Samia Fattel-Fazenda

Chemoprotective effect of caffeic acid phenethyl ester on promotion in a medium-term rat hepatocarcinogenesis assay.

Caffeic acid phenethyl ester (CAPE), a natural honeybee product exhibits a spectrum of biological activities including anti‐microbial, anti‐inflammatory, antioxidant and anti‐tumoral actions. CAPE is also chemopreventive against intestinal, colon and skin cancer. Our aim was to extend the study of its chemoprotective features to the promotion of hepatocarcinogenesis. Male Wistar rats were subjected to a protocol under a modified promotion regimen of the resistant hepatocyte model. The altered hepatic foci (AHF) were quantitatively analyzed by histochemistry and image processing. When given during promotion, CAPE (20 mg/kg) decreased the expression of number and area γ‐glutamyl transpeptidase (GGT) positive AHF by 91% and 97%, respectively. When GGT expression was analyzed by RT‐PCR, CAPE drastically decreased and prevented expression of almost all GGT transcripts at this stage of the carcinogenic process. Glutathione S‐transferase placental form (GST‐P), another protein marker for preneoplastic lesions was measured by Western blot and a decrease of 82% was observed. Additionally, we evaluated the effect of CAPE on the expression of nuclear factor NF‐κB and found an 85% decrease in nuclear localization of the p65 subunit of NF‐κB; however, their repressor, IκBα was not modified. Our results showed that CAPE given during promotion in hepatocarcinogenesis protects against induction of GGT‐positive AHF, GST‐P protein, GGT mRNA expression and translocation of p65. This phenomenon was independent of IκBα degradation.

Antigenotoxic, antimutagenic and ROS scavenging activities of a Rhoeo discolor ethanolic crude extract.

Rhoeo discolor is a legendary plant used for treatment of superficial mycoses in Mexican traditional medicine. Despite its extended use, it is not known whether it has side-effects. An ethanolic crude extract from Rhoeo discolor was prepared, its mutagenic capacity was investigated by the Ames test, and its genotoxic activity in primary liver cell cultures using the unscheduled DNA synthesis assay. This extract was not mutagenic when tested with Salmonella typhimurium strains TA97, TA98 and TA100, and it did not elicit unscheduled DNA synthesis in hepatocyte cultures. In addition, we explored the antimutagenic and antigenotoxic activities of the extract and its ROS scavenger behaviour. Our results show that Rhoeo extract is antimutagenic for S. typhimurium strain TA102 pretreated with ROS-generating mutagen norfloxacin in the Ames test, and protects liver cell cultures against diethylnitrosamine induction of unscheduled DNA synthesis even at 1.9 ng per dish, which was the lowest dose tested. A free radical scavenging test was used in order to explore the antioxidant capacity of Rhoeo extract, as compared with three commercial well-known antioxidants quercetin, ascorbic acid and tocopherol. Rhoeo extract showed less radical scavenging effect than quercetin, but similar to that of alpha-tocopherol and more than ascorbic acid. It is important to note that this extract was neither mutagenic in S. typhimurium nor genotoxic in liver cell culture, even at concentrations as high as four- and 166-fold of those needed for maximal antimutagenic or chemoprotective activities, respectively.

The differential NF-kB modulation by S-adenosyl-L-methionine, N-acetylcysteine and quercetin on the promotion stage of chemical hepatocarcinogenesis

S-adenosylmethionine (SAM), N-acetylcysteine (NAC) and quercetin exhibit a chemoprotective effect. Likely this effect is mediated by counteracting, oxidative stress and NF-kB activation. To test this hypothesis F344 rats were subjected to hepatocarcinogenesis with or without antioxidants. NAC decreased foci in number and area, SAM and quercetin decreased area. Lipid-peroxidation was decreased by antioxidants, but only SAM increased glutathione. SAM, in its regulation from IKK downwards, abolished the NF-kB activation. NAC decreased IKK and IkB-a phosphorylation, and Rel-A/p65 and NF-kB binding, though the last two were affected with less intensity compared to the NF-kB inhibitor. Quercetin decreased Rel-A/p65, without modifying upstream signalling. Although all antioxidants inhibited oxidative stress as shown by reduction of lipid peroxidation, not all exerted the same effect on NF-kB signalling pathway and only SAM increased GSH. The mechanisms exerted by SAM in the reduction of foci makes this compound a potential liver cancer therapeutic agent.

