Caffeine, Trigonelline and Chlorogenic Acid: Modulation of miRNA expression in Fibrosis-associated Hepatocarcinogenesis.

• Grant number: 16/12015-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: January 01, 2017
• End date: February 29, 2020
• Field of knowledge: Health Sciences - Nutrition
• Principal Investigator: Luís Fernando Barbisan
• Grantee: Guilherme Ribeiro Romualdo
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Recently, hepatic fibrosis and carcinogenesis have been linked to altered expression of several miRNAs. In contrast, studies indicate that coffee drinking is associated with a 40% lower risk of developing fibrosis or liver cancer. Thus, the present project will evaluate whether caffeine alone or associated with trigoneline and/or chlorogenic acid (CGA): (A) attenuates fibrosis-associated hepatocarcinogenesis (FAH) in mice; (B) attenuates the fibrosis in co-culture of malignant hepatocytes and hepatic stellate cells (HSCs) and (C) alters the miRNAs global expression profile and the expression of target genes of differentially expressed miRNAs in the liver. In vivo: for FAH model, C3H/He male mice will receive single dose of diethylnitrosamine (intraperitoneal [i.p.], 50 mg/Kg body weight [b.w.]) at the postnatal day (PND) 14. From the 8th to 16th week, the animals receive three weekly doses of carbon tetrachloride (i.p., initial dose 0.25 mg/Kg b.w., and maximum dose 1.25 mg/Kg b.w.). In addition, from the 7th to the 17th week (1x/day, 5x/week), animals will receive by gavage caffeine (50 mg/Kg b.w./day); caffeine and trigonelline (50 and 25 mg/Kg b.w./day , respectively); caffeine and CGA (50 and 25 mg/Kg b.w./day); caffeine, trigonelline and CGA (50, 25 and 25 mg/Kg b.w./day) or just vehicle (distilled water). The animals will be euthanized at 17th week. Liver samples will be collected for histological analysis: histopathological evaluation (HE), analysis of collagen content (Picrosirius Red) and immunohistochemistry to activated HSCs (desmin and ±-SMA), macrophage (F4/80), preneoplastic liver lesions (CK8/18), cell proliferation (Ki-67) and apoptosis (cleaved caspase-3) markers. Other liver samples will be used for global miRNA expression (nCounter miRNA Expression Assay). After identification of differentially expressed miRNAs, bioinformatics prediction of targets genes will be performed. The list of predicted genes will be used to evaluate the expression of target mRNAs by RT-qPCR. In vitro: Co-culture of malignant hepatocytes (C3A) and human HSCs (LX-2) will be performed. Initially, the activation of HSCs will be performed by exposing them to lipopolysaccharide (LPS). In sequence, the co-culture will be exposed to caffeine (10, 5 or 2.5 mM), trigonelline (5, 2.5 or 25.1 mM) or CGA (5, 2.5 or 25.1 mM) alone or in combination for 24 to 48 hours. Samples of co-culture will be collected for histological analysis: HE, Picrosirius Red and immunofluorescence for HSCs counting. Other samples will be used for collagen I determination and quantification of pro-inflammatory cytokines (IL-6, TNF-±, TGFb-1, I-17A and IL-23) by ELISA. Moreover, the gene expression of collagen ±1(I), ±-SMA, TGF²-1, MMP-2, MMP-9, MMP-1 and TIMP-1 will be evaluated by qRT-PCR. The data will be statistically analyzed by ANOVA or Kruskal-Wallis test. Significant differences will be assumed when p<0.05.

Scientific publications

(8)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

RAMOS CAETANO, BRUNNO FELIPE; TABLAS, MARIANA BAPTISTA; ROMUALDO, GUILHERME RIBEIRO; MARCHESAN RODRIGUES, MARIA APARECIDA; BARBISAN, LUIS FERNANDO. Early molecular events associated with liver and colon sub-acute responses to 1,2-dimethylhydrazine: Potential implications on preneoplastic and neoplastic lesion development. Toxicology Letters, v. 329, p. 67-79, SEP 1 2020. (16/12015-0, 14/24762-0, 14/21951-6)

ROMUALDO, GUILHERME RIBEIRO; LEROY, KAAT; COSTA, CICERO JULIO SILVA; PRATA, GABRIEL BACIL; VANDERBORGHT, BART; DA SILVA, TEREZA CRISTINA; BARBISAN, LUIS FERNANDO; ANDRAUS, WELLINGTON; DEVISSCHER, LINDSEY; CAMARA, NIELS OLSEN SARAIVA; et al. In Vivo and In Vitro Models of Hepatocellular Carcinoma: Current Strategies for Translational Modeling. CANCERS, v. 13, n. 21, NOV 2021. (16/14420-0, 16/12015-0, 18/10953-9)

