The resveratrol analogue trimethoxystilbene inhibits cancer cell growth by inducing multipolar cell mitosis.

Natural compounds are extensively studied for their potential use in traditional and non-traditional medicine. Several natural and synthetic Resveratrol analogues have shown interesting biological activities in the field of cancer chemoprevention. In the present study, we have focused on the ability of Resveratrol and two methoxylated derivatives (Trimethoxystilbene and Pterostilbene) to inhibit human cancer cell growth particularly analyzing their ability to interfere with tubulin dynamics at mitosis. We show that Trimethoxystilbene, differently from Resveratrol and Pterostilbene, alters microtubule polymerization dynamics in HeLa cells specifically inducing multipolar spindles and mitotic arrest coupled to a reduction of cell growth and an increase in apoptotic death by mitotic catastrophe. This work demonstrates that the structural modification of Rsv causes substantial changes in the mechanism of action of the derivatives. The presence of three extra methyl groups renders Trimethoxy very efficient in impairing cell proliferation by inducing mitotic catastrophe in cancer cells. © 2016 Wiley Periodicals, Inc.

Resveratrol and its methoxy-derivatives as modulators of DNA damage induced by ionising radiation.

Various naturally occurring stilbene-like compounds that are related to resveratrol (RSV) possess some of the beneficial effects of the parent molecule and provide even further benefits. Therefore, a series of methoxylated analogues of RSV were prepared with the aim of increasing antitumour and proapoptotic activity. In a previous article, we studied two methoxy-derivatives, pterostilbene (PTERO) and trimethoxystilbene (TRIMETHOXY), in which the first was formed by the substitution of two hydroxyl groups with two methoxy groups (trans-3,5-dimethoxy-4'-hydroxystilbene) and the second was formed by the replacement of all three OH groups with methoxy groups (trans-3,5,4'-trimethoxystilbene). Both methoxy-derivatives showed stronger antioxidant activity when compared with RSV. In the present article, we focused on the analysis of the ability of RSV and its two methoxylated derivatives to protect proliferating non-tumoural cells from the damage induced by ionising radiation (IR). First we showed that the methoxy derivatives, contrary to their parental compound, are unable to affect topoisomerase enzyme and consequently are not clastogenic per se Second we showed that both PTERO and TRIMETHOXY more efficiently reduce the chromosome damage induced by IR. Furthermore, TRIMETHOXY, but not PTERO, causes a delay in cell proliferation, particularly in mitosis progression increasing the number of cells in metaphase at the expense of prophases and ana/telophases.

Epidemiological, clinical, and molecular study of a cohort of Italian Parkinson disease patients: association with glutathione-S-transferase and DNA repair gene polymorphisms.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders whose etiology is multifactorial including both hereditary and environmental factors. Currently, pathogenic mutations in at least five genes have been implicated in familial PD generally accounting for less than 10 % of all PD cases in most populations. It has been suggested that polymorphisms in other genes such as those encoding enzymes involved in oxidative metabolism and detoxification could be involved in predisposition to PD since oxidative stress in dopaminergic neurons is thought to be of central importance in the pathogenesis of the disease. The aim of our work was to study the association of genetic polymorphisms in genes involved in oxidative metabolism and detoxification mechanism, namely GSTM1, GSTT1, GSTP1, and those involved in DNA damage repair, OGG1 and XRCC1, in an Italian cohort of sporadic PD patients. We did not detect any association between GSTT1 and GTTM1 null polymorphisms and PD, whereas the 104GSTP1 polymorphism was associated with PD in male patients but not in females. Furthermore, we detected a protective effect of wild type genotype of XRCC1 in women.

Effects of resveratrol on topoisomerase II-α activity: induction of micronuclei and inhibition of chromosome segregation in CHO-K1 cells.

In recent years, a great interest has emerged in resveratrol (RSV) activity in the prevention of various pathologies including cancer. We recently showed that RSV is able to interfere with topoisomerase II-α (TOPO2) activity in cancer cells, thus inducing a delay in S-phase progression with concomitant phosphorylation of the histone H2AX. TOPO2 is mainly active in proliferating cells and is involved in the resolution of supercoiled DNA and chromosome segregation during mitosis. Here, we studied the effects of RSV in CHO-K1 cells concerning to chromosome damage and segregation as a consequence of TOPO2 inhibition. We show an increase in micronuclei and in polyploid and endoreduplicated cells due to incorrect chromosome segregation. Furthermore, since incomplete segregation can also affect the normal distribution of mitotic figures, we checked mitosis progression showing an increase in metaphase in relation to ana-telophase after RSV treatment. On the whole, our data show that RSV affects chromosome stability and segregation in proliferating cells, probably interfering with TOPO2 activity.

Resveratrol acts as a topoisomerase II poison in human glioma cells.

Recently, we demonstrated that Resveratrol (RSV), a well known natural stilbene, is able to induce a delay in S progression with a concomitant increase in γH2AX expression in U87 glioma cells. Furthermore, we showed that it inhibits the ability of recombinant human topoisomerase IIα to decatenate kDNA in vitro. Because proliferating tumor cells express topoisomerases at high levels and these enzymes are important targets of some of the most successful anticancer drugs, we tested whether RSV is able to poison topoisomerase IIα in glioma cells. Then, we monitored the increase of micronuclei in RSV treated U87 cells as a consequence of the conversion of TOPOII/DNA cleavable complexes to permanent DNA damage. Finally, we assayed the ability of RSV in modulating the expression of target proteins involved in DNA damage signalling, namely ATR, ATM, Chk1, Chk2 and γH2AX. Through a molecular modelling here we show that RSV binds at the TOPOII/DNA interface thus establishing several hydrogen bonds. Moreover, we show that RSV poisons TOPOIIα so inducing DNA damage; ATM, Chk2 and γH2AX are involved in the DNA damage signalling after RSV treatment.

DNA repair capacity and acute radiotherapy adverse effects in Italian breast cancer patients.

Therapeutic exposure to ionising radiation can induce normal tissue side effects which consistently differ among individuals suggesting a possible genetic control. One approach to elucidate the underlying mechanisms is to analyse the relation between genetic traits, biomarkers of in vitro DNA damage and side toxicity in vivo. 43 breast cancer (BC) patients receiving radiotherapy after a breast-conserving surgery were recruited together with 34 age- and sex-matched healthy controls. Adverse tissue reactions were recorded as indicators of radiotherapy susceptibility. All blood samples from both patients (35) and controls (34) were irradiated in vitro and DNA primary damage and repair kinetic were measured through Comet assay. All study subjects were genotyped for XRCC1, OGG1 and XRCC3 gene polymorphisms. In our small groups we found a positive association between XRCC1 variant allele (399Gln) and the occurrence of breast cancer [p=0.01; OR=2.41, 95%CI (1.24-4.66)]. BC patients showed a higher degree of basal (p<0.001) and X-ray induced DNA damage (p<0.01) when compared to healthy controls. A reduced repair ability was found in BC patients showing high degrees of tissue side effects as classified by Radiation Morbidity Scoring Scheme. BC patients showed an impairment of their DNA repair capacity associated with the development of radiation sensitivity but not with polymorphisms in any of the considered genes.

DNA damage and related modifier genes in Italian cystic fibrosis patients.

Cystic Fibrosis (CF) is an autosomal recessive multisystemic disorder showing a highly heterogeneous phenotype, even among siblings carrying identical CFTR mutations. Moreover, oxidative stress is of central importance in the pathogenesis of cystic fibrosis. The present study seeks to value the presence of oxidative damage in CF patients and the possible modifier effect of repair and glutathione-S-transferase genes. We analysed the presence of DNA damage in leukocytes of 63 CF patients at an Italian CF centre and 63 controls, through the alkaline Comet assay to detect DNA strand breaks. Furthermore, controls and 93 CF subjects were genotyped for 5 genes by RFLP-PCR (XRCC1,0GG1,GSTP1) and PCR assay (GSTM1, GSTT1). No difference in Comet assay values was observed comparing controls to CF patients, although CF subjects showed slightly higher mean values. The crude Odds-Ratio (OR) was higher than one for XRCC1 and GSTP1 genotypes and liver status and for XRCC1 and OGG1 genotypes and pancreatic insufficiency, but in all cases the p-values were not significant. In this case-control study, neither DNA damage ñor gene polymorphisms seem to influence CF manifestation.

