Panx1 knockout promotes preneoplastic aberrant crypt foci development in a chemically induced model of mouse colon carcinogenesis.

Colorectal cancer, which is the third leading cause of cancer-related deaths worldwide, is a multistep disease, featuring preneoplastic aberrant crypt foci (ACF) as the early morphological manifestation. The roles of hemichannel-forming transmembrane Pannexin 1 (Panx1) protein have not been investigated in the context of colon carcinogenesis yet, although it has contrasting roles in other cancer types. Thus, this study was conducted to examine the effects of Panx1 knockout (Panx1-/- ) on the early events of chemically induced colon carcinogenesis in mouse. Wild type (WT) and Panx1-/- female C57BL6J mice were submitted to a chemically induced model of colon carcinogenesis by receiving six intraperitoneal administrations of 1,2-dimethylhydrazine (DMH) carcinogen. Animals were euthanized 8 h (week 7) or 30 weeks (week 37) after the last DMH administration in order to evaluate sub-acute colon toxicity outcomes or the burden of ACF, respectively. At week 7, Panx1 genetic ablation increased DMH-induced genotoxicity in peripheral blood cells, malondialdehyde levels in the colon, and apoptosis (cleaved caspase-3) in colonic crypts. Of note, at week 37, Panx1-/- animals showed an increase in aberrant crypts (AC), ACF mean number, and ACF multiplicity (AC per ACF) by 56%, 57% and 20%, respectively. In essence, our findings indicate that Panx1 genetic ablation promotes preneoplastic ACF development during chemically induced mouse colon carcinogenesis, and a protective role of Panx1 is postulated.

The Implications of Connexin 43 Deficiency during the Early Stages of Chemically Induced Mouse Colon Carcinogenesis.

Colorectal cancer (CRC), associated with an increased intake of processed red meats, saturated fats, and simple carbohydrates accompanied by low dietary fiber, fruits, and vegetables consumption, presents a high epidemiological burden. Connexin43 (Cx43) protein, which forms gap junctions or hemichannels, has tumor suppressor or oncogenic activities in a cancer type- and stage-dependent manner. Cx43 expression varies during colon carcinogenesis, and its functional role is not fully understood. Thus, we evaluated the implications of Cx43 heterologous deletion (Cx43+/-) during the early stages of a chemically induced model of colon carcinogenesis. Female C57BL/6J mice (wild-type or Cx43+/-) were submitted to a colon carcinogenesis model induced by 1,2 dimethylhydrazine (DMH). Mice were euthanized eight hours (week 7) or 30 weeks (week 37) after the last DMH administration to evaluate subacute colon toxicity outcomes or the burden of (pre)neoplastic lesions, respectively. At week 7, Cx43 deficiency inferred no alterations in the DMH-induced increase in systemic (peripheral blood), in situ (colonocytes) DNA damage, and apoptosis in the colonocytes. At week 30, Cx43+/- mice presented an increase in preneoplastic aberrant crypt foci (ACF) multiplicity, while no alterations were observed in colorectal adenoma (CRA) occurrence, multiplicity, volume, proliferation, growth, and ß-catenin immunoexpression. Similarly, an in silico analysis of human CRA showed decreased mRNA expression of Cx43 with no correlation with proliferation, apoptosis, and ß-catenin markers. These findings indicate the discrete role of Cx43 in the early stages of chemically induced mouse colon carcinogenesis.

Hepatocellular Carcinoma in a Free-Ranging Three-Toed Sloth (Bradypus variegatus).

The increasing interest of tumors in wildlife is important for biodiversity conservation and for monitoring environmental agents and/or contaminants with potential impact on human health. Here we described the occurrence of hepatocellular carcinoma (HCC) in noncirrhotic liver of a free-ranging three-toed sloth (Bradypus variegatus) from the Atlantic Forest biome in Brazil. The HCC showed a moderate mononuclear inflammatory infiltrate within the tumor tissue but with no inflammation and fibrosis in the adjacent liver tissue. Upon immunohistochemistry, neoplastic cells were diffusely positive for HepPar-1 and glutamine-synthetase presenting an irregular and random immunostaining pattern; ß-catenin was positive in the cytoplasmic membrane of malignant hepatocytes; and cytokeratin 19 immunostaining was restricted to bile duct epithelial cells. The liver tissue was negative for HBV-like and HCV-like viruses assessed by molecular tests. The potential similarity of pathogenesis may reinforce the need for research on environmental and/or infectious agents associated with HCC that may contribute to the understanding of cancer in wildlife.

