In vitro Anti-inflammatory Evaluation of 6-Thioguanine and 6-Hydroxy-2-Mercaptopurine as A Potential Treatment for Rheumatoid Arthritis

Nurul Syuhada Nordin, Yong Yoke Keong, Siti Farah Md Tohid, Zetty Nadia Md Zain, Muhammad Nazrul Hakim


BACKGROUND: Eventhough 6-Mercaptopurine act as a major drug for rheumatoid arthritis (RA) treatment, however, its toxicity become a limitation. Therefore, this current study investigated whether 6-hydroxy-2-mercaptopurine (6H2MP) and 6-thioguanine (6TG) compounds are purine nucleoside analogues as a potential treatment of RA. The objective was to evaluate the therapeutics effects, especially the anti-inflammatory potential of 6H2MP and 6TG in the lipopolysaccharides (LPS)-induced RAW264.7 cells and phorbol myristate acetate (PMA)-induced HIG-82 cells.

METHODS: Macrophage cells (RAW264.7) and rabbit synoviocytes (HIG-82) cells were induced using LPS and PMA to evaluate the anti-inflammatory potential of 6H2MP and 6TG. The cytotoxicity assessment was done by using MTT assay, while enzyme-linked immunosorbent assay (ELISA) was used to determine the anti‑inflammatory potential, including tumour necrosis factor (TNF-α), interleukins (IL-1β, and IL-6).

RESULTS: Upon LPS-induced, RAW 264.7 macrophages demonstrated that 6H2MP and 6TG could suppress the production of nitric oxide (NO) in vitro. The half-maximal inhibitory concentration (IC50) value of 6TG and 6H2MP were 10.73 and 13.31, respectively. Further studied in PMA-induced HIG-82 synovial fibroblast cells showed that 6H2MP and 6TG also suppressed the release of NO, Prostaglandin E2 (PGE2), and inflammatory cytokines such as TNF-α, IL-1β and IL-6. The 6TG is more effective to reduce inflammatory reactions compared to 6H2MP, by the lower dose needed compared to 6H2MP in all experiments except in PGE2.

CONCLUSION: The inhibition of inflammatory mediators is an important mechanism by which 6TG and 6H2MP may alleviate pain and articular inflammation. These results indicated that 6H2MP and 6TG are effective candidates for ameliorating inflammatory-associated complications.

KEYWORDS: anti-inflammatory, HIG-82 cells, RAW264.7 cells, 6-Thioguanine, 6-Hydroxy-2-Mercaptopurine

Full Text:



Lee SC, Kwon YW, Park JY, Park SY, Lee JH, Park SD. (2017). Antioxidant and anti-inflammatory effects of herbal formula SC-E3 in lipopolysaccharide-stimulated RAW 264.7 macrophages. eCAM. 2017; 2017: 1725246, CrossRef.

Chen J, Er HM, Mohamed SM, Chen YS. In vitro anti-inflammatory activity of fractionated Euphorbia hirta aqueous extract on rabbit synovial fibroblasts. Biomed J. 2015; 38: 301-6, CrossRef.

Petit E, Langouet S, Akhdar H, Nicolas-Nicolaz C, Guillouzo A, Morel F. Differential toxic effects of azathioprine, 6-mercaptopurine and 6-thioguanine on human hepatocytes. Toxicol in vitro. 2008; 22: 632-42, CrossRef.

Karim H, Hashemi J, Larsson C, Moshfegh A, Fotoohi AK, Albertioni F. The pattern of gene expression and gene dose profiles of 6-Mercaptopurine-and 6-Thioguanine-resistant human leukemia cells. Biochem Biophys Res Commun. 2011; 411: 156-61, CrossRef.

Deshpande AR, Abreu MT. Optimizing therapy with 6-mercaptopurine and azathioprine: To measure or not to measure? Therap Adv Gastroenterol. 2010; 3: 275-9, CrossRef.