A novel resveratrol derivative induces mitotic arrest, centrosome fragmentation and cancer cell death by inhibiting γ-tubulin.

BACKGROUND: Resveratrol and its natural stilbene-containing derivatives have been extensively investigated as potential chemotherapeutic agents. The synthetic manipulation of the stilbene scaffold has led to the generation of new analogues with improved anticancer activity and better bioavailability. In the present study we investigated the anticancer activity of a novel trimethoxystilbene derivative (3,4,4'-trimethoxylstilbene), where two methoxyl groups are adjacent on the benzene ring (ortho configuration), and compared its activity to 3,5,4'-trimethoxylstilbene, whose methoxyl groups are in meta configuration. RESULTS: We provide evidence that the presence of the two methoxyl groups in ortho configuration renders 3,4,4'-trimethoxystilbene more efficient than the meta isomer in inhibiting cell proliferation and producing apoptotic death in colorectal cancer cells. Confocal microscopy of α- and γ-tubulin staining shows that the novel compound strongly depolymerizes the mitotic spindle and produces fragmentation of the pericentrosomal material. Computer assisted docking studies indicate that both molecules potentially interact with γ-tubulin, and that 3,4,4'-trimethoxystilbene is likely to establish stronger interactions with the protein. CONCLUSIONS: These findings demonstrate the ortho configuration confers higher specificity for γ-tubulin with respect to α-tubulin on 3,4,4' trimethoxystilbene, allowing it to be defined as a new γ-tubulin inhibitor. A strong interaction with γ-tubulin might be a defining feature of molecules with high anticancer activity, as shown for the 3,4,4' isomer.

Resveratrol and X rays affect gap junction intercellular communications in human glioblastoma cells.

Resveratrol (3,4',5-trihydroxystilbene) is a polyphenol synthesized by a wide variety of plant species in response to injury, UV irradiation and fungal attack. Many studies have revealed a variety of resveratrol intracellular targets whose modulation gives rise to overlapping responses leading to growth arrest and death. Many authors have reported different human cancer cell lines, treated with resveratrol at micromolar concentrations, arrested their proliferative cycle in the G1/S boundary or in the S phase and this cell cycle arrest was followed by apoptotic death. Less is known about the ability of resveratrol to modify the effect of radiation exposure in normal and cancer cells. Considering that controlled exposure to ionizing radiation is one of the most used treatments in cancer patients and that these schedules are not always effective in medical practice, as in the case of glioma patients, the testing of combined treatment protocols (resveratrol and ionizing radiation) could be of interest, opening the door to future studies which would examine the pharmacological activity of resveratrol. In this study we have looked into whether resveratrol is able to modulate cell cycle progression in human glioblastoma cells and to regulate GJs expression in cancer cells. With this aim in mind we have performed a cytofluorimetric multiparameter assay to quantify the presence of GJs in U87 glioma cells treated with resveratrol and/or X rays. We report that resveratrol induces a delay in cell cycle progression and both alone and in combination with X rays is able to enhance gap junction Intercellular Communications.

Molecular biomonitoring of a population of nurses handling antineoplastic drugs.

Many antineoplastic drugs have been found to have carcinogenic, mutagenic and teratogenic activity and so hospital personnel handling these substances are potentially exposed to health risk. Understanding this risk derived from protracted occupational exposure has great relevance even if the workers normally adopt individual and environmental protective measures. To address this question we have studied the presence of DNA and chromosome damage in a population of nurses employed in Italian oncology units and in matched controls. We used the comet assay to evidence the presence of DNA strand breaks, due to both acute and chronic exposure, and the micronucleus (MN) test, which is a measure of clastogenic and aneugenic events. Furthermore, since the individual response to the exogenous insults may be genetically determined, we studied the possible influence of single nucleotide polymorphism in XRCC1 and XRCC3 DNA repair genes on induced genetic damage. We also considered the effects of confounding factors like smoking, age and gender. The results indicated that the exposed subjects had significantly high levels of genetic damage. Age and gender were associated with increased values in MN, both in control and in exposed groups; the smoking habit affects MN frequency in controls, but not in workers. Furthermore we found that exposed subjects bearing at least one XRCC1 variant allele (399Gln) show higher values of MN. The present data provide the evidence to show that occupational exposure to antineoplastic drugs, even if in safety controlled conditions, represents a serious health risk. Furthermore we have shown that the presence of XRCC1 genetic polymorphism could contribute to increase the genetic damage in susceptible individuals who are occupationally exposed to dangerous substances.

Assessment of the genotoxic risk of Punica granatum L. (Punicaceae) whole fruit extracts.

Punica granatum L. (Punicaceae) whole fruit extracts, have been used in Cuban traditional medicine as an effective drug for the treatment of respiratory diseases. This species showed interesting anti-viral activity, e.g. aqueous or hydroalcoholic extracts of whole fruits have proved highly active against the influenza virus. However, some toxic properties of this extract have also been reported and, to date, very little is known about its genotoxic properties. In the present study, the genotoxicity of a Punica granatum (pomegranate) whole fruit extract was assessed using different in vitro and in vivo assays that detect DNA damage at different expression levels. Results from reversion and gene-conversion test in microorganisms, sister chromatid exchanges, micronuclei and sperm-shape abnormality assays in mice, clearly showed that the hydroalcoholic extract of P. granatum whole fruits is genotoxic when tested both in vitro and in vivo.

A multi-biomarker analysis of the antioxidant efficacy of Parkinson's disease therapy.

Substantial evidences suggest that reactive oxygen species participate in the normal aging process and in cancer and neurodegenerative age-related diseases. Parkinson's disease (PD), one of the most common oxidative stress-associated pathology in aging people, is treated with a standard pharmacological protocol consisting in a combined therapy l-dopa plus an inhibitor of dopa-decarboxylase, such as carbidopa. The therapy is well validated for the ability to restoring dopaminergic neurotransmission in PD patients, while l-dopa and carbidopa ability in modulating oxidative stress is currently under discussion. Our aim was to evaluate the impact of l-dopa and carbidopa on several biomarkers of exogenously-induced oxidative stress to validate the overall antioxidant effectiveness of the therapy. For this purpose we used peripheral blood lymphocytes from healthy donors treated in vitro with l-dopa and carbidopa and then challenged by different concentrations of H2O2. Glutathione (GSH, GSSG, GSH/GSSG), malondialdehyde (TBARs), protein carbonyls as well as DNA damage (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and micronuclei (MN)), modulation was evaluated. Our results show that l-dopa, but not carbidopa, decreases the markers of lipid and protein oxidation and increases the total content of glutathione. Both l-dopa and carbidopa (alone or in combination) are able to counteract the formation of 8-oxodG and to reduce H2O2-induced micronuclei.

Influence of glutathione S-transferase polymorphisms on genotoxic effects induced by tobacco smoke.

Genotoxicity of tobacco smoke has long been investigated and tobacco smoke is considered to be one of the principal human carcinogens. Although its role in DNA-damage induction and cancer development has been documented, the mechanisms by which this happens are not well understood. Many chemical constituents of tobacco smoke are enzymatically metabolized by phase-I and phase-II enzymes, but modifications in coding and regulating sequences of these genes could influence their ability to detoxify these compounds. In this work, we studied several enzymes involved in the metabolism of xenobiotics, viz. the glutathione S-transferases (GST) M1, T1, P1 and A1, with respect to their influence on the genotoxic effects induced by cigarette smoking. We assessed the genotoxic effects of tobacco smoke on peripheral blood lymphocytes of 72 healthy caucasians by use of the chromosomal aberration (CA) assay and the micronucleus (MN) test. Genotypes of GST M1, T1, P1 and A1 were determined by means of the polymerase chain reaction and methods based on restriction fragment length polymorphism (RFLP). We found that smoke and gender are the two variables that most influence the DNA damage. In particular, we observed that female smokers seem to be more sensitive than male smokers, having a significantly higher frequency of CAs. Moreover, a significant increase in frequency of micronuclei in bi-nucleated cells (BNMN) was found in smokers, but not in non-smokers. This increase seems to be influenced not only by age and gender, but also by genetic constitution. Subjects carrying GSTM1-null genotype seemed to have an higher susceptibility to DNA damage induced by tobacco smoke than GSTM1-positive ones. When considering a combination of GST genotypes, we found a lower BNMN frequency in subjects with GSTP1 variant allele plus GSTM1-positive genotypes, while the most damaged cells are found in subjects bearing GSTM1-null plus GSTP1-wild type. Our results suggest that investigation of the association between several gene polymorphisms and important endpoints of DNA damage could contribute to better understanding the role of gene-gene interaction.

DNA damage repair and genetic polymorphisms: assessment of individual sensitivity and repair capacity.

PURPOSE: To study the repair capacity after X-ray irradiation in human peripheral blood cells of healthy subjects, in relation to their genotypes. METHODS AND MATERIALS: The peripheral blood of 50 healthy subjects was irradiated in vitro with 2 Gy of X rays and the induced DNA damage was measured by Comet assay immediately after irradiation. DNA repair was detected by analyzing the cells at defined time intervals after the exposure. Furthermore, all subjects were genotyped for XRCC1, OGG1, and XPC genes. RESULTS: After X-ray irradiation, persons bearing XRCC1 homozygous variant (codon 399) genotype exhibited significantly lower Tail DNA values than those bearing wild-type and heterozygous genotypes. These results are also confirmed at 30 and 60 min after irradiation. Furthermore, XPC heterozygous subjects (variant codon 939) showed lower residual DNA damage 60 min after irradiation compared with wild-type and homozygous genotypes. CONCLUSION: The results of the present study show that polymorphisms in DNA repair genes could influence individual DNA repair capacity.

Resveratrol affects DNA damage induced by ionizing radiation in human lymphocytes in vitro.

Resveratrol (3,4',5-trihydroxystilbene; RSV) acts on cancer cells in several ways, inducing cell cycle delay and apoptotic death, and enhancing ionizing radiation (IR)-mediated responses. However, fewer studies have examined RSV effects on normal cells. We have treated human lymphocytes in vitro with RSV, either alone or combined with IR, to evaluate its potential use as a radioprotector. We measured the effects of RSV on induction of DNA damage, repair kinetics, and modulation of histone deacetylase activity.

Xanthium strumarium extract inhibits mammalian cell proliferation through mitotic spindle disruption mediated by xanthatin.

ETHNOPHARMACOLOGICAL RELEVANCE: Xanthium strumarium L. is a member of the Asteraceae family popularly used with multiple therapeutic purposes. Whole extracts of this plant have shown anti-mitotic activity in vitro suggesting that some components could induce mitotic arrest in proliferating cells. AIM OF THE SUDY: Aim of the present work was to characterize the anti-mitotic properties of the X. strumarium whole extract and to isolate and purify active molecule(s). MATERIALS AND METHODS: The capacity of the whole extract to inhibit mitotic progression in mammalian cultured cells was investigated to identify its anti-mitotic activity. Isolation of active component(s) was performed using a bioassay-guided multistep separation procedure in which whole extract was submitted to a progressive process of fractionation and fractions were challenged for their anti-mitotic activity. RESULTS: Our results show for the first time that X. strumarium whole extract inhibits assembly of the mitotic spindle and spindle-pole separation, thereby heavily affecting mitosis, impairing the metaphase to anaphase transition and inducing apoptosis. The purification procedure led to a fraction with an anti-mitotic activity comparable to that of the whole extract. Chemical analysis of this fraction showed that its major component was xanthatin. CONCLUSIONS: The present work shows a new activity of X. strumarium extract, i.e. the alteration of the mitotic apparatus in cultured cells that may be responsible for the anti-proliferative activity of the extract. Anti-mitotic activity is shown to be mainly exerted by xanthatin.

Resveratrol affects X-ray induced apoptosis and cell cycle delay in human cells in vitro.

Many studies in human and mammalian cells have shown the involvement of resveratrol in the modulation of several biological processes, including the regulation of carcinogenesis. However, the molecular mechanisms underlying its antitumorigenic activity are still not defined. There is great interest in developing new agents that can modify the efficacy of radiation therapy. The aim of the present study was to investigate the cellular response to treatments with X rays and resveratrol, alone or in combination, in terms of DNA damage, cell cycle delays and induction of apoptosis. Lymphoblastoid cells AHH-1 were treated and analysed at successive sampling times in order to study the induction of DNA breaks using the Comet assay and the induction of apoptosis and cell cycle modulation through cytofluorimetric analysis. Our results suggest that resveratrol causes the induction of apoptosis and a block of cell cycle progression at an early step of S-phase. Furthermore, resveratrol mitigates the apoptotic clearance of irradiated cells and prevents the G2 phase cell cycle arrest induced by X-rays.

