Carboxyamidotriazole exerts anti‑inflammatory activity in lipopolysaccharide‑induced RAW264.7 macrophages by inhibiting NF‑κB and MAPKs pathways

Lu,Shan; Duan,Mengyuan; Guo,Zehao; Zhou,Yongting; Wu,Danwei; Zhang,Xiaojuan; Wang,Yicheng; Ye,Caiying; Ju,Rui; Li,Juan; Zhang,Dechang; Zhu,Lei

doi:10.3892/etm.2020.8889

Carboxyamidotriazole exerts anti‑inflammatory activity in lipopolysaccharide‑induced RAW264.7 macrophages by inhibiting NF‑κB and MAPKs pathways

Authors:
- Shan Lu
- Mengyuan Duan
- Zehao Guo
- Yongting Zhou
- Danwei Wu
- Xiaojuan Zhang
- Yicheng Wang
- Caiying Ye
- Rui Ju
- Juan Li
- Dechang Zhang
- Lei Zhu
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Affiliations: Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, P.R. China, Department of Pharmacy, Beijing Jishuitan Hospital, Beijing 100035, P.R. China
Published online on: June 12, 2020 https://doi.org/10.3892/etm.2020.8889
Pages: 1455-1466
Copyright: © Lu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Carboxyamidotriazole (CAI), originally developed as a non-cytotoxic anti-cancer drug, was shown to have anti‑inflammatory activity according to recent studies in a number of animal models of inflammation. However, its mechanism of action has not been characterized. Therefore, the present study was performed to identify the anti‑inflammatory action of CAI in lipopolysaccharide (LPS)‑induced RAW 264.7 macrophages and to identify the signal transduction pathways involved. The in vitro results revealed that CAI had no direct effect on the activity of cyclooxygenase (COX), suggesting a different anti‑inflammatory mechanism compared with that of COX‑inhibiting non‑steroidal anti‑inflammatory drugs. Further investigation in RAW264.7 macrophages revealed that CAI decreased the production of nitric oxide via decreasing the LPS‑stimulated expression of inducible nitric oxide synthase, and downregulated both mRNA and protein expression levels of the cytokines tumor necrosis factor‑α, interleukin (IL)‑1β, and IL‑6. CAI also significantly reduced the increased DNA‑binding activity of nuclear factor (NF)‑κB induced by LPS stimulation. With respect to the mechanisms involved on NF‑κB activity, CAI exhibited suppression of the phosphorylation and degradation of the inhibitor of nuclear factor‑κBα (IκB), and decreased the phosphorylation levels of the p65 subunit and its subsequent nuclear translocation. In addition, CAI significantly decreased the phosphorylated forms of p38, JNK and ERK, which were increased following LPS stimulation, while the total expression levels of p38, JNK and ERK remained unaltered. The results in the present study indicate that CAI alleviates the inflammatory responses of RAW 264.7 macrophages in response to LPS stimulation via attenuating the activation of NF‑κB and MAPK signaling pathways and decreasing the levels of pro‑inflammatory mediators. This offers a novel perspective for understanding the anti‑inflammatory mechanism of CAI and suggests its potential use as a therapeutic treatment in inflammatory diseases with excessive macrophage activation.