Evidence that the Anticarcinogenic Effect of Caffeic Acid Phenethyl Ester in the Resistant Hepatocyte Model Involves Modifications of Cytochrome P450

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, shows anticarcinogenic properties in the modified resistant hepatocyte model when administered before initiation or promotion of hepatocarcinogenesis process; however, information about the mechanism underlying this chemoprotection is limited. The aim of this work was to characterize the effect of CAPE on cytochrome P450 (CYP), which is involved in diethylnitrosamine (DEN) metabolism during the initiation stage of chemical hepatocarcinogenesis. Male Fischer-344 rats were treated as in the modified resistant hepatocyte model. Liver samples were obtained at four different times: at 12 h after pretreatment with CAPE and at 12 and 24 h and 25 days after DEN administration. Liver damage was determined by histology with hematoxylin and eosin, measurement of total CYP levels and enzyme activity, and gamma-glutamyl transpeptidase-positive (GGT+) staining of hepatocyte foci. CAPE administration prevented DEN-induced necrosis at 24 h. It also decreased O-dealkylation of 7-ethoxy-resorufin (EROD), O-dealkylation of 7-methoxyresorufin (MROD), and 7-pentoxy-resorufin activities at 12 h after its administration and EROD and MROD activities at 12 h after administration of DEN. CAPE treatment decreased GGT+ foci by 59% on day 25. Our results suggest that CAPE modifies the enzymatic activity of CYP isoforms involved in the activation of DEN, such as CYP1A1/2 and CYP2B1/2. These findings describe an alternative mechanism for understanding the ability of CAPE to protect against chemical hepatocarcinogenesis.

Tumor promoting and co-carcinogenic effects in medium-term rat hepatocarcinogenesis are not modified by co-administration of 12 pesticides in mixture at acceptable daily intake.

The purpose of this investigation was to evaluate the possible influence of a mixture of pesticides on medium-term carcinogenesis using improved hepatocarcinogenesis protocols. We performed a 12 commercially available pesticides combination with alachlor, atrazine, carbofuran, chlorpyrifos, diazinon, dicofol, endosulfan, iprodione, mancozeb, maneb, procymidone and rotenone. The mixture was given at 1-fold and 10-fold the acceptable daily intake (ADI) level in a set of Solt-Farber-derived protocols involving diethylnitrosamine, 2-acetylaminofluorene treatments and a partial hepatectomy. Co-carcinogenic effect and promoting activity were evaluated using gamma-glutamyl transpeptidase (GGT) positive altered hepatocyte foci, as well, protein and mRNA levels of glutathione S-transferase P (GSTP) in liver extracts as molecular biomarkers of carcinogenic effects. The pesticide treatments when compared to vehicle treatments always produced the same number of hepatocyte lesions and an equal GSTP expression on liver extracts independently of carcinogenic-protocol utilized. On this base, we concluded that the pesticide mixture evaluated in this report does not have tumor promoting activity or co-carcinogenic effect in the rat medium-term liver carcinogenesis. Altogether these data contribute to the confidence that the ADI represents a safe intake level to mixture of pesticides at dietary exposure.

The transmembrane transporter ABCC3 participates in liver cancer progression and is a potential biomarker.

The poor prognosis, few available treatment options, and multidrug resistance present in hepatocellular carcinoma are major problems, and new early biomarkers are needed to reduce the liver cancer death rate. ATP-binding cassette sub-family C member 3 (Abcc3) is overexpressed in different cancers and is associated with multidrug resistance and a carcinogenic stem cell phenotype. We present evidence for the first time that ABCC3 is a potential sanguine biomarker and anticancer target in hepatocellular carcinoma. Abcc3 mRNA expression was elevated in liver nodules and tumors in rat hepatocarcinogenesis model. Accordingly, the ABCC3 protein was preferentially overexpressed within the nodules and tumors during hepatocellular carcinoma progression and was secreted into the bloodstream of rat hepatocarcinogenesis model. The ABCC3 protein was expressed in human hepatoma cells and, importantly, was also present in HepG2- and Huh7-conditioned media. Furthermore, ABCC3 was overexpressed in human hepatocellular carcinoma. This report is the first to describe liver overexpression of Abcc3 during the cancer initiation, promotion, and progression periods in rat hepatocarcinogenesis model and in human hepatocellular carcinoma.