Phytochemical screening and effect of Viscum album L. on monoamine oxidase A and B activity and serotonin, dopamine and serotonin receptor 5-HTR1A levels in Galleria mellonealla (Lepidoptera).

ETHNOPHARMACOLOGICAL RELEVANCE: Viscum album L. (European mistletoe), a member of the Santalaceae, is a hemiparasitic, evergreen shrub growing on deciduous and coniferous trees. In traditional and folk medicine, mistletoe was used for the treatment of central nervous system disorders such as epilepsy, hysteria, insomnia, nervous excitability, neuralgia, headache, dizziness and fatigue. However, relatively little is known of its neuropharmacological activity. AIM OF THE STUDY: The aim of the present study was to evaluate the effect of treatment with aqueous and hydroethanolic extracts from Viscum album L. parasitizing birch, linden and pine, on MAO-A and MAO-B activity as well as serotonin, dopamine and serotonin receptor 5-HTR1A levels in Galleria mellonella (Lepidoptera) larvae. MATERIALS AND METHODS: The phytochemical composition of the extracts was characterised using UPLC-DAD-ESI-MS/MS. To investigate the neuropharmacological activity of Viscum album L. extracts, Galleria mellonella (Lepidoptera) larvae were used as a model organism. The inhibitory potential of the extracts against MAO-A and MAO-B was determined by fluorometry. The serotonin, dopamine and serotonin receptor 5-HTR1A levels in larvae hemolymph after treatment were quantified by ELISA. RESULTS: UPLC-DAD-ESI-MS/MS analysis allowed the identification of 88 compounds, either full or in part. Most of the characterised phytochemicals were flavonoids, hydroxycinnamic acids and lignans. Screening found that aqueous and hydroethanolic mistletoe extracts inhibited the enzymatic activity of either MAO-A or MAO-B or both. Additionally, mistletoe extract administration increased the levels of serotonin and serotonin receptor 5-HTR1A. None of the tested extracts had any significant effect on dopamine level. CONCLUSIONS: A key novel finding was that the aqueous and hydroethanolic extracts from Viscum album L. inhibited monoamine oxidase activity and increased the levels of serotonin and serotonin receptor 5-HTR1A in Galleria mellonella (Lepidoptera) larvae. These properties may be due to the presence of phenolic constituents, particularly flavonoids. Further research based on bioassay-guided fractionation of mistletoe is needed to identify CNS-active molecules.

New camptothecin derivatives for generalized oncological chemotherapy: Synthesis, stereochemistry and biology.

Derivatives of SN38 were synthesized that were either monosubstituted at C-5 or C-9 or disubstituted at both C-5 and C-9. Substitution to C-5 led to the generation of pairs of diastereomers (2c-2 h) in a one-pot reaction and was readily separable by HPLC. The absolute configurations of C-5 were established by electronic circular dichroism experiments. Compounds were tested in vitro against human cancer cell lines as well as a normal cell line. The impact of compounds 2a-2j on cancer cells is significant and the IC50 values against the normal cell line are several times higher than that of SN38. Using the Mannich reaction we obtained a new innovative group of derivatives with unique biological properties that preserves the high cytotoxicity in cancer cells and eliminates the acute toxicity to non-neoplastic cells, which can be considered a breakthrough in chemotherapy with the use of topoisomerase I inhibitors from the camptothecin family.

Fluoroaryl analogs of sulforaphane - A group of compounds of anticancer and antimicrobial activity.

A series of new sulforaphane analogs bearing various (poly)fluoroaryl substituents bonded to the sulfinyl sulfur atom in place of the original methyl group and having different number of methylene groups in the central alkyl chain were synthesized and fully characterized. The new compounds were tested in vitro for their anticancer, antibacterial, antifungal and antiviral properties. Some of them demonstrated a much higher anticancer activity against selected lines of cancer: skin (MALME-3M), colon (HT-29) and breast (MCF7 and MDA-MB-231) cells than that exhibited by native sulforaphane (SFN). Related lines of untransformed (normal) cells, taken from the same organs as the cancer ones, i.e. MALME3, CRL-1790 and MCF10, respectively, were checked, which allowed for the determination of the selectivity indexes (SI). In certain cases, the latter exceeded 3.2. Concerning the antibacterial activity, gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) were susceptible to some newly synthesized SFN analogs, while the selected probiotic strains were from 10 to 100 fold more resistant to them, which gives a possibility of protection of symbiont strains during a potential therapy with such compounds. The antifungal activity of the new compounds possessing the fluorophenyl substituent was found to be higher than the activity of the parent SFN. In turn, most of the new compounds showed generally no anti-HIV activity. The influence of the particular structural differences in the new molecules, analogs of SFN, on their biological activity is discussed.

In Vitro Evaluation of Sulforaphane and a Natural Analog as Potent Inducers of 5-Fluorouracil Anticancer Activity.

Isothiocyanates (R-NCS) are sulphur-containing phytochemicals. The main source are plants of the Brassicaceae family. The best known plant-derived isothiocyanate is sulforaphane that has exhibited anticancer activity in both in vivo and in vitro studies. Recent attempts to expand their use in cancer therapy involve combining them with standard chemotherapeutics in order to increase their therapeutic efficacy. The aim of this paper is to determine the impact of sulforaphane and its natural analog alyssin on the anticancer activity of the well-known anticancer drug 5-fluorouracil. The type of drug-drug interactions was determined in prostate and colon cancer cell lines. Confocal microscopy, western blot and flow cytometry methods were employed to determine the mechanism of cytotoxic and cytostatic action of the combinations. The study revealed that additive or synergistic interactions were observed between 5-fluorouracil and both isothiocyanates, which enhanced the anticancer activity of 5-fluorouracil, particularly in colon cancer cell lines. An increased cytostatic effect was observed in case of alyssin while for sulforaphane the synergistic interaction with 5-fluorouracil involved an intensification of apoptotic cell death.

