Morphological and molecular pathology of CCL4-induced hepatic fibrosis in connexin43-deficient mice.

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4) -induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis.

Inhibition of ascitic ehrlich tumor cell growth by intraperitoneal injection of Pfaffia paniculata (Brazilian ginseng) butanolic residue

This study aimed to investigate the effects of the administration of butanolic residue (BR) of Pfaffia paniculata by intraperitoneal route to Ehrlich ascitis tumor bearing mice. Initially, a toxicity study of P. paniculata BR was performed in which doses of 12.5; 25 and 50mg/Kg were administered by intraperitoneal injection for seven days to Swiss mice. The treatment did not show toxicity. Then, Swiss male mice received, by intraperitoneal injection, once a day, 12.5; 25 or 50mg/Kg of P. paniculata BR for seven days. This protocol started in the same day of tumor inoculation with 5X10(6) cells i.p. The treatment with butanolic residue of P.paniculata i.p caused a significant increase in the ascitic volume; however, a significant decrease in tumor cells number per ml (p<0.05) was observed in P. paniculata treated mice that was followed by a numerical (although non-significant) decrease in the total numbers of tumor cells in the collected ascitic fluid. These results indicated a tumor cell inhibitory effect by P. paniculata butanolic residue in this experimental system, and indicate that topical application of this residue can be useful to control the cancer growth.

Neste estudo, foi avaliado o efeito do tratamento intraperitoneal com Resíduo Butanólico de Pfaffia paniculata, sobre o crescimento do Tumor de Ehrlich, forma ascítica. Foram utilizados dois grupos de 15 camundongos cada, sendo um grupo controle e o outro grupo tratado com RB 50mg/Kg. Todos os animais foram inoculados intraperitonealmente, com 5X10(6) células tumorais O tratamento iniciou-se no mesmo dia da inoculação do tumor. Assim, os animais receberam diariamente, por via intraperitoneal, 0,1 ml de RB na concentrações 50 mg/Kg, ou PBS como controle. Após 7 dias da inoculação do tumor, os animais foram eutanasiados e foi colhido o fluído ascítico total, para a contagem do número de células tumorais presentes neste fluído e estudo da morfologia destas células . Neste experimento observou-se aumento significante da quantidade de fluido ascítico nos animais tratados com RB, e diminuição significativa em relação ao número de células tumorais/ml e células tumorais totais, presentes no fluído ascítico, comparativamente com os animais controle. Estes resultados sugerem efeito inibitório tópico do RB levando à morte as células neoplásicas.

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.

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.

Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content.

Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.

Effects of peripheral-type benzodiazepine receptor ligands on Ehrlich tumor cell proliferation.

Peripheral-type benzodiazepine receptors have been found throughout the body, and particularly, in high numbers, in neoplastic tissues such as the ovary, liver, colon, breast, prostate and brain cancer. Peripheral-type benzodiazepine receptor expression has been associated with tumor malignity, and its subcellular localization is important to define its function in tumor cells. We investigated the presence of peripheral-type benzodiazepine receptors in Ehrlich tumor cells, and the in vitro effects of peripheral-type benzodiazepine receptors ligands on tumor cell proliferation. Our results demonstrate the presence of peripheral-type benzodiazepine receptor in the nucleus of Ehrlich tumor cells (85.53+/-12.60%). They also show that diazepam and Ro5-4864 (peripheral-type benzodiazepine receptor agonists) but not clonazepam (a molecule with low affinity for the peripheral-type benzodiazepine receptor) decreased the percentage of tumor cells in G0-G1 phases and increased that of cells in S-G2-M phases. The effects of those agonists were prevented by PK11195 (a peripheral-type benzodiazepine receptor antagonist) that did not produce effects by itself. Altogether, these data suggest that the presence of peripheral-type benzodiazepine receptor within the nucleus of Ehrlich tumor cells is associated with tumor malignity and proliferation capacity.

Effects of Pfaffia paniculata (Brazilian ginseng) extract on macrophage activity.

The roots of Pfaffia paniculata (Brazilian ginseng) have been indicated for the treatment of several diseases and as an analgesic and antiinflamatory drug. Treatment of mice with 200 mg/kg of the powdered root of P. paniculata reduced the Ehrlich ascitic volume [Matsuzaki, P., Akisue, G., Salgado Oloris, S.C., Gorniak, S.L., Zaidan Dagli, M.L., 2003. Effect of Pffafia paniculata (Brazilian ginseng) on the Ehrlich tumor on its ascitic form. Life Sciences, Dec 19; 74 (5), 573-579.]. One of the putative means to control the Ehrlich tumor growth is by increasing macrophage activity [Kleeb, S.R., Xavier, J.G., Frussa-Filho, R., Dagli, M.L.Z., 1997. Effect of haloperidol on the development of the solid Ehrlich tumor in mice. Life Sciences, 60 (4/5), 69-742.]. The aim of this study was to investigate experimentally the effects of the methanolic extract of P. paniculata roots on macrophage activity. Male mice received, by gavage, once a day, different doses (100, 250, or 500 mg/kg) of the methanolic extract of P. paniculata or filtered water, as control, for 10 days. Macrophage activity was evaluated through the phagocytosis index (PI), spreading index (SI), production of peroxide oxigen and nitric oxide. The peritoneal cells were activated with ip inoculation of Ehrlich ascitic cells, 24 h before the macrophage harvesting. The methanolic extract raised significantly the SI of mice from group of 500 mg/kg in comparison with the control group and group of 100 mg/kg. This raise of SI possibly induced the higher phagocytic activity observed in the experimental situation. Increased macrophage activity may be one of the effects contributing to inhibition of the Ehrlich ascitic tumor growth in mice.

Antineoplastic effects of butanolic residue of Pfaffia paniculata.

We have previously reported a reduction in the accumulation of ascitic fluid in Ehrlich tumor-bearing mice following treatment with the powdered roots of Pfaffia paniculata. The aim of this study was to investigate which extracts from these roots presented antineoplastic properties. Thus, the effects of the ethanolic extract, butanolic residue, or aqueous residue from Pfaffia paniculata on animal survival and tumor growth in mice bearing this tumor were studied. Butanolic residue-treated mice survived longer than untreated mice. This result points to an antineoplastic effect exerted by the butanolic fraction from the roots of P. paniculata on this tumor model.

Inhibitory effects of Pfaffia paniculata (Brazilian ginseng) on preneoplastic and neoplastic lesions in a mouse hepatocarcinogenesis model.

Studies have been demonstrating Pfaffia paniculata root (Brazilian ginseng) anticarcinogenic activities. We evaluated its chemopreventive effects on preneoplastic hepatic lesions. BALB/c aged-15 days received 10mug/g of diethylnitrosamine carcinogen, i.p. They were fed with the powdered root added to the diet: 0.5, 2 or 10% during 27 weeks. After being sacrificed, the macroscopic lesions in the livers were examined. Preneoplastic or neoplastic lesions were measured, quantified and classified morphologically. The treatment reduced the incidence, mean area and number of lesions, indicating an inhibitory effect of these roots on hepatocarcinogenesis promotion or progression steps.

Effect of Pfaffia paniculata (Brazilian ginseng) on the Ehrlich tumor in its ascitic form.

The roots of Pfaffia paniculata (Brazilian ginseng) have been indicated for the treatment of several diseases, among which the cancer. The purpose of this study was to investigate experimentally the possible antineoplastic effect of this root. Firstly, a toxicity study was performed in which the doses of 400 and 200 mg/Kg of the powdered root were administered by gavage for 10 days to BALB/cICB mice. The mice did not lose weight during the treatment. No increase in serum alanine-aminotransferase neither histopathological alteration (liver, kidney and spleen) was observed in mice treated with P. paniculata. The effect of this root on the ascitic Ehrlich tumor in BALB/cICB mice was then investigated. Male mice received, by gavage, once a day, 200 mg/Kg of the powdered root of P. paniculata or distilled water, as control, for 20 days. This protocol started 10 days before tumor inoculation with 5 x 10(6) cells i.p., and lasted until 10 days after. The ascitic tumor was evaluated by the quantification of the volume of the ascitic fluid, relative number of tumor cells and total number of tumor cells. A decrease in the total ascitic volume was observed in P. paniculata treated mice, that was followed by a numerical decrease in the total number of Ehrlich tumor cells. These results may indicate that P. paniculata anti-inflammatory effects were responsible by the decrease in the total ascitic fluid. In addition, the presence of tumor-cell inhibitory factors in P. paniculata roots is in agreement with other in vitro studies. The mechanisms of such tumor inhibition should be further investigated.