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1	Krishnamoorthy S and Honn KV: Inflammation and disease progression. Cancer Metastasis Rev. 25:481–491. 2006.PubMed/NCBI View Article : Google Scholar
2	Kawai T and Akira S: TLR signaling. Cell Death Differ. 13:816–825. 2006.PubMed/NCBI View Article : Google Scholar
3	Siebert S, Tsoukas A, Robertson J and McInnes I: Cytokines as therapeutic targets in rheumatoid arthritis and other inflammatory diseases. Pharmacol Rev. 67:280–309. 2015.PubMed/NCBI View Article : Google Scholar
4	Jang KJ, Choi SH, Yu GJ, Hong SH, Chung YH, Kim CH, Yoon HM, Kim GY, Kim BW and Choi YH: Anti-inflammatory potential of total saponins derived from the roots of panax ginseng in lipopolysaccharide-activated RAW 264.7 macrophages. Exp Ther Med. 11:1109–1115. 2016.PubMed/NCBI View Article : Google Scholar
5	Wei X, Song H, Yin L, Rizzo MG, Sidhu R, Covey DF, Ory DS and Semenkovich CF: Fatty acid synthesis configures the plasma membrane for inflammation in diabetes. Nature. 539:294–298. 2016.PubMed/NCBI View Article : Google Scholar
6	Zhu L, Wei W, Zheng YQ and Jia XY: Effects and mechanisms of total glucosides of paeony on joint damage in rat collagen-induced arthritis. Inflamm Res. 54:211–220. 2005.PubMed/NCBI View Article : Google Scholar
7	Diakos CI, Charles KA, McMillan DC and Clarke SJ: Cancer-related inflammation and treatment effectiveness. Lancet Oncol. 15:e493–e503. 2014.PubMed/NCBI View Article : Google Scholar
8	Ferrucci L and Fabbri E: Inflammageing: Chronic inflammation in ageing, cardiovascular disease, and frailty. Nat Rev Cardiol. 15:505–522. 2018.PubMed/NCBI View Article : Google Scholar
9	Kohn EC, Sandeen MA and Liotta LA: In vivo efficacy of a novel inhibitor of selected signal transduction pathways including calcium, arachidonate, and inositol phosphates. Cancer Res. 52:3208–3212. 1992.PubMed/NCBI
10	Kohn EC, Felder CC, Jacobs W, Holmes KA, Day A, Freer R and Liotta LA: Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI. Cancer Res. 54:935–942. 1994.PubMed/NCBI
11	Wasilenko WJ, Palad AJ, Somers KD, Blackmore PF, Kohn EC, Rhim JS, Wright GL Jr and Schellhammer PF: Effects of the calcium influx inhibitor carboxyamido-triazole on the proliferation and invasiveness of human prostate tumor cell lines. Int J Cancer. 68:259–264. 1996.PubMed/NCBI View Article : Google Scholar
12	Moody TW, Chiles J, Moody E, Sieczkiewicz GJ and Kohn EC: CAI inhibits the growth of small cell lung cancer cells. Lung Cancer. 39:279–288. 2003.PubMed/NCBI View Article : Google Scholar
13	Hussain MM, Kotz H, Minasian L, Premkumar A, Sarosy G, Reed E, Zhai S, Steinberg SM, Raggio M, Oliver VK, et al: Phase II trial of carboxyamidotriazole in patients with relapsed epithelial ovarian cancer. J Clin Oncol. 21:4356–4363. 2003.PubMed/NCBI View Article : Google Scholar
14	Dutcher JP, Leon L, Manola J, Friedland DM, Roth B and Wilding G: Eastern Cooperative Oncology Group. Phase II study of carboxyamidotriazole in patients with advanced renal cell carcinoma refractory to immunotherapy: E4896, an Eastern cooperative oncology group study. Cancer. 104:2392–2399. 2005.PubMed/NCBI View Article : Google Scholar
15	Mikkelsen T, Lush R, Grossman SA, Carson KA, Fisher JD, Alavi JB and Rosenfeld S: Phase II clinical and pharmacologic study of radiation therapy and carboxyamido-triazole (CAI) in adults with newly diagnosed glioblastoma multiforme. Invest New Drugs. 25:259–263. 2007.PubMed/NCBI View Article : Google Scholar
16	Ju R, Wu D, Guo L, Li J, Ye C and Zhang D: Inhibition of pro-inflammatory cytokines in tumour associated macrophages is a potential anti-cancer mechanism of carboxyamidotriazole. Eur J Cancer. 48:1085–1095. 2012.PubMed/NCBI View Article : Google Scholar
17	Guo L, Ye C, Chen W, Ye H, Zheng R, Li J, Yang H, Yu X and Zhang D: Anti-inflammatory and analgesic potency of carboxyamidotriazole, a tumorostatic agent. J Pharmacol Exp Ther. 325:10–16. 2008.PubMed/NCBI View Article : Google Scholar
18	Zhu L, Li J, Guo L, Yu X, Wu D, Luo L, Zhu L, Chen W, Chen C, Ye C and Zhang D: Activation of NALP1 inflammasomes in rats with adjuvant arthritis; a novel therapeutic target of carboxyamidotriazole in a model of rheumatoid arthritis. Br J Pharmacol. 172:3446–3459. 2015.PubMed/NCBI View Article : Google Scholar
19	Zhu L, Li J, Guo L, Yu XL, Wu DW, Chen W, Chen C, Du XW, Zhang DC and Ye CY: Therapeutic effect of carboxyamidotriazole on adjuvant arthritis in rats. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 38:49–54. 2016.PubMed/NCBI View Article : Google Scholar
20	Du X, Chen W, Wang Y, Chen C, Guo L, Ju R, Li J, Zhang D, Zhu L and Ye C: Therapeutic efficacy of carboxyamidotriazole on 2,4,6-trinitrobenzene sulfonic acid-induced colitis model is associated with the inhibition of NLRP3 inflammasome and NF-κB activation. Int Immunopharmacol. 45:16–25. 2017.PubMed/NCBI View Article : Google Scholar
21	Jeon HL, Yoo JM, Lee BD, Lee SJ, Sohn EJ and Kim MR: Anti-inflammatory and antioxidant actions of N-arachidonoyl serotonin in RAW264.7 cells. Pharmacology. 97:195–206. 2016.PubMed/NCBI View Article : Google Scholar
22	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
23	Guo T, Lin Q, Li X, Nie Y, Wang L, Shi L, Xu W, Hu T, Guo T and Luo F: Octacosanol attenuates inflammation in both RAW264.7 macrophages and a mouse model of colitis. J Agric Food Chem. 65:3647–3658. 2017.PubMed/NCBI View Article : Google Scholar
24	Fengyang L, Yunhe F, Bo L, Zhicheng L, Depeng L, Dejie L, Wen Z, Yongguo C, Naisheng Z, Xichen Z and Zhengtao Y: Stevioside suppressed inflammatory cytokine secretion by downregulation of NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 cells. Inflammation. 35:1669–1675. 2012.PubMed/NCBI View Article : Google Scholar
25	Choi WS, Shin PG, Lee JH and Kim GD: The regulatory effect of veratric acid on NO production in LPS-stimulated RAW264.7 macrophage cells. Cell Immunol. 280:164–170. 2012.PubMed/NCBI View Article : Google Scholar
26	Choi WS, Seo YB, Shin PG, Kim WY, Lee SY, Choi YJ and Kim GD: Veratric acid inhibits iNOS expression through the regulation of PI3K activation and histone acetylation in LPS-stimulated RAW264.7 cells. Int J Mol Med. 35:202–210. 2015.PubMed/NCBI View Article : Google Scholar
27	Bacchi S, Palumbo P, Sponta A and Coppolino MF: Clinical pharmacology of non-steroidal anti-inflammatory drugs: A review. Antiinflamm Antiallergy Agents Med Chem. 11:52–64. 2012.PubMed/NCBI View Article : Google Scholar
28	Viatour P, Merville MP, Bours V and Chariot A: Phosphorylation of NF-kappaB and IkappaB proteins: Implications in cancer and inflammation. Trends Biochem Sci. 30:43–52. 2005.PubMed/NCBI View Article : Google Scholar
29	Seger R and Krebs EG: The MAPK signaling cascade. FASEB J. 9:726–735. 1995.PubMed/NCBI
30	Nemoto S, DiDonato JA and Lin A: Coordinate regulation of IkappaB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappaB-inducing kinase. Mol Cell Biol. 18:7336–7343. 1998.PubMed/NCBI View Article : Google Scholar
31	Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, Lee L and Isakson P: Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci USA. 91:12013–12017. 1994.PubMed/NCBI View Article : Google Scholar
32	Zhu L, Duan MY, Zhang XJ, Li J, JU R, Guo L, Lu S and Ye CY: Inhibitory effects of carboxyamidotriazole on cytokines production by peritoneal macrophages from adjuvant arthritis rats. Basic Clin Med. 38:798–802. 2018.
33	Zhang X and Mosser DM: Macrophage activation by endogenous danger signals. J Pathol. 214:161–178. 2008.PubMed/NCBI View Article : Google Scholar
34	Rim HK, Cho W, Sung SH and Lee KT: Nodakenin suppresses lipopolysaccharide-induced inflammatory responses in macrophage cells by inhibiting tumor necrosis factor receptor-associated factor 6 and nuclear factor-κB pathways and protects mice from lethal endotoxin shock. J Pharmacol Exp Ther. 342:654–664. 2012.PubMed/NCBI View Article : Google Scholar
35	Bogdan C: Nitric oxide and the immune response. Nat Immunol. 2:907–916. 2001.PubMed/NCBI View Article : Google Scholar
36	Davis KL, Martin E, Turko IV and Murad F: Novel effects of nitric oxide. Annu Rev Pharmacol Toxicol. 41:203–236. 2001.PubMed/NCBI View Article : Google Scholar
37	Aktan F: iNOS-mediated nitric oxide production and its regulation. Life Sci. 75:639–653. 2004.PubMed/NCBI View Article : Google Scholar
38	Farlik M, Reutterer B, Schindler C, Greten F, Vogl C, Müller M and Decker T: Nonconventional initiation complex assembly by STAT and NF-kappaB transcription factors regulates nitric oxide synthase expression. Immunity. 33:25–34. 2010.PubMed/NCBI View Article : Google Scholar
39	Kalliolias GD and Ivashkiv LB: TNF biology, pathogenic mechanisms and emerging therapeutic strategies. Nat Rev Rheumatol. 12:49–62. 2016.PubMed/NCBI View Article : Google Scholar
40	Ulevitch RJ and Tobias PS: Recognition of gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol. 11:19–22. 1999.PubMed/NCBI View Article : Google Scholar
41	Aderem A and Ulevitch RJ: Toll-like receptors in the induction of the innate immune response. Nature. 406:782–787. 2000.PubMed/NCBI View Article : Google Scholar
42	Bonizzi G and Karin M: The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25:280–288. 2004.PubMed/NCBI View Article : Google Scholar
43	Oeckinghaus A, Hayden MS and Ghosh S: Crosstalk in NF-κB signaling pathways. Nat Immunol. 12:695–708. 2011.PubMed/NCBI View Article : Google Scholar
44	Li Q and Verma IM: NF-kappaB regulation in the immune system. Nat Rev Immunol. 2:725–734. 2002.PubMed/NCBI View Article : Google Scholar
45	Shibata W, Maeda S, Hikiba Y, Yanai A, Ohmae T, Sakamoto K, Nakagawa H, Ogura K and Omata M: Cutting edge: The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks inflammatory injury in murine colitis. J Immunol. 179:2681–2685. 2007.PubMed/NCBI View Article : Google Scholar
46	Johnson GL and Lapadat R: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 298:1911–1912. 2002.PubMed/NCBI View Article : Google Scholar
47	Strnisková M, Barancík M and Ravingerová T: Mitogen-activated protein kinases and their role in regulation of cellular processes. Gen Physiol Biophys. 21:231–255. 2002.PubMed/NCBI
48	Kristof AS, Marks-Konczalik J and Moss J: Mitogen-activated protein kinases mediate activator protein-1-dependent human inducible nitric-oxide synthase promoter activation. J Biol Chem. 276:8445–8452. 2001.PubMed/NCBI View Article : Google Scholar
49	Tanoue T and Nishida E: Docking interactions in the mitogen-activated protein kinase cascades. Pharmacol Ther. 93:193–202. 2002.PubMed/NCBI View Article : Google Scholar