Kim I, Choi YS, Song JH, Choi EA, Park S, Lee EJ, et al. A drug‐repositioning screen for primary pancreatic ductal adenocarcinoma cells identifies 6‐thioguanine as an effective therapeutic agent for TPMT‐low cancer cells. Mol Oncol. 2018; 12: 1526-39, CrossRef.

Shin JH, Ryu JH, Kang MJ, Hwang CR, Han J, Kang D. Short-term heating reduces the anti-inflammatory effects of fresh raw garlic extracts on the LPS-induced production of NO and pro-inflammatory cytokines by downregulating allicin activity in RAW 264.7 macrophages. Food Chem. Toxicol. 2013; 58: 545-51, CrossRef.

Chiong HS, Yong YK, Ahmad Z, Sulaiman MR, Zakaria ZA, Yuen KH, et al. (2013). Cytoprotective and enhanced anti-inflammatory activities of liposomal piroxicam formulation in lipopolysaccharide-stimulated RAW 264.7 macrophages. Int J Nanomedicine. 2013; 8, 1245-55, CrossRef.

Seidman EG. Clinical use and practical application of TPMT enzyme and 6-mercaptopurine metabolite monitoring in IBD. Rev Gastroenterol Disord. 2003; 3 (Suppl 1): S30-8, PMID.

Kurakula K, Hamers AA, van Loenen P, de Vries CJ. 6-Mercaptopurine reduces cytokine and Muc5ac expression involving inhibition of NFκB activation in airway epithelial cells. Respir Res. 2015; 16: 73, CrossRef.

Murthuza S, Manjunatha BK. In vitro and in vivo evaluation of anti-inflammatory potency of Mesua ferrea, Saraca asoca, Viscum album & Anthocephalus cadamba in murine macrophages raw 264.7 cell lines and Wistar albino rats. Beni-Suef Univ J Basic Appl Sci. 2018; 7: 719-23, CrossRef.

Romanowicz GE, He W, Nielsen M, Frost MC. Novel device for continuous spatial control and temporal delivery of nitric oxide for in vitro cell culture. Redox Biol. 2013; 1: 332-9, CrossRef.

Joo T, Sowndhararajan K, Hong S, Lee J, Park SY, Kim S, et al. Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumila L. Saudi J Biol Sci. 2014; 21: 427-35, CrossRef.

Sosroseno W, Bird PS, Seymour GJ. Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material. Anaerobe. 2011; 17: 246-51, CrossRef.

Villalonga N, David M, Bielańska J, González T, Parra D, Soler C, et al. Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1. 3 voltage-dependent potassium channels. Biochem Pharmacol. 2010; 80: 858-66, CrossRef.

Bombardo M, Malagola E, Chen R, Rudnicka A, Graf R, Sonda S. Ibuprofen and diclofenac treatments reduce proliferation of pancreatic acinar cells upon inflammatory injury and mitogenic stimulation. Br J Pharmacol. 2018; 175: 335-47, CrossRef.

Kaminska B. MAPK signalling pathways as molecular targets for anti-inflammatory therapy - From molecular mechanisms to therapeutic benefits. Biochim Biophys Acta Proteins Proteom. 2005; 1754: 253-62, CrossRef.

Al-Nimer MSM, Ali EA. Dual effects of interaction between meloxicam, diclofenac sodium or tramadol and nitrogen species radicals: In vitro comparative study. Int J Pharmacol. 2009; 5: 86-9, CrossRef.

Marinković G, Hamers AA, de Vries CJ, de Waard V. 6-Mercaptopurine reduces macrophage activation and gut epithelium proliferation through inhibition of GTPase Rac1. Inflamm Bowel Dis. 2014; 20: 1487-95, CrossRef.

Marei H, Malliri A. Rac1 in human diseases : The therapeutic potential of targeting Rac1 signaling regulatory mechanisms. Small GTPases. 2017; 8: 139-63, CrossRef.

Stefanovic‐Racic M, Stadler J, Georgescu HI, Evans CH. Nitric oxide and energy production in articular chondrocytes. J Cell Physiol. 1994; 159: 274-80, CrossRef.

Dey P, Panga V, Raghunathan S. A cytokine signalling network for the regulation of inducible nitric oxide synthase expression in rheumatoid arthritis. PLoS One. 2016; 11: e0161306, CrossRef.

Casnici C, Lattuada D, Tonna N, Crotta K, Storini C, Bianco F, et al. Optimized "in vitro" culture conditions for human rheumatoid arthritis synovial fibroblasts. Mediators Inflamm. 2014; 2014: 702057, CrossRef.

Kumar DA, Settu K, Raju KVN, Kumanan K, Manohar BM, Puvanakrishnan R. Inhibition of nitric oxide and caspase-3 mediated apoptosis by a tetrapeptide derivative (PEP1261) in cultured synovial fibroblasts from collagen-induced arthritis. Mol Cell Biochem. 2006; 282: 125-39, CrossRef.

Paduch R, Kandefer-Szerszeń M. Nitric oxide (NO) and cyclooxygenase-2 (COX-2) cross-talk in co-cultures of tumor spheroids with normal cells. Cancer Microenviron. 2011; 4: 187-98, CrossRef.

Palmer G, Gabay C, Imhof BA. Leukocyte migration to rheumatoid joints: enzymes take over. Arthritis Rheumatol. 2006; 54: 2707-10, CrossRef.

Gallelli L, Galasso O, Falcone D, Southworth S, Greco M, Ventura V, et al. The effects of nonsteroidal anti-inflammatory drugs on clinical outcomes, synovial fluid cytokine concentration and signal transduction pathways in knee osteoarthritis. A randomized open label trial. Osteoarthr Cartil. 2013; 21: 1400-8, CrossRef.

Yeo NK, Park WJ, Eom DW, Oh MY, Lee JH. Effects of azathioprine and its metabolites on inflammatory cytokines in human nasal polyp organ cultures. Int Forum Allergy Rhinol. 2019; 9: 648-55, CrossRef.

Kverka M, Rossmann P, Tlaskalova-Hogenova H, Klimesova K, Jharap B, de Boer NK, et al. Safety and efficacy of the immunosuppressive agent 6-tioguanine in murine model of acute and chronic colitis. BMC Gastroenterol. 2011; 11: 1-9. 47, CrossRef.

Tan YY, Epstein LB, Armstrong RD. In vitro evaluation of 6-thioguanine and α-interferon as a therapeutic combination in HL-60 and natural killer cells. Cancer Res. 1989; 49: 4431-4, PMID.

Yarur AJ, Abreu MT, Deshpande AR, Kerman DH, Sussman DA. Therapeutic drug monitoring in patients with inflammatory bowel disease. World J Gastroenterol. 2014; 20: 3475-84, CrossRef.

Shivashankara AR, Rao S, George T, Abraham S, Colin MD, Palatty PL, et al. Chapter 15: Tea (Camellia sinensis L. Kuntze) as hepatoprotective agent: a revisit. In: Dietary Interventions in Liver Disease. Cambride: Academic Press; 2019. p. 183-92, CrossRef.

Tidén AK, Sjögren T, Svensson M, Bernlind A, Senthilmohan R, Auchère F, Lundquist S. 2-thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation. J Biol Chem. 2011; 286: 37578-89, CrossRef.

Hachiya M, Osawa Y, Akashi M. Role of TNF-α in regulation of myeloperoxidase expression in irradiated HL60 promyelocytic cells. Biochim Biophys Acta Mol Cell Res. 2000; 1495: 237-49, CrossRef.

Matsuno H, Yudoh K, Katayama R, Nakazawa F, Uzuki M, Sawai T, et al. The role of TNF‐α in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera. Rheumatology. 2002; 41: 329-37, CrossRef.

Abu-Soud HM, Hazen SL. Nitric oxide modulates the catalytic activity of myeloperoxidase. J Biol Chem. 2000; 275: 5425-30, CrossRef.


Copyright (c) 2021 The Prodia Education and Research Institute

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.


Indexed by:






The Prodia Education and Research Institute