α-Hexylcinnamaldehyde synergistically increases doxorubicin cytotoxicity towards human cancer cell lines

α-Hexylcinnamaldehyde (HCA), a compound derived from cinnamaldehyde, was evaluated for its potential chemosensitizing properties

Antimutagenic and antioxidant activity of a protein fraction from aerial parts of Urtica dioica

Abstract Context: Urtica dioica L. (Urticaceae), stinging nettle, has been employed as a folklore remedy for a wide spectrum of ailments, including urinary disorders, prostatic hyperplasia, and liver diseases. It has been also used traditionally for cancer treatment. Object: To evaluate the potential chemopreventive properties of a protein fraction from the aerial part of Urtica dioica (namely UDHL30). Materials and methods: UDHL30 has been tested for the antimutagenic activity in bacteria (50-800 μg/plate; Ames test by the preincubation method) and for the cytotoxicity on human hepatoma HepG2 cells (0.06-2 mg/mL; 24 and 48 h incubation). Moreover, the antioxidant activity of UDHL30 (0.1-1200 μg/mL; ABTS and superoxide-radical scavenger assays) was evaluated as potential protective mechanisms. Results: UDHL30 was not cytotoxic on HepG2 cells up to 2 mg/mL; conversely, it exhibited a strong antimutagenic activity against the mutagen 2-aminoanthracene (2AA) in all strains tested (maximum inhibition of 56, 78, and 61% in TA98, TA100, and WP2uvrA strains, respectively, at 800 μg/plate). In addition, a remarkable scavenging activity against ABTS radical and superoxide anion (IC50 values of 19.9 ± 1.0 μg/mL and 75.3 ± 0.9 μg/mL, respectively) was produced. Discussion and conclusions: UDHL30 possesses antimutagenic and radical scavenging properties. Being 2AA a pro-carcinogenic agent, we hypothesize that the antimutagenicity of UDHL30 can be due to the inhibition of CYP450-isoenzymes, involved in the mutagen bioactivation. The radical scavenger ability could contribute to 2AA-antimutagenicity. These data encourage further studies in order to better define the potential usefulness of UDHL30 in chemoprevention

Natural and naturally-derived compounds as new chemopreventive agents

Chemoprevention is an approach based on the use of natural or synthetic compounds to inhibit, suppress or reverse the development and progression of cancer. In order to overcome the cancer disease, the identification of chemopreventive compounds is of particular interest. Among them, antimutagens prevent the mutagen-induced DNA-injury or promote the repair and/or the reversion of damage. In addition to antimutagenicity, some agents also act as chemosensitizers, by increasing the effectiveness of cancer chemotherapy and radiotherapy, when used in combination with chemotherapeutical agents. This approach is very interesting to prevent the development of multidrug resistance (MDR), which makes cancer cells not-sensitive to a broad range of drugs. In this context, present study was aimed at evaluating the potential chemopreventive properties of some natural and naturally-derived compounds, particularly the sesquiterpenes β-caryophyllene (CRY) and β-caryophyllene oxide (CRYO), and the aldehyde α-hexylcinnamal (HCA). The antimutagenic activity was evaluated by the reverse bacterial mutation assay (Ames test), on different strains of Salmonella typhimurium and Escherichia coli, both in absence and presence of the S9-metabolic activation system. As mutagens, 2- nitrofluorene (2NF), sodium azide (SA), methyl methanesulfonate (MMS), 2-aminoanthracene (2AA), benzo[a]pyrene (BaP), 4-nitroquinoline N-oxide (4NQO), 1-nitropyrene (1NP), 1,8-dinitropyrene (1,8-DNP) and a sample of condensed smoke (CSC) from standard 3R4F cigarette were used. In addition to antimutagenicity studies, the potential chemosensitizing properties of CRY, CRYO and HCA and their ability to interfere with ABC-transporter function were evaluated, in Caco-2, CEM/ADR5000 and CCRF/CEM human cancer cells. For each compound, low concentrations (IC10 and IC20) were assayed in order to verify their potential additive, synergistic or antagonistic effects with the anticancer doxorubicin. The nature and the extent of the interaction were evaluated by the combination index (CI) and the isobologram analysis, respectively; conversely, the potential enhancement of drug effectiveness was quantified by cytotoxicity enhancement ratio (RR). The interaction between test compounds and ABC-transporters was studied by the rhodamine 123 assay. HCA exhibited an antimutagenic activity against different nitro-compounds and in various experimental protocols, suggesting the involvement of both desmutagenic and bioantimutagenic mechanisms. The sesquiterpenes CRY and CRYO resulted able to inhibit the mutagenicity of CSC, although with different potency and specificity: CRYO was the most potent compound, acting at concentrations about ten-times lower than CRY. The antimutagenicity was highlighted in different strains and in all experimental protocols, suggesting the overlapping of various protective mechanisms; the inhibition of CSC-induced oxidative stress seems to be likely and deserves further investigations. In human cancer cells, the substances produced cytotoxic effects at high concentrations both in resistant and in sensitive cell lines: HCA was the most effective substance, especially in the sensitive CCRF-CEM cells. All the compounds synergistically acted with doxorubicin, although HCA was the most potent: IC20 HCA increased the doxorubicin cytotoxicity of about six, seven and fourthy-seven folds, in Caco-2, CEM/ADR5000, and CCRF-CEM, respectively. In addition, a remarkable inhibition of ABCtrasporter was produced by HCA in the cancer cells tested: the effect was higher than that of the standard inhibitior verapamil. Also CRY and CRYO inhibited the ABC transporters but with lower potency than verapamil. The antimutagenic and chemosensitizing properties of β-caryophyllene, β-caryophyllene oxide and the α-hexylcinnamaldehyde deserves attention and represent a starting point to better evaluate their potential applications in the field of chemoprevention

Discovery of Chalcone-Based Hybrid Structures as High Affinity and Site-Specific Inhibitors against SARS-CoV-2: A Comprehensive Structural Analysis Based on Various Host-Based and Viral Targets

Previous studies indicated that natural-based chalcones have significant inhibitory effects on the coronavirus enzymes 3CLpro and PLpro as well as modulation of some host-based antiviral targets (HBATs). In this study, a comprehensive computational and structural study was performed to investigate the affinity of our compound library consisting of 757 chalcone-based structures (CHA-1 to CHA-757) for inhibiting the 3CLpro and PLpro enzymes and against twelve selected host-based targets. Our results indicated that CHA-12 (VUF 4819) is the most potent and multi-target inhibitor in our chemical library over all viral and host-based targets. Correspondingly, CHA-384 and its congeners containing ureide moieties were found to be potent and selective 3CLpro inhibitors, and benzotriazole moiety in CHA-37 was found to be a main fragment for inhibiting the 3CLpro and PLpro. Surprisingly, our results indicate that the ureide and sulfonamide moieties are integral fragments for the optimum 3CLpro inhibition while occupying the S1 and S3 subsites, which is fully consistent with recent reports on the site-specific 3CLpro inhibitors. Finding the multi-target inhibitor CHA-12, previously reported as an LTD4 antagonist for the treatment of inflammatory pulmonary diseases, prompted us to suggest it as a concomitant agent for relieving respiratory symptoms and suppressing COVID-19 infection

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

Body composition and metabolic status of Italian and Spanish university students: relationship with fruit and vegetable consumption

Most university students do not follow recommendations for fruit and vegetable intake, with a consequent increase in the prevalence of cardiovascular disease (CVD) risk factors. The aim of this study was to compare obesity prevalence and biomarkers of metabolic status between Italian and Spanish university students, in relation with the consumption of fruits and vegetables. Food consumption, adherence to a Mediterranean diet (MD), level of physical activity (PA), blood glucose, total cholesterol, triglycerides and ketones, blood pressure, and body composition were evaluated. Among CVD risk factors, only glucose was significantly higher in Spaniards (SP), and only 3.1% of SP presented ketosis. SP had a higher percentage of energy from fat. Although adherence to MD and fruit and vegetable consumption did not differ between Italians and SP, students who consumed at least four servings of fruit and vegetables (FV group) showed better values for pressure and metabolic parameters than the no FV group. We observed an association between consumption of fruit and PA. Students who consumed more vegetables than fruit reported a better body composition profile and lower glucose concentrations. As previously suggested, in addition to PA, two servings of fruit and three servings of vegetables per day should be recommended

Regulation of CREB activation by p38 mitogen activated protein kinase during human primary erythroblast differentiation.

Among the molecular events underlying erythroid differentiation, we analyzed the signalling pathway leading to cAMP response element binding (CREB) nuclear transcription factor activation. Normal donor blood light density cells differentiated to pro-erythroblasts during the proliferative phase (10 days) of the Human Erithroblast Massive Amplification (HEMA) culture, and to orthochromatic erythroblasts, during the differentiative phase (4 additional days) of the culture. Since erythropoietin was present all over the culture, also pro-erythroblasts left in proliferative medium for 14 days continued their maturation without reaching the final steps of differentiation. p38 Mitogen Activated Protein Kinase (p38 MAPK) and CREB maximal activation occurred upon 4 days of differentiation induction, whereas a lower activation was detectable in the cells maintained in parallel in proliferative medium (14 days). Interestingly, when SB203580, a specific p38 MAPK inhibitor, was added to the culture the percentage of differentiated cells decreased along with p38 MAPK and CREB phosphorylation. All in all, our results evidence a role for p38 MAPK in activating CREB metabolic pathway in the events leading to erythroid differentiation

Genotoxicity assessment of piperitenone oxide: an in vitro and in silico evaluation

Piperitenone oxide, a natural flavouring agent also known as rotundifolone, has been studied for the genotoxicity assessment by an integrated in vitro and in silico experimental approach, including the bacterial reverse mutation assay, the micronucleus test, the comet assay and the computational prediction by Toxtree and VEGA tools. Under our experimental conditions, the monoterpene showed to induce both point mutations (i.e. frameshift, base-substitution and/or oxidative damage) and DNA damage (i.e. clastogenic or aneuploidic damage, or single-strand breaks). Computational prediction for piperitenone oxide agreed with the toxicological data, and highlighted the presence of the epoxide function and the α,β-unsaturated carbonyl as possible structural alerts for DNA damage. However, improving the toxicological libraries for natural occurring compounds is required in order to favour the applicability of in silico models to the toxicological predictions. Further in vivo evaluations are strictly needed in order to evaluate the role of the bioavailability of the substance and the metabolic fate on its genotoxicity profile. To the best of our knowledge, these data represent the first evaluation of the genotoxicity for this flavour compound and suggest the need of further studies to assess the safety of piperitenone oxide as either flavour or fragrance chemicals