Novel gammaherpesvirus associated with primary gastric T-cell lymphoma in a free-ranging giant armadillo in Brazil.

The number of viral-associated neoplasms reported in wildlife has increased over the last decades, likely because of growing research efforts and a potentially greater burden of carcinogenic pathogens. Herein, we describe a primary gastric T-cell lymphoma in one free-ranging giant armadillo (Priodontes maximus) from Brazilian Pantanal infected by a novel gammaherpesvirus, proposed as Cingulatid gammaherpesvirus 1 (CiHV-1). By chromogenic in situ hybridisation against Epstein-Barr virus some neoplastic cells were labeled. Subsequently, a molecular screening was carried out to detect the occurrence of this pathogen in other giant armadillos in the same region. Overall, this novel virus was detected in 14.3% (3/21) of the tested giant armadillos. We suggest this herpesvirus, the first in Xenarthra, as a plausible aetiology of the neoplasm. The implications of CiHV-1 for this species are uncertain; while no outbreaks of disease have been recorded, the present study raises concerns. Further research is warranted to assess the real significance of CiHV-1 and its potential oncogenic role in this species.

In Vivo and In Vitro Models of Hepatocellular Carcinoma: Current Strategies for Translational Modeling.

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related death globally. HCC is a complex multistep disease and usually emerges in the setting of chronic liver diseases. The molecular pathogenesis of HCC varies according to the etiology, mainly caused by chronic hepatitis B and C virus infections, chronic alcohol consumption, aflatoxin-contaminated food, and non-alcoholic fatty liver disease associated with metabolic syndrome or diabetes mellitus. The establishment of HCC models has become essential for both basic and translational research to improve our understanding of the pathophysiology and unravel new molecular drivers of this disease. The ideal model should recapitulate key events observed during hepatocarcinogenesis and HCC progression in view of establishing effective diagnostic and therapeutic strategies to be translated into clinical practice. Despite considerable efforts currently devoted to liver cancer research, only a few anti-HCC drugs are available, and patient prognosis and survival are still poor. The present paper provides a state-of-the-art overview of in vivo and in vitro models used for translational modeling of HCC with a specific focus on their key molecular hallmarks.

Immunoexpression and Prognostic Significance of Multidrug Resistance Markers in Feline Mammary Carcinomas.

Feline mammary carcinomas (FMCs) are commonly characterized by high clinical aggressiveness and poor prognosis. FMCs share many features with the corresponding human disease, allowing the comparative investigation of tumour biology and therapeutic strategies, including multidrug resistance (MDR) mechanisms. Although transporting/binding proteins, including permeability glycoprotein (P-gp), lung resistance protein (LRP) and metallothionein (MT), are frequently associated with tumour aggressiveness and unresponsiveness to chemotherapy in human breast cancer, they have not been analysed in FMCs. We investigated the immunoexpression of P-gp, LRP and MT in FMCs and their correlation with clinicopathological parameters and overall survival (OS) time in 46 FMCs, with a median follow-up period of 289 days. These markers were co-expressed in 85% of tumours. P-gp was expressed in 93.4% of FMCs and was positively associated with tumour grade (P = 0.049). While unequivocally observed in all FMCs, LRP immunoexpression did not correlate with any clinicopathological parameters or OS. Expression of MT was significant in triple-negative basal- and normal-like molecular subtypes of FMCs (P = 0.023). The concurrent expression of MDR proteins indicates the potential existence of chemotherapy resistance-related mechanisms in FMCs. The positive association between P-gp and MT immunoexpression and aggressive phenotypes could open new therapeutic and translational strategies for FMCs.

Genetic variants of BRCA1 and BRCA2 genes in cats with mammary gland carcinoma.