Magnetic polymer microcapsules loaded with Nile Red fluorescent dye.

Fabrication of multifunctional smart vehicles for drug delivery is a fascinating challenge of multidisciplinary research at the crossroads of materials science, physics and biology. We demonstrate a prototypical microcapsule system that is capable of encapsulating hydrophobic molecules and at the same time reveals magnetic properties. The microcapsules are prepared using a templated synthesis approach where the molecules to be encapsulated (Nile Red) are present in the organic droplets that are suspended in the polymerization solution which also contains magnetic nanoparticles. The polymer (polypyrrole) grows on the surface of organic droplets encapsulating the fluorescent dye in the core of the formed microcapsule which incorporates the nanoparticles into its wall. For characterization of the resulting structures a range of complementary physicochemical methodology is used including optical and electron microscopy, magnetometry, 1H NMR and spectroscopy in the visible and X-ray spectral ranges. Moreover, the microcapsules have been examined in biological environment in in vitro and in vivo studies.

Autophagic cell death and premature senescence: New mechanism of 5-fluorouracil and sulforaphane synergistic anticancer effect in MDA-MB-231 triple negative breast cancer cell line.

In view of the need for new, more effective therapies for the triple negative breast cancer treatment, the aim of the study was to evaluate the anticancer activity and mechanism of action of the sulforaphane and 5-fluorouracil combination in the triple negative breast cancer cell line MDA-MB-231. Changes in the number of live cells after alone and sequential treatment were determined by the MTT test. The Chou and Talaly method was used to identify the type of interaction. Confocal microscopy, flow cytometry, western blot and spectrophotometry were used to examine apoptosis, autophagy and premature senescence. The western blot method was applied to measure the level of enzymes that are crucial for the 5-fluorouracil activity. Sulforaphane and 5-fluorouracil have been shown to interact synergistically in the breast cancerMDA-MB-231 cell line, resulting in a significant reduction of the number of live cells compared to alone treatments. Sulforaphane has decreased the level of thymidylate synthetase, which was also observed in the case of the sequential sulforaphane and 5-fluorouracil treatment. Studies of the interaction mechanism have revealed that sulforaphane and 5-fluorouracil act synergistically in the MDA-MB-231 cells by inducing autophagic cell death and premature senescence.

Sulforaphane Regulates NFE2L2/Nrf2-Dependent Xenobiotic Metabolism Phase II and Phase III Enzymes Differently in Human Colorectal Cancer and Untransformed Epithelial Colon Cells.

Sulforaphane (SFN), a naturally occurring chemopreventive and anticancer agent, is a nuclear factor, erythroid 2-like 2 (NFE2L2/Nrf2) inducer. Nrf2 plays a critical role in coordinating the cell defense system by initiating the transcription of cytoprotective genes, including detoxification enzymes such as NAD(P)H quinone dehydrogenase 1 (NQO1) and transport proteins such as ATP-binding cassette, subfamily C (CFTR/MRP). Recently, the essential role of Nrf2 in tumor development and progression and in the development of multidrug resistance in cancer cells has been highlighted. The aim of this study was to compare the effect of SFN on the Nrf2 system and the Nrf2-target enzymes NQO1 and MRP in human untransformed epithelial colon CRL-1790 cells and in HT-29 and Caco-2 colorectal cancer cells to elucidate the role of SFN in cancer prevention and treatment. We have demonstrated that SFN has excellent cytoprotective properties in CRL-1790 cells, as it induced Nrf2-dependent expression of MRP1 and NQO1. SFN induced Nrf2 target enzyme activity in HT-29 and Caco-2 cancer cells but regulated the Nrf2/ARE signaling pathway differently in cancer and untransformed cells.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely.

The Comparison of MTT and CVS Assays for the Assessment of Anticancer Agent Interactions.

Multiple in vitro tests are widely applied to assess the anticancer activity of new compounds, including their combinations and interactions with other drugs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most commonly used assays to assess the efficacy and interactions of anticancer agents. However, it can be significantly influenced by compounds that modify cell metabolism and reaction conditions. Therefore, several assays are sometimes used to screen for potential anticancer drugs. However, the majority of drug interactions are evaluated only with this single method. The aim of our studies was to verify whether the choice of an assay has an impact on determining the type of interaction and to identify the source of discrepancies. We compared the accuracy of MTT and CVS (crystal violet staining) assays in the interaction of two compounds characterized by similar anticancer activity: isothiocyanates (ITCs) and Selol. Confocal microscopy studies were carried out to assess the influence of these compounds on the reactive oxygen species (ROS) level, mitochondrial membrane potential, dead-to-live cell ratio and MTT-tetrazolium salt reduction rate. The MTT assay was less reliable than CVS. The MTT test of Selol and 2-oxoheptyl ITC, which affected the ROS level and MTT reduction rate, gave false negative (2-oxoheptyl ITC) or false positive (Selol) results. As a consequence, the MTT assay identified an antagonistic interaction between Selol and ITC, while the metabolism-independent CVS test identified an additive or synergistic interaction. In this paper, we show for the first time that the test assay may change the interpretation of the compound interaction. Therefore, the test method should be chosen with caution, considering the mechanism of action of the compound.

New enantiomeric fluorine-containing derivatives of sulforaphane: synthesis, absolute configurations and biological activity.