ROMUALDO, GUILHERME R.; SILVA, ELIZANGELA DOS ANJOS; DA SILVA, TEREZA C.; ALOIA, THIAGO P. A.; NOGUEIRA, MARINA S.; DE CASTRO, INAR A.; VINKEN, MATHIEU; BARBISAN, LUIS F.; COGLIATI, BRUNO. Burdock (Arctium lappa L.) root attenuates preneoplastic lesion development in a diet and thioacetamide-induced model of steatohepatitis-associated hepatocarcinogenesis. ENVIRONMENTAL TOXICOLOGY, v. 35, n. 4, p. 10-pg., 2019-12-05. (16/12015-0)

ROMUALDO, GUILHERME R.; SILVA, ELIZANGELA DOS ANJOS; DA SILVA, TEREZA C.; ALOIA, THIAGO P. A.; NOGUEIRA, MARINA S.; DE CASTRO, INAR A.; VINKEN, MATHIEU; BARBISAN, LUIS F.; COGLIATI, BRUNO. Burdock (Arctium lappa L.) root attenuates preneoplastic lesion development in a diet and thioacetamide-induced model of steatohepatitis-associated hepatocarcinogenesis. ENVIRONMENTAL TOXICOLOGY, v. 35, n. 4, DEC 2019. (16/12015-0)

SARMIENTO-MACHADO, LUIS MANUEL; ROMUALDO, GUILHERME RIBEIRO; ZAPATERINI, JOYCE REGINA; TABLAS, MARIANA BAPTISTA; HENRIQUE FERNANDES, ANA ANGELICA; MORENO, FERNANDO SALVADOR; BARBISAN, LUIS FERNANDO. Protective Effects of Dietary Capsaicin on the Initiation Step of a Two-Stage Hepatocarcinogenesis Rat Model. NUTRITION AND CANCER-AN INTERNATIONAL JOURNAL, v. 73, n. 5, p. 817-828, MAR 10 2021. (18/19432-1, 16/12015-0)

ROMUALDO, GUILHERME RIBEIRO; ROCHA, ARIANE BARTOLOMEU; VINKEN, MATHIEU; COGLIATI, BRUNO; MORENO, FERNANDO SALVADOR; GARCIA CHAVES, MARIA ANGEL; BARBISAN, LUIS FERNANDO. Drinking for protection? Epidemiological and experimental evidence on the beneficial effects of coffee or major coffee compounds against gastrointestinal and liver carcinogenesis. Food Research International, v. 123, p. 567-589, SEP 2019. (16/12015-0, 16/14420-0, 17/26217-7)

ROMUALDO, GUILHERME RIBEIRO; PRATA, GABRIEL BACIL; DA SILVA, TEREZA CRISTINA; EVANGELISTA, ADRIANE FEIJO; REIS, RUI MANUEL; VINKEN, MATHIEU; MORENO, FERNANDO SALVADOR; COGLIATI, BRUNO; BARBISAN, LUIS FERNANDO. The combination of coffee compounds attenuates early fibrosis-associated hepatocarcinogenesis in mice: involvement of miRNA profile modulation. JOURNAL OF NUTRITIONAL BIOCHEMISTRY, v. 85, NOV 2020. (16/14420-0, 16/12015-0, 17/16596-0)

BARTOLOMEU, ARIANE ROCHA; ROMUALDO, GUILHERME RIBEIRO; LISON, CARMEN GRINAN; BESHARAT, ZEIN MERSINI; MARCHAL CORRALES, JUAN ANTONIO; GARCIA CHAVES, MARIA ANGEL; BARBISAN, LUIS FERNANDO. Caffeine and Chlorogenic Acid Combination Attenuate Early-Stage Chemically Induced Colon Carcinogenesis in Mice: Involvement of oncomiR miR-21a-5p. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, v. 23, n. 11, p. 16-pg., 2022-06-01. (17/26217-7, 16/12015-0, 16/14420-0)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

ROMUALDO, Guilherme Ribeiro. Caffeine, trigonelline and chlorogenic acid: modulation of mirna expression during fibrosis-associated hepatocarcinogenesis. 2020. Doctoral Thesis - Universidade Estadual Paulista (Unesp). Faculdade de Medicina. Botucatu Botucatu.