Mammary tumours are the first and third most incident neoplasm in women and cats, respectively. Approximately 85% of feline mammary gland tumours are malignant and aggressive, especially the triple-negative and HER-2+ molecular subtypes. Triple-negative basal-like feline mammary carcinomas (FMCs) are considered suitable models due to the clinical and morphological similarities with human basal-like triple-negative breast cancer (TNBC). In women, TNBC has a poor prognosis and is often associated with mutations in the tumour suppressor genes BRCA1 and BRCA2. In light of this, the aim of the present investigation was to screen somatic and germline variants of BRCA1 and BRCA2 in nine female cats bearing FMCs. Matched whole blood and FMC samples were obtained for genetic analysis. Additional tumour samples were obtained for histopathological and immunohistochemical evaluation. Genomic DNA was isolated and 27 exonic regions of BRCA1 and BRCA2 genes were amplified and screened by next-generation sequencing. A somatic variant with high functional impact was found in exon 11 of BRCA2 at a frequency of 4.34% in one FMC-bearing cat. Four germline variants with moderate impact were detected in three of the nine FMC-bearing cats and were restricted to exon 9 of BRCA1. It is concluded that the germline genetic variants found in one-third of FMC-bearing animals might be associated with a higher risk of hereditary mammary carcinogenesis.

Sorafenib reduces steatosis-induced fibrogenesis in a human 3D co-culture model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) affects around 25% of the worldwide population. Non-alcoholic steatohepatitis (NASH), the more progressive variant of NAFLD, is characterized by steatosis, cellular ballooning, lobular inflammation, and may culminate on hepatic stellate cell (HSC) activation, thus increasing the risk for fibrosis, cirrhosis, and HCC development. Conversely, the antifibrotic effects of sorafenib, an FDA-approved drug for HCC treatment, have been demonstrated in 2D cell cultures and animal models, but its mechanisms in a NAFLD-related microenvironment in vitro requires further investigation. Thus, a human 3D co-culture model of fatty hepatocytes and HSC was established by culturing hepatoma C3A cells, pre-treated with 1.32 mM oleic acid, with HSC LX-2 cells. The fatty C3A/LX-2 spheroids showed morphological and molecular hallmarks of altered lipid metabolism and steatosis-induced fibrogenesis, similarly to the human disease. Sorafenib (15 µM) for 72 hours reduced fatty spheroid viability, and upregulated the expression of lipid oxidation- and hydrolysis-related genes, CPT1 and LIPC, respectively. Sorafenib also inhibited steatosis-induced fibrogenesis by downregulating COL1A1, TGFB1, PDGF, and TIMP1 and by decreasing protein levels of IL-6, TGF-ß1, and TNF-α in fatty spheroids. The demonstration of the antifibrotic properties of sorafenib on steatosis-induced fibrogenesis in a 3D in vitro model of NAFLD supports its clinical use as a therapeutic agent for the treatment of NAFLD/NASH patients.

The combination of coffee compounds attenuates early fibrosis-associated hepatocarcinogenesis in mice: involvement of miRNA profile modulation.

Aberrant microRNA expression implicates on hepatocellular carcinoma (HCC) development. Conversely, coffee consumption reduces by ~40% the risk for fibrosis/cirrhosis and HCC, while decaffeinated coffee does not. It is currently unknown whether these protective effects are related to caffeine (CAF), or to its combination with other common and/or highly bioavailable coffee compounds, such as trigonelline (TRI) and chlorogenic acid (CGA). We evaluated whether CAF individually or combined with TRI and/or CGA alleviates fibrosis-associated hepatocarcinogenesis, examining the involvement of miRNA profile modulation. Then, male C3H/HeJ mice were submitted to a diethylnitrosamine/carbon tetrachloride-induced model. Animals received CAF (50 mg/kg), CAF+TRI (50 and 25 mg/kg), CAF+CGA (50 and 25 mg/kg) or CAF+TRI+CGA (50, 25 and 25 mg/kg), intragastrically, 5×/week, for 10 weeks. Only CAF+TRI+CGA combination reduced the incidence, number and proliferation (Ki-67) of hepatocellular preneoplastic foci while enhanced apoptosis (cleaved caspase-3) in adjacent parenchyma. CAF+TRI+CGA treatment also decreased hepatic oxidative stress and enhanced the antioxidant Nrf2 axis. CAF+TRI+CGA had the most pronounced effects on decreasing hepatic pro-inflammatory IL-17 and NFκB, contributing to reduce CD68-positive macrophage number, stellate cell activation, and collagen deposition. In agreement, CAF+TRI+CGA upregulated tumor suppressors miR-144-3p, miR-376a-3p and antifibrotic miR-15b-5p, frequently deregulated in human HCC. CAF+TRI+CGA reduced the hepatic protein levels of pro-proliferative EGFR (miR-144-3p target), antiapoptotic Bcl-2 family members (miR-15b-5p targets), and the number of PCNA (miR-376a-3p target) positive hepatocytes in preneoplastic foci. Our results suggest that the combination of most common and highly bioavailable coffee compounds, rather than CAF individually, attenuates fibrosis-associated hepatocarcinogenesis by modulating miRNA expression profile.

