDs may also show a Translational Relevance potential usefulness in cancer treatment. Parent compounds, diclofenac and sulindac, have been investigated for their Occupational and environmental exposure to polycyclic potential chemopreventive properties and have been found aromatic hydrocarbons (PAH) has been suggested to pro- particularly useful for prevention and treatment in in vitro and voke inflammatory disorders and cancer. The molecular in vivo models of colon cancer in addition to other cancer types pathway/mechanisms invoked by PAH-mediated inflam- (16, 17). Recent work on ACS 15 and 18 has also shown mation remain to be clarified. Our article contributed to the antiangiogenic activities of the compounds in models of tumor- identification of both the molecular pathway and regulation driven angiogenesis (14). Our laboratory has shown the ability of PAH metabolism in both human hepatoma cells, which is of sulindac as well as the dithiolethione 5-(p-methoxyphenyl)- the active site for carcinogenesis, and colon adenocarcino- 1,2-dithiole-3-thione (ADT)8, to regulate the aryl hydrocarbon ma cells, which is the first exposure site for environmental receptor (AhR) pathway and phase I enzymes (18). The AhR is xenobiotics. the key transcriptional controller of carcinogen metabolizing The dithiolethione-NSAIDs in our study are newly syn- pathways involving the genes that encode phase I and phase II thesized drugs, which conjugate the dithiolethione moiety enzymes. Inhibiting the activation of procarcinogens by phase I with NSAIDs. We found the novel compounds not only enzymes through suppression of the AhR signaling pathway inhibited the PAH activation of phase 1enzymes induced has been suggested as an important mechanism for cancer by PAHs but also increased the detoxification of phase 2 prevention (19, 20). Based on the above data, we hypothesized enzymes.We are the first to identify the dual function prop- that the novel compounds, ACS 15 and ACS 18, could have erty of S-NSAIDs, which modulated carcinogen toxicity potential usefulness as chemoprotective compounds through mediated by the aryl hydrocarbon receptor as a molecular regulation of the AhR pathway and associated enzymes. We mechanism/pathway toward chemoprotection against en- show that both ACS 15 and 18 blocked carcinogen-induced vironmental xenobiotics. phase I enzyme activity in vitro, and that this activity was regulated through inhibition of AhR-mediated transcription. In addition, ACS 18 increased the expression of several phase II without affecting the functional suppression of prostaglandins genes. This dual action of reduced carcinogen activation (9). Additionally, an H2S-releasing derivative of mesalamine simultaneous with increased carcinogen detoxification supports reduced visceral sensitivity and pain perception in a rat model of the view that the S-NSAID model may be a valuable tool for irritable bowel syndrome (10). Finally, H2S may also play a role chemoprotection against environmental carcinogens. in cardio protection (11). These observations, as well as the fact that many NSAIDs reduce endogenous H2S formation (9), which may contribute to their tendency to cause gastric damage, led to the hypothesis that dithiolethione-modified NSAIDs Materials and Methods (S-NSAID) may serve as valuable chemoprotective drugs. The newly synthesized, novel compounds, S-diclofenac Materials. HepG2 human hepatocellular carcinoma and LS180 and S-sulindac, are composed of an H2S-releasing dithiole- human colorectal adenocarcinoma cell lines were from American Type thione moiety, ADT-OH covalently conjugated with the parent Culture Collection. RPMI 1640, Eagle’s MEM with Earle’s balanced salt NSAID by an ester linkage (Fig. 1). The chemical synthesis of solution,