Age-related changes of the noradrenergic and acetylcholinesterase reactive nerve fibres innervating the pigeon bursa of Fabricius.

Age-dependent changes in the innervation of the pigeon (Columba livia, L.) bursa of Fabricius, from hatching to 120 days of age, were studied by fluorescence-histochemical and neurochemical methods for demonstrating noradrenergic and acetylcholinesterase (AChE)-reactive nerve fibres respectively. The distribution of both nerve fibre types was largely perivascular. Furthermore, a few isolated nerve fiber profiles were observed beneath the bursal epithelium, in the interfollicular septa and in the follicular cortex. No nerve fibre profiles reaching the medulla of the lymphoid follicles were observed. In addition to nerve fibres, AChE reactive neuron-like cells were encountered within the capsule and interfollicular septa. AChE reactivity was also found in dendritic-like cells localized in the cortical and cortico-medullary border. No changes in the density of perivascular noradrenergic innervation were noticeable during the ages studied, whereas the density of AChE-reactive fibres supplying vessels reached the adult pattern at 30 days, and then remained unvaried. The density of non-perivascular nerve fiber profiles, specially the AChE reactive type, increased until 30 days, remained unchanged until 75 days and then increased with aging (90-120 days). The interrelationship between the autonomic nervous system and the immune system is discussed

β-caryophyllene and low-doses of doxorubicin against liver cancer cells: a “metronomic chemotherapy”

Cholangiocarcinoma and hepatocellular carcinoma are primary liver cancers, both representing a growing challenge due to their increasing morbidity and mortality. A “metronomic chemotherapy”, consisting of the repeated administration of low and/or continuous doses of anti-neoplastic drugs, represents an alternative approach to the standard chemotherapy [1]. Numerous natural substances exhibited in vitro chemosensitizing features: in particular, the natural sesquiterpene β-caryophyllene (CRY) has been proved to increase the cytotoxicity of doxorubicin (DOXO) in leukemic cells [2]. Hence, our aim has been to evaluate the ability of CRY to enhance the efficacy of low-dose DOXO in human liver cancer cells, by applying a metronomic protocol. To this end, human liver HepG2 and CCA cells have been used as models of hepatocellular carcinoma and cholangiocarcinoma. The metronomic protocol was based on a 2h low-time exposition to the test substances, followed by 72h incubation for restoring. This scheduling has been applied 3 times and cytotoxicity was measured by MTT assay. Both the substances alone (CRY 1-100 μg/ml; DOXO 1-500 μg/ml) and the combination of DOXO with a nontoxic concentration of CRY were assessed. We found that the repeated treatments with low concentrations produced a significant potentiation (about 30 %) of DOXO cytotoxicity in HepG2. The combination with CRY increased the DOXO activity, reaching a 70 % inhibition of cell viability at 50 μg/ml after 2 repeated treatments. Similar effects were found in CCA, although repeated treatments induced no additional potentiation. These results highlight a possible role of CRY as a chemosensitizing agent for DOXO-based chemotherapy of liver cancer

β-caryophyllene and low-doses of doxorubicin against liver cancer cells: a “metronomic chemotherapy”

Cholangiocarcinoma and hepatocellular carcinoma are primary liver cancers, both representing a growing challenge due to their increasing morbidity and mortality. A “metronomic chemotherapy”, consisting of the repeated administration of low and/or continuous doses of anti-neoplastic drugs, represents an alternative approach to the standard chemotherapy [1]. Numerous natural substances exhibited in vitro chemosensitizing features: in particular, the natural sesquiterpene β-caryophyllene (CRY) has been proved to increase the cytotoxicity of doxorubicin (DOXO) in leukemic cells [2]. Hence, our aim has been to evaluate the ability of CRY to enhance the efficacy of low-dose DOXO in human liver cancer cells, by applying a metronomic protocol. To this end, human liver HepG2 and CCA cells have been used as models of hepatocellular carcinoma and cholangiocarcinoma. The metronomic protocol was based on a 2h low-time exposition to the test substances, followed by 72h incubation for restoring. This scheduling has been applied 3 times and cytotoxicity was measured by MTT assay. Both the substances alone (CRY 1-100 μg/ml; DOXO 1-500 μg/ml) and the combination of DOXO with a nontoxic concentration of CRY were assessed. We found that the repeated treatments with low concentrations produced a significant potentiation (about 30 %) of DOXO cytotoxicity in HepG2. The combination with CRY increased the DOXO activity, reaching a 70 % inhibition of cell viability at 50 μg/ml after 2 repeated treatments. Similar effects were found in CCA, although repeated treatments induced no additional potentiation. These results highlight a possible role of CRY as a chemosensitizing agent for DOXO-based chemotherapy of liver cancer

Modulation of STAT3 signaling, cell redox defenses and cell cycle checkpoints by β-caryophyllene in cholangiocarcinoma cells: possible mechanisms accounting for doxorubicin chemosensitization and chemoprevention