Evaluation of levodopa and carbidopa antioxidant activity in normal human lymphocytes in vitro: implication for oxidative stress in Parkinson's disease.

The main pathochemical hallmark of Parkinson's disease (PD) is the loss of dopamine in the striatum of the brain, and the oral administration of levodopa (L-dopa) is a treatment that partially restores the dopaminergic transmission. In vitro assays have demonstrated both toxic and protective effects of L-dopa on dopaminergic cells, while in vivo studies have not provided any convincing data. The peripheral metabolic pathways significantly decrease the amount of L-dopa reaching the brain; therefore, all of the current commercial formulations require an association with an inhibitor of dopa-decarboxylase, such as carbidopa. However, the dosage and the actual effectiveness of carbidopa have not yet been well defined. PD patients exhibit a reduced efficiency of the endogenous antioxidant system, and peripheral blood lymphocytes (PBLs) represent a dopaminergic system for use as a cellular model to study the pharmacological treatments of neurodegenerative disorders in addition to analysing the systemic oxidative stress. According to our previous studies demonstrating a protective effect of both L-dopa and carbidopa on neuroblastoma cells in vitro, we used the PBLs of healthy donors to evaluate the modulation of DNA damage by different concentrations of L-dopa and carbidopa in the presence of oxidative stress that was exogenously induced by H2O2. We utilised a TAS assay to evaluate the in vitro direct scavenging activity of L-dopa and carbidopa and analysed the expression of genes that were involved in cellular oxidative metabolism. Our data demonstrate the antioxidant capacity of L-dopa and carbidopa and their ability to protect DNA against oxidative-induced damage that derives from different mechanisms of action.

Resveratrol affects in a different way primary versus fixed DNA damage induced by H(2)O(2) in mammalian cells in vitro.

Resveratrol (3,5,4'-trihydroxystilbene) is a natural occurring molecule, synthesized by plants in response to different stresses. Recent literature data seem to converge in indicating Resveratrol as an agent possessing protective effects against oxidative stresses through different mechanisms. Furthermore conflicting data are present in relation to its activity of free radical scavenger. Here we studied the antioxidant activity actually exerted by the agent against reactive oxygen species induced by H(2)O(2) treatments in CHO cells. Our attention has been focused on two major potential mechanisms: scavenging activity and interference with oxidative metabolism, by the analysis of three important targets: intracellular oxidation (Dichlorofluorescein Test), primary DNA damage (Comet Assay) and fixed DNA damage (chromosomal aberrations). Cells were treated with a single H(2)O(2) dose (2x10(-4) M) in order to induce Reactive Oxygen Species and than challenged with Resveratrol to test its ability in modulating damage. Two experimental protocols have been applied: (i) simultaneous treatment and (ii) a 3 h Resveratrol pre-treatment. In our experimental conditions Resveratrol does not appear able, 'per se', to induce primary DNA damage whereas a slight increase in endogenous oxidation and chromosomal aberrations at the highest dose have to be noticed. In combined treatments the molecule appears to differently affect primary and fixed DNA damage.

Assessment of the potential genotoxic risk of Phyllantus orbicularis HBK aqueous extract using in vitro and in vivo assays.

Phyllanthus orbicularis HBK is an endemic Cuban plant whose aqueous extract has been proposed as an effective drug for the treatment of viral diseases. In addition, antimutagenic properties of this extract have also been reported. In the present study, the genotoxicity of this plant extract was assessed using different in vitro and in vivo assays. Results from SOS gene induction, gene reversion and conversion, and SMART assays clearly show that P. orbicularis aqueous extract does not induce either primary DNA damage or mutation. Additionally, no statistically significant difference was found in the percentage of chromosomal aberrations in Chinese hamster ovarian (CHO) cells treated with the plant extract. On the contrary, micronuclei and abnormal anaphase were induced by this extract in CHO cells. This genotoxic effect was related to a high cytotoxicity. Single spots were detected in the SMART assay. These results point to a possible aneugenic effect of the P. orbicularis aqueous extract at cytotoxic doses which are much higher than those seen by their antiviral and antimutagenic activities.