Calyptranthes grandifolia O.Berg (Myrtaceae) ethanolic extract inhibits TNF-α gene expression and cytokine release in vitro

Dexheimer,Geórgia  Muccillo; Becker Delving,Luciana   Knabben de Oliveira; de Oliveira,Henrique   Sulzbach; Biolchi,Vanderlei; Goettert,Márcia  Inês; Pozzobon,Adriane

doi:10.3892/mmr.2017.6319

Calyptranthes grandifolia O.Berg (Myrtaceae) ethanolic extract inhibits TNF-α gene expression and cytokine release in vitro

Authors:
- Geórgia Muccillo Dexheimer
- Luciana Knabben de Oliveira Becker Delving
- Henrique Sulzbach de Oliveira
- Vanderlei Biolchi
- Márcia Inês Goettert
- Adriane Pozzobon
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Affiliations: Biotechnology Program, Univates, RS 95900‑000, Brazil, Molecular Biology Laboratory and Medical School, Univates, Lajeado, RS 95900‑000, Brazil
Published online on: March 14, 2017 https://doi.org/10.3892/mmr.2017.6319
Pages: 2873-2880

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Abstract

Anti-tumor therapies based on anti-inflammatory effects have been considered in cancer treatment. Survival, proliferation and, resultantly, invasion and metastasis of tumor cells are regulated by local inflammatory mediators. Primary inflammatory cytokines, such as tumor necrosis factor (TNF), are targets for anticancer therapy. Several anti‑inflammatory agents isolated from natural products are becoming important chemopreventive and therapeutic agents for cancer. The present study aimed to investigate the expression of TNF‑α, nuclear factor‑κΒ (NF‑κΒ) and p38α mitogen-activated protein kinase (p38α) genes, associated with proliferation and inflammation in the Caco‑2 cell line treated with ethanolic and hexanic extracts of Calyptranthes grandifolia O.Berg (Myrtaceae). Caco‑2 cells were cultured and treated with plant extract at different concentrations (25, 50, 100 and 200 µg/ml) and stimulated with lipopolysaccharide (LPS). For gene expression, analysis was performed by total RNA extraction followed by synthesis of complementary DNA and analysis by quantitative polymerase chain reaction. The release of TNF‑α cytokine was evaluated by ELISA in RAW 264.7 murine macrophages activated by LPS. Among the evaluated genes, there was a decrease in TNF-α expression at 100 and 200 µg/ml concentrations only with the ethanolic extract (P<0.025). The p38α gene exhibited a tendency to increase expression only when treated with ethanolic extract and the NF‑κΒ gene did not significantly differ compared with the positive control when treated with either analyzed extract. The inhibition of TNF-α cytokine in the RAW 264.7 cell line was significant (P<0.05) in ethanolic extract at 200 µg/ml compared with the positive control (LPS 1 µg/ml). In conclusion, the ethanolic extract may exhibit an anti‑inflammatory activity by inhibiting TNF‑α. However, further studies are required to confirm its potential anti-inflammatory effects.

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1	Germano G, Allavena P and Mantovani A: Cytokines as key component of cancer-related inflammation. Cytokine. 43:374–379. 2008. View Article : Google Scholar : PubMed/NCBI
2	Bartlett DL, Ma G, Alexander HR, Libutti SK and Fraker DL: Isolated limb reperfusion with tumor necrosis factor and melphalan in patients with extremity melanoma after failure of isolated limb perfusion with chemotherapeutics. Cancer. 80:2084–2090. 1997. View Article : Google Scholar : PubMed/NCBI
3	Madhusudan S, Muthuramalingam SR, Braybrooke JP, Wilner S, Kaur K, Han C, Hoare S, Balkwill F and Ganesan TS: Study of etanercept, a tumor necrosis factor-alpha inhibitor, in recurrent ovarian cancer. J ClinOncol. 23:5950–5959. 2005. View Article : Google Scholar
4	Harrison ML, Obermueller E, Maisey NR, Hoare S, Edmonds K, Li NF, Chao D, Hall K, Lee C, Timotheadou E, et al: Tumor necrosis factor alpha as a new target for renal cell carcinoma: Two sequential phase II trials of infliximab at standard and high dose. J ClinOncol. 25:4542–4549. 2007. View Article : Google Scholar
5	Garg A and Aggarwal BB: Nuclear transcription factor-kappaB as a target for cancer drug development. Leukemia. 16:1053–1068. 2002. View Article : Google Scholar : PubMed/NCBI
6	Beg AA and Baltimore D: An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science. 274:782–784. 1996. View Article : Google Scholar : PubMed/NCBI
7	Stroh C, Held J, Samraj AK and Schulze-Osthoff K: Specific inhibition of transcription factor NF-kappaB through intracellular protein delivery of IkappaBalpha by the Herpes virus protein VP22. Oncogene. 22:5367–5373. 2003. View Article : Google Scholar : PubMed/NCBI
8	Dolado I and Nebreda AR: Regulation of tumorigenesis by p38α MAP kinase. Topics Curr Genet. 20:99–128. 2008. View Article : Google Scholar
9	Brandão HN, David JP, Couto RD, Nascimento JAP and David JM: Química e farmacologia de quimioterápicos antineoplásicos derivados de plantas. Quím Nova. 33:1359–1369. 2010. View Article : Google Scholar
10	Aravindaram K and Yang NS: Anti-inflammatory plant natural products for cancer therapy. Planta Med. 76:1103–1117. 2010. View Article : Google Scholar : PubMed/NCBI
11	Bhanot A, Sharma R and Noolvi MN: Natural sources as potential anti-cancer agents: A review. Int J Phytomed. 3:9–26. 2011.
12	Newman DJ, Cragg GM and Snader KM: The influence of natural products upon drug discovery. Nat Prod Rep. 17:215–234. 2000. View Article : Google Scholar : PubMed/NCBI
13	Newman DJ and Cragg GM: Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod. 75:311–335. 2012. View Article : Google Scholar : PubMed/NCBI
14	Souza-Moreira TM, Moreira RRD, Sacramento LVS and Pietro RCLR: Histochemical, phytochemical and biological screening of Pliniacauliflora (Mart.) Kausel (Myrtaceae) leaves. RevBras Farmacogn. 20:48–53. 2010. View Article : Google Scholar
15	Zhang P, Zhang E, Xiao M, Chen C and Xu W: Enhanced chemical and biological activities of a newly biosynthesized eugenol glycoconjugate, eugenol α-D-glucopyranoside. Appl Microbiol Biotechnol. 97:1043–1050. 2013. View Article : Google Scholar : PubMed/NCBI
16	Rizzo LY, Longato GB, Ruiz AL, Tinti SV, Possenti A, Vendramini-Costa DB, Sartoratto A, Figueira GM, Silva FL, Eberlin MN, et al: In vitro, in vivo and in silico analysis of the anticancer and estrogen-like activity of guava leaf extracts. Curr Med Chem. 21:2322–2330. 2014. View Article : Google Scholar : PubMed/NCBI
17	Sultana B, Anwar F, Mushtaq M, Aslam M and Ijaz S: In vitro antimutagenic, antioxidant activities and total phenolics of clove (Syzygiumaromaticum L.) seed extracts. Pak J Pharm Sci. 27:893–899. 2014.PubMed/NCBI
18	Liu H, Schmitz JC, Wei J, Cao S, Beumer JH, Strychor S, Cheng L, Liu M, Wang C, Wu N, et al: Clove extract inhibits tumor growth and promotes cell cycle arrest and apoptosis. Oncol Res. 21:247–259. 2014. View Article : Google Scholar : PubMed/NCBI
19	Ye CL, Liu Y and Wei DZ: Antioxidant and anticancer activity of 3′-formyl-4′,6′-dihydroxy-2′-methoxy-5′-methylchalcone and (2S)-8-formyl-5-hydroxy-7-methoxy-6-methylflavanone. J Pharm Pharmacol. 59:553–559. 2007. View Article : Google Scholar : PubMed/NCBI
20	Aisha AF, Ismail Z, Abu-Salah KM, Siddiqui JM, Ghafar G and Majid Abdul AM: Syzygium campanulatum korth methanolic extract inhibits angiogenesis and tumor growth in nude mice. BMC Complement Altern Med. 13:1682013. View Article : Google Scholar : PubMed/NCBI
21	Pascoal AC, Ehrenfried CA, Lopez BG, de Araujo TM, Pascoal VD, Gilioli R, Ruiz AL, Carvalho JE, Stefanello ME and Salvador MJ: Antiproliferative activity and induction of apoptosis IN PC-3 cells by the chalcone cardamonin from Campomanesia adamantium (Myrtaceae) in a bioactivity-guided study. Molecules. 19:1843–1855. 2014. View Article : Google Scholar : PubMed/NCBI
22	Michel MCP, Guimarães AG, Paula CA, Rezende SA, Sobral MEG and Guimarães DAS: Extracts from the leaves of Campomanesia velutina inhibits production of LPS/INF-γ induced inflammatory mediators in J774A.1 cells and exerts anti-inflammatory and antinociceptive effects in vivo. Rev Bras Farmacogn. 23:927–936. 2013. View Article : Google Scholar
23	Jeong D, Yang WS, Yang Y, Nam G, Kim JH, Yoon DH, Noh HJ, Lee S, Kim TW, Sung GH and Cho JY: In vitro and in vivo anti-inflammatory effect of Rhodomyrtus tomentosa methanol extract. J Ethnopharmacol. 146:205–213. 2013. View Article : Google Scholar : PubMed/NCBI
24	Flores G, Dastmalchi K, Wu SB, Whalen K, Dabo AJ, Reynertson KA, Foronjy RFD, Armiento JM and Kennelly EJ: Phenolic-rich extract from the Costa Rican guava (Psidiumfriedrichsthalianum) pulp with antioxidant and anti-inflammatory activity. Potential for COPD therapy. Food Chem. 141:889–895. 2013. View Article : Google Scholar : PubMed/NCBI
25	Serafino A, Vallebona Sinibaldi P, Andreola F, Zonfrillo M, Mercuri L, Federici M, Rasi G, Garaci E and Pierimarchi P: Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response. BMC Immunol. 9:2008. View Article : Google Scholar : PubMed/NCBI
26	Santos FA, Rao VSN and Silveira ER: Anti-inflammatory and analgesic activities of the essential oil of Psidium guianense. Fitoterapia. 68:65–68. 1997.
27	Sharma R, Kishore N, Hussein A and Lall N: Antibacterial and anti-inflammatory effects of Syzygiumjambos L. (Alston) and isolated compounds on acne vulgaris. BMC Complement Altern Med. 13:2922013. View Article : Google Scholar : PubMed/NCBI
28	Gourdeau H, Leblond L, Hamelin B, Desputeau C, Dong K, Kianicka I, Custeau D, Boudreau C, Geerts L, Cai SX, et al: Antivascular and antitumor evaluation of 2-amino-4-(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes, a novel series of anticancer agents. Mol Cancer Ther. 3:1375–1384. 2004.PubMed/NCBI
29	Chetan BS, Nimesh MS, Manish PP and Ranjan GP: Microwave assisted synthesis of novel 4H-chromene derivatives bearing phenoxypyrazole and their antimicrobial activity assess. J Serb ChemSoc. 77:1165–1174. 2012. View Article : Google Scholar
30	Mladenović M, Mihailović M, Bogojević D, Matić S, Nićiforović N, Mihailović V, Vuković N, Sukdolak S and Solujić S: In vitro antioxidant of selected 4-Hydroxy-chromene-2-one derivatives-SAR, QSAR and DFT studies. Int J MolSci. 12:2822–2841. 2011. View Article : Google Scholar
31	Cheng JF, Ishikawa A, Ono Y, Arrhenius T and Nadzan A: Novel chromene derivatives as TNF-alpha inhibitors. Bioorg Med Chem Lett. 13:3647–3650. 2003. View Article : Google Scholar : PubMed/NCBI
32	Thareja S, Verma A, Kalra A, Gosain S, Rewatkar PV and Kokil GR: Novel chromeneimidazole derivatives as antifungal compounds: Synthesis and in vitro evaluation. Acta Pol Pharm. 67:423–427. 2010.PubMed/NCBI
33	Correa RGC, Silva ML, Maia JGS, Gottlieb OR, Mourão JC, Marx MC, Morais AA, Moura LL and Magalhães MT: Acta Amaz. 2:531972.
34	Silva ML, Luz AIR, Zoghbi MGB, Ramos LS and Maia JGS: Essential oil variation in Calyptranthes spruceana. Phytochem. 23:2515–2516. 1984. View Article : Google Scholar
35	Maia JGS, Zoghbi MGB and Andrade EHA: Aromatic Plants in the Amazon and their Essential Oils. Emílio Goeldi Paraense Museum (Adolpho Ducke Series): Belém. 2001.(Portuguese).
36	Nanthakumar NN, Fusunyan RD, Sanderson I and Walker WA: Inflammation in the developing human intestine: A possible pathophysiologic contribution to necrotizing enterocolitis. Proc Natl Acad Sci USA. 97:6043–6048. 2000. View Article : Google Scholar : PubMed/NCBI
37	Strober W: Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2001. View Article : Google Scholar
38	Yang J, Yang Q, Yu S and Zhang X: Evaluation and validation of suitable reference genes for reverse transcription-quantitative polymerase chain reaction studies in cholangiocarcinoma patients and cell lines. Oncol Lett. 11:2673–2681. 2016.PubMed/NCBI
39	Piana C, Wirth M, Gerbes S, Viernstein H, Gabor F and Toegel S: Validation of reference genes for qPCR studies on Caco-2 cell differentiation. Eur J Pharm Biopharm. 69:1187–1192. 2008. View Article : Google Scholar : PubMed/NCBI
40	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. View Article : Google Scholar : PubMed/NCBI
41	Rozen S and Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 132:365–386. 2000.PubMed/NCBI
42	Herath TD, Darveau RP, Seneviratne CJ, Wang CY, Wang Y and Jin L: Tetra- and penta-acylated lipid A structures of Porphyromonasgingivalis LPS differentially activate TLR4-mediated NF-κB signal transduction cascade and immuno-inflammatory response in human gingival fibroblasts. PLoS One. 8:e584962013. View Article : Google Scholar : PubMed/NCBI
43	Ferreira LC, Grabe-guimarães A, de Paula CA, Michel MC, Guimarães RG, Rezende SA, de Souza Filho JD and Saúde-Guimarães DA: Anti-inflammatory and antinociceptive activities of Campomanesia adamantium. J Ethnopharmacol. 9:100–108. 2013. View Article : Google Scholar
44	Li LJ, Yu LJ, Li YC, Liu MY and Wu ZZ: In vitro anti-inflammatory and free radical scavenging activities of flavans from Ilex centrochinensis. Zhongguo Zhong Yao Za Zhi. 40:1523–1528. 2015.(In Chinese). PubMed/NCBI
45	Perkins ND: Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol. 8:49–62. 2007. View Article : Google Scholar : PubMed/NCBI
46	Foo SY and Nolan GP: NF-kappaB to the rescue: RELs, apoptosis and cellular transformation. Trends Genet. 15:229–235. 1999. View Article : Google Scholar : PubMed/NCBI
47	Clément JF, Meloche S and Servant MJ: The IKK-related kinases: From innate immunity to oncogenesis. Cell Res. 18:889–899. 2008. View Article : Google Scholar : PubMed/NCBI
48	Vallabhapurapu S and Karin M: Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol. 27:693–733. 2009. View Article : Google Scholar : PubMed/NCBI
49	Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E and Ben-Neriah Y: NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature. 23:461–466. 2004. View Article : Google Scholar
50	Wolska A, Lech-Maranda E and Robak T: Toll-like receptors and their role in hematologic malignancies. CurrMol Med. 9:324–335. 2009.
51	Surh YJ: Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer. 3:768–780. 2003. View Article : Google Scholar : PubMed/NCBI
52	Aggarwal BB and Shishodia S: Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol. 71:1397–1421. 2006. View Article : Google Scholar : PubMed/NCBI
53	Hu MC, Wang YP, Mikhail A, Qiu WR and Tan TH: Murine p38-delta mitogen activated protein kinase, a developmentally regulated protein kinase that is activated by stress and proinflammatory cytokines. J BiolChem. 274:7095–7102. 1999.
54	Enslen H, Raingeaud J and Davis RJ: Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J BiolChem. 273:1741–1748. 1998.
55	Zarubin T and Han J: Activation and signaling of the p38 MAP kinase pathway. Cell Res. 15:11–18. 2005. View Article : Google Scholar : PubMed/NCBI
56	Porras A and Guerrero C: Role of p38α in apoptosis: Implication in cancer development and therapy. Atlas Genet Cytogenet Oncol Haematol. 15:316–326. 2011.
57	Campbell J, Ciesielski CJ, Hunt AE, Horwood NJ, Beech JT, Hayes LA, Denys A, Feldmann M, Brennan FM and Foxwell BM: A novel mechanism for TNF-α regulation by p38 MAPK: Involvement of NF-kappaB with implications for therapy in rheumatoid arthritis. J Immunol. 173:6928–6937. 2004. View Article : Google Scholar : PubMed/NCBI
58	Héron-Milhavet L and Leroith D: Insulin-like growth factor I induces MDM2-dependent degradation of p53 via the p38 MAPK pathway in response to DNA damage. J BiolChem. 3:15600–15606. 2002.
59	Okamoto S, Krainc D, Sherman K and Lipton SA: Antiapoptotic role of the p38 mitogen-activated protein kinase-myocyte enhancer factor 2 transcription factor pathway during neuronal differentiation. Proc Natl AcadSci USA. 20:7561–7566. 2000. View Article : Google Scholar
60	Park JM, Greten FR, Li ZW and Karin M: Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science. 20:2048–2051. 2002. View Article : Google Scholar
61	Zhang X, Shan P, Alam J, Davis RJ, Flavell RA and Lee PJ: Carbon monoxide modulates Fas/Fas ligand, caspases, and Bcl-2 family proteins via the p38alpha mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury. J BiolChem. 13:22061–22070. 2003.
62	Comes F, Matrone A, Lastella P, Nico B, Susca FC, Bagnulo R, Ingravallo G, Modica S, Lo Sasso G, Moschetta A, et al: A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells. Cell Death Differ. 14:693–702. 2007. View Article : Google Scholar : PubMed/NCBI
63	Schewe DM and Aguirre-Ghiso JA: ATF6alpha-Rheb-mTOR signaling promotes survival of dormant tumor cells in vivo. Proc Natl Acad Sci USA. 29:10519–10524. 2008. View Article : Google Scholar
64	Geran RI, Greenberg NH, McDonald MM, Schumacher AM and Abott BJ: Protocols for screening chemical agents and natural products against animal tumour and other biological systems. Cancer Chemother Rep. 3:17–19. 1972.
65	Suffness M and Pezzuto JM: Assays related to cancer drug discovery. In: Hostettmann K (ed), Methods in Plant Biochemistry. Assays for Bioactivity. 6:71–133. 1990.
66	Mensor LL, Menezes FS, Leitão GG, Reis AS, Santos TC, Coube CS and Leitão SG: Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res. 15:127–130. 2001. View Article : Google Scholar : PubMed/NCBI