Fibrosis-associated hepatocarcinogenesis revisited: Establishing standard medium-term chemically-induced male and female models.

Hepatocellular carcinoma causes ~10% of all cancer-related deaths worldwide, usually emerging in a background of liver fibrosis/cirrhosis (70%-90% of cases). Chemically-induced mouse models for fibrosis-associated hepatocarcinogenesis are widely-applied, resembling the corresponding human disease. Nonetheless, a long time is necessary for the development of preneoplastic/neoplastic lesions. Thus, we proposed an early fibrosis-associated hepatocarcinogenesis model for male and female mice separately, focusing on reducing the experimental time for preneoplastic/neoplastic lesions development and establishing standard models for both sexes. Then, two-week old susceptible C3H/HeJ male and female mice (n = 8 animals/sex/group) received a single dose of diethylnitrosamine (DEN, 10 or 50 mg/Kg). During 2 months, mice received 3 weekly doses of carbon tetrachloride (CCl4, 10% corn oil solution, 0.25 to 1.50 µL/g b.wt.) and they were euthanized at week 17. DEN/CCl4 protocols for males and females displayed clear liver fibrosis, featuring collagen accumulation and hepatic stellate cell activation (α-SMA). In addition, liver from males displayed increased CD68+ macrophage number, COX-2 protein expression and IL-6 levels. The DEN/CCl4 models in both sexes impaired antioxidant defense as well as enhanced hepatocyte proliferation and apoptosis. Moreover, DEN/CCl4-treated male and female developed multiple preneoplastic altered hepatocyte foci and hepatocellular adenomas. As expected, the models showed clear male bias. Therefore, we established standard and suitable fibrosis-associated hepatocarcinogenesis models for male and female mice, shortening the experimental time for the development of hepatocellular preneoplastic/neoplastic lesions in comparison to other classical models.

Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice.

While gap junctions mediate intercellular communication and support liver homeostasis, connexin hemichannels are preferentially opened by pathological stimuli, including inflammation and oxidative stress. The latter are essential features of non-alcoholic steatohepatitis. In this study, it was investigated whether connexin32 and connexin43 hemichannels play a role in non-alcoholic steatohepatitis. Mice were fed a choline-deficient high-fat diet or normal diet for 8 weeks. Thereafter, TAT-Gap24 or TAT-Gap19, specific inhibitors of hemichannels composed of connexin32 and connexin43, respectively, were administered for 2 weeks. Subsequently, histopathological examination was carried out and various indicators of inflammation, liver damage and oxidative stress were tested. In addition, whole transcriptome microarray analysis of liver tissue was performed. Channel specificity of TAT-Gap24 and TAT-Gap19 was examined in vitro by fluorescence recovery after photobleaching analysis and measurement of extracellular release of adenosine triphosphate. TAT-Gap24 and TAT-Gap19 were shown to be hemichannel-specific in cultured primary hepatocytes. Diet-fed animals treated with TAT-Gap24 or TAT-Gap19 displayed decreased amounts of liver lipids and inflammatory markers, and augmented levels of superoxide dismutase, which was supported by the microarray results. These findings show the involvement of connexin32 and connexin43 hemichannels in non-alcoholic steatohepatitis and, simultaneously, suggest a role as potential drug targets in non-alcoholic steatohepatitis.

Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice.

Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure.

Connexins and pannexins in liver damage.

Connexins and pannexins are key players in the control of cellular communication and thus in the maintenance of tissue homeostasis. Inherent to this function these proteins are frequently involved in pathological processes. The present paper reviews the role of connexins and pannexins in liver toxicity and disease. As they act both as sensors and effectors in these deleterious events connexins and pannexins could represent a set of novel clinical diagnostic biomarkers and drug targets.

Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells.

Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng) show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A-F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27(KIP1) overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27(KIP1) overexpression, besides induction of apoptosis through caspase-3 activation.

Strategies, models and biomarkers in experimental non-alcoholic fatty liver disease research.

Non-alcoholic fatty liver disease encompasses a spectrum of liver diseases, including simple steatosis, steatohepatitis, liver fibrosis and cirrhosis and hepatocellular carcinoma. Non-alcoholic fatty liver disease is currently the most dominant chronic liver disease in Western countries due to the fact that hepatic steatosis is associated with insulin resistance, type 2 diabetes mellitus, obesity, metabolic syndrome and drug-induced injury. A variety of chemicals, mainly drugs, and diets is known to cause hepatic steatosis in humans and rodents. Experimental non-alcoholic fatty liver disease models rely on the application of a diet or the administration of drugs to laboratory animals or the exposure of hepatic cell lines to these drugs. More recently, genetically modified rodents or zebrafish have been introduced as non-alcoholic fatty liver disease models. Considerable interest now lies in the discovery and development of novel non-invasive biomarkers of non-alcoholic fatty liver disease, with specific focus on hepatic steatosis. Experimental diagnostic biomarkers of non-alcoholic fatty liver disease, such as (epi)genetic parameters and '-omics'-based read-outs are still in their infancy, but show great promise. In this paper, the array of tools and models for the study of liver steatosis is discussed. Furthermore, the current state-of-art regarding experimental biomarkers such as epigenetic, genetic, transcriptomic, proteomic and metabonomic biomarkers will be reviewed.

Cell proliferation and expression of connexins differ in melanotic and amelanotic canine oral melanomas.

Melanoma is a malignant neoplasm occurring in several animal species, and is the most frequently found tumor in the oral cavity in dogs. Melanomas are classified into two types: melanotic and amelanotic. Prior research suggests that human amelanotic melanomas are more aggressive than their melanotic counterparts. This study evaluates the behavior of canine melanotic and amelanotic oral cavity melanomas and quantifies cell proliferation and the expression of connexins. Twenty-five melanomas (16 melanotic and 9 amelanotic) were collected from dogs during clinical procedures at the Veterinary Hospital of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil. After diagnosis, dogs were followed until death or euthanasia. Histopathology confirmed the gross melanotic or amelanotic characteristics and tumors were classified according to the WHO. HMB45 or Melan A immunostainings were performed to confirm the diagnosis of amelanotic melanomas. Cell proliferation was quantified both by counting mitotic figures and PCNA positive nuclei. Expressions of connexins 26 and 43 were evaluated by immunohistochemistry, qRT-PCR and Western blot. Dogs bearing amelanotic melanomas presented a shorter lifespan in comparison to those with melanotic melanomas. Cell proliferation was significantly higher in amelanotic melanomas. Expressions of Connexins 26 and 43 were significantly reduced in amelanotic melanomas. The results presented here suggest that oral cavity melanotic and amelanotic melanomas differ regarding their behavior, cell proliferation and connexin expression in dogs, indicating a higher aggressiveness of amelanotic variants.

Pfaffia paniculata (Brazilian ginseng) roots decrease proliferation and increase apoptosis but do not affect cell communication in murine hepatocarcinogenesis.

Pfaffia paniculata (Brazilian ginseng) roots and/or its extracts have shown anti-neoplastic, chemopreventive, and anti-angiogenic properties. The aim of this work was to investigate the chemopreventive mechanisms of this root in mice submitted to the infant model of hepatocarcinogenesis, evaluating the effects on cellular proliferation, apoptosis, and intercellular communication. Fifteen-day-old BALB/c male mice were given, i.p., 10mug/g of the carcinogen N-nitrosodiethylamine (DEN). Animals were separated into three groups at weaning and were given different concentrations of powdered P. paniculata root (0%, 2%, or 10%) added to commercial food for 27 weeks. Control group (CT) was not exposed to the carcinogen and was given ration without the root. After euthanasia, the animals' liver and body weight were measured. Liver fragments were sampled to study intercellular communication, molecular biology, and histopathological analysis. Cellular proliferation was evaluated by immunohistochemistry for PCNA, apoptosis was evaluated by apoptotic bodies count and alkaline comet technique, and intercellular communication by diffusion of lucifer yellow dye, immunofluorescence, western blot and real-time PCR for connexins 26 and 32. Chronic treatment with powdered P. paniculata root reduced cellular proliferation and increased apoptosis in the 2% group. Animals in the 10% group had an increase in apoptosis with chronic inflammatory process. Intercellular communication showed no alterations in any of the groups analyzed. These results indicate that chemopreventive effects of P. paniculata are related to the control of cellular proliferation and apoptosis, but not to cell communication and/or connexin expression, and are directly influenced by the root concentration.

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian ginseng) on cultured human breast cancer cell line MCF-7.

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line, as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated with butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations.

Histological alterations in the livers of Cx43-deficient mice submitted to a cholestasis model.

Gap junction intercellular communication capacity and connexin expression are reportedly involved in cell proliferation. To understand the participation of connexins in biliary duct hyperplasia, a cholestasis model was applied to mice with heterologous deletion of Gja 1, the connexin 43 (Cx43) gene. Heterozygous (Cx43+/-) knockout (KO) and wild-type mice (Cx43+/+) (WT) were submitted to bile duct ligation and euthanized at different time points (48 h, 7 days, and 14 days) after surgery. After euthanasia, the macroscopic and microscopic liver alterations were examined. A histomorphometric study of the livers was performed. For this purpose, a grid containing 100 points was applied to each liver section. The volumetric fraction of bile ducts, hepatocytes, arterioles, and terminal hepatic vein were quantified. Cell proliferation was also quantified by western blot PCNA. High mortality was observed in both genotypes. The heterologous deletion of Cx43 did not affect the biliary duct hyperplasia or most of the other parameters analyzed; however, the Cx43-deficient mice showed decrease in hepatic vein angiogenesis in comparison with the wild-type mice 48 h after surgery. In conclusion, our results indicate that the Cx43 gene heterologous deletion does not affect the biliary duct hyperplasia; on the other hand, connexin 43 deficiencies do affect the hepatic vein angiogenesis, although other studies to understand the details of this influence will be necessary.

Pfaffia paniculata (Brazilian ginseng) methanolic extract reduces angiogenesis in mice.

Pfaffia paniculata (Brazilian ginseng) roots have been indicated for the treatment of several diseases. Our studies have shown that P. paniculata roots present antineoplastic effects and cancer chemopreventive activity in a mouse hepatocarcinogenesis model. The purpose of this study was to investigate the effects of the Brazilian ginseng on corneal angiogenesis in mice. We first conducted a toxicological study employing 250, 500, or 1000mg/kg/day of the methanolic extract of P. paniculata roots by gavage to BALB/c mice. Animals did not lose weight during the treatment nor presented histopathological alterations. The effect of this root on angiogenesis in the cornea of BALB/c mice was then assessed. Male mice were treated, by gavage, once a day, with doses of 250, 500, or 1000mg/kg of methanolic extract of P. paniculata powdered root for 10 days; filtered water was used as control. Corneal cauterization was accomplished by the contact of a silver nitrate crystal on the central area of the cornea, in the 5th day of treatment with P. paniculata, which continued thereafter; the animals were euthanized on the 6th day after cauterization. Newly formed blood vessels were filled with India ink, and the corneas were routinely processed. Blood vessels were quantified in an image analysis system. A smaller total area of neovascularization in the mouse cornea was observed in animals treated with 1000mg/kg of the methanolic extract of P. paniculata. These results indicate an antiangiogenic effect of this extract. The mechanisms of this antiangiogenic activity of P. paniculata should be further investigated.