Cholangiocarcinoma (CCA) is an aggressive group of biliary tract cancers, characterized by late diagnosis, low effective chemotherapies, multidrug resistance, and poor outcomes. In the attempt to identify new therapeutic strategies for CCA, we studied the antiproliferative activity of a combination between doxorubicin and the natural sesquiterpene β-caryophyllene in cholangiocarcinoma Mz-ChA-1 cells and nonmalignant H69 cholangiocytes, under both long-term and metronomic schedules. The modulation of STAT3 signaling, oxidative stress, DNA damage response, cell cycle progression and apoptosis was investigated as possible mechanisms of action. β-caryophyllene was able to synergize the cytotoxicity of low dose doxorubicin in Mz-ChA-1 cells, while producing cytoprotective effects in H69 cholangiocytes, mainly after a long-term exposure of 24 h. The mechanistic analysis highlighted that the sesquiterpene induced a cell cycle arrest in G2/M phase along with the doxorubicin-induced accumulation in S phase, reduced the γH2AX and GSH levels without affecting GSSG. ROS amount was partly lowered by the combination in Mz-ChA-1 cells, while increased in H69 cells. A lowered expression of doxorubicin-induced STAT3 activation was found in the presence of β-caryophyllene in both cancer and normal cholangiocytes. These networking effects resulted in an increased apoptosis rate in Mz-ChA-1 cells, despite a lowering in H69 cholangiocytes. This evidence highlighted a possible role of STAT3 as a final effector of a complex network regulated by β-caryophyllene, which leads to an enhanced doxorubicin-sensitivity of cholangiocarcinoma cells and a lowered chemotherapy toxicity in nonmalignant cholangiocytes, thus strengthening the interest for this natural sesquiterpene as a dual-acting chemosensitizing and chemopreventive agent

Role of Cassia angustifolia on hepatic toxicity in Wistar rats

Background. Cassia angustifolia L. (senna) is traditionally used as a laxative for the short-term treatment of acute constipation and as a purgative before diagnostic endoscopy. Among several adverse reactions in literature, hepatotoxicity (Sonmez et al., Gastroenterol. Belg., 2005;68:385-7) and portal vein thrombosis (Soyuncu et al., Clin. Toxicol. Phila, 2008;46:774-7) have been reported. Present study was aimed to evaluate the long-term effects of C. angustifolia by both in vivo (oral administration to rats) and in vitro (liver cell cultures) approaches. Materials and methods. In vivo, experiments were performed in Wistar rats, after oral administration of senna leaf extract (12 and 58 mg/kg/day) for 4 or 8 weeks. Serum was used for biochemical analysis. Liver samples were used for hystomorphological and immunohistochemical examination (Gaudio E. et al., Gastroenterology. 2006;130:1270-82) along with the determination of oxidative stress parameters. Cytotoxicity was assessed on Buffalo normal Rat Liver cell line (BRL-3A) by the Trypan blue assay and the MTT reduction method after 24 h of exposure. Results. In Wistar rats the extract did not induce any significant change in animal body weight, food and water consumption, enzyme activities, hystomorphological hepatic characteristics, levels of reduced glutathione, and MDA formation, at either time or dosage level. In BRL-3A cells the substance induced concentration-dependent cytotoxicity. Conclusions. C. angustifolia, at doses about 10 and 50 times higher than those used in humans (during a lapse of time corresponding to a chronic administration) does not affect liver morphology and hepatic function indices in rats. In vitro senna extract is cytotoxic at concentrations at least 2000 fold higher than those obtainable in vivo. Nevertheless, in humans the safety of C. angustifolia should be further monitored, in terms of patient-related factors

Role of acute administration of Chelidonium majus alone and in association with acetaminophen in rat liver

Aerial parts of Chelidonium majus (CM) are used to treat liver and gallbladder disorders, however several cases of hepatotoxicity following use of CM preparations are reported (Moro et al, 2009). Previous studies showed that high doses of CM do not alter morpho-functional rat liver parameters (Mazzanti et al. 2009). We aimed to evaluate if the co-treatment of CM and sub-toxic doses of acetaminophen can induce rat liver damage. Rats were treated as follows: 1) received vehicle at 0, 12, 24, 36 and 37h; 2) received vehicle at 0, 12, 24, 36 vehicle and acetaminophen at 37h; 3) received CM at 0, 12, 24, 36 and vehicle at 37h; 4) received CM at 0, 12, 24, 36 and acetaminophen at 37h. 24 hours after last treatment animals were sacrified and blood samples were collected to perform biochemical analysis and liver samples were underwent histomorphological and immunohistochemical examination. CM or acetaminophen did not alter body weight whereas total bilirubin, AST and ALT were slightly modified. Neither CM nor acetaminophen altered the hepatic structure while both substances induced a weak inflammatory cells infiltration. The co-administration of CM and acetaminophen did not modify the picture caused by the single treatments. Expression of α-SMA in hepatic stellate cells was negative in all experimental groups. In conclusion, the co-administration of C.majus and acetaminophen, does not have any synergistic effect in this experimental model. In humans, the safety of CM should be further monitored, taking into account the possibility of idiosyncratic reactions and/or other patient-related factors

Chemopreventive potential of caryophyllane sesquiterpenes: an overview of preliminary evidence

Chemoprevention is referred to as a strategy to inhibit, suppress, or reverse tumor development and progression in healthy people along with high-risk subjects and oncologic patients through using pharmacological or natural substances. Numerous phytochemicals have been widely described in the literature to possess chemopreventive properties, although their clinical usefulness remains to be defined. Among them, caryophyllane sesquiterpenes are natural compounds widely occurring in nature kingdoms, especially in plants, fungi, and marine environments. Several structures, characterized by a common caryophyllane skeleton with further rearrangements, have been identified, but those isolated from plant essential oils, including β-caryophyllene, β-caryophyllene oxide, α-humulene, and isocaryophyllene, have attracted the greatest pharmacological attention. Emerging evidence has outlined a complex polypharmacological profile of caryophyllane sesquiterpenes characterized by blocking, suppressing, chemosensitizing, and cytoprotective properties, which suggests a possible usefulness of these natural substances in cancer chemoprevention for both preventive and adjuvant purposes. In the present review, the scientific knowledge about the chemopreventive properties of caryophyllane sesquiterpenes and the mechanisms involved have been collected and discussed; moreover, possible structure-activity relationships have been highlighted. Although further high-quality studies are required, the promising preclinical findings and the safe pharmacological profile encourage further studies to define a clinical usefulness of caryophyllane sesquiterpenes in primary, secondary, or tertiary chemoprevention

Natural sesquiterpenes β-caryophyllene and β-caryophyllene oxide as chemopreventive agents for cholangiocarcinoma

Cholangiocarcinoma (CCA) represents a particular type of liver cancer originating from the epithelial cells lining the intrahepatic and extrahepatic biliary tree. It is often clinically silent until it becomes an advanced disease with obstructive symptoms and a poor prognosis for patients [1]. In this context, searching for alternative therapeutic strategies becomes a pivotal goal in the battle against liver cancer. In present research, β-caryophyllene (CRY) and β-caryophyllene oxide (CRYO), two natural sesquiterpenes with protective properties [2], have been investigated for their ability to inhibit the growth of the human Mz-ChA-1 cholangiocarcinoma cells from extrahepatic bile ducts and the H69 non-malignant cholagiocytes. The cells were exposed to the test compounds for 24 h, then the cytotoxicity was measured by MTT assay [3]; doxorubicin was included as a standard cytotoxic agents. Both sesquiterpenes reduced the cholangiocytes proliferation. Particularly, CRY produced a marked cytotoxicity in the Mz-ChA-1cells at concentrations lower than that required for CRYO. Surprisingly, the antiproliferative effects produced by both CRY and CRYO were higher than that of the standard doxoru- bicin. In the non-malignant cholangiocytes, in spite of a low cytotoxicity of CRYO, CRY reduced the cell proliferation already at lower concentrations, acting in a similar way to doxorubicin. Being CRYO an epoxide analogue of CRY, the different cytotoxicity found could be due to their structural features and bioavailability. Present results encourage further studies on CRY and CRYO as chemopreventive agents for cholangiocarcinoma. This work was supported by grants from Sapienza University Project 2014. Dr. Antonella Di Sotto was financed by “Enrico and Enrica Sovena” Foundation

Multifaceted Roles of GSK-3 in Cancer and Autophagy-Related Diseases

GSK-3 is a ubiquitously expressed serine/threonine kinase existing as GSK-3α and GSK-3β isoforms, both active under basal conditions and inactivated upon phosphorylation by different upstream kinases. Initially discovered as a regulator of glycogen synthesis, GSK-3 is also involved in several signaling pathways controlling many different key functions. Here, we discuss recent advances regarding (i) GSK-3 structure, function, regulation, and involvement in several cancers, including hepatocarcinoma, cholangiocarcinoma, breast cancer, prostate cancer, leukemia, and melanoma (active GSK-3 has been shown to induce apoptosis in some cases or inhibit apoptosis in other cases and to induce cancer progression or inhibit tumor cell proliferation, suggesting that different GSK-3 modulators may address different specific targets); (ii) GSK-3 involvement in autophagy modulation, reviewing signaling pathways involved in neurodegenerative and liver diseases; (iii) GSK-3 role in oxidative stress and autophagic cell death, focusing on liver injury; (iv) GSK-3 as a possible therapeutic target of natural substances and synthetic inhibitors in many diseases; and (v) GSK-3 role as modulator of mammalian aging, related to metabolic alterations characterizing senescent cells and age-related diseases. Studies summarized here underline the GSK-3 multifaceted role and indicate such kinase as a molecular target in different pathologies, including diseases associated with autophagy dysregulation

Differential redox state contributes to sex disparities in the response to influenza virus infection in male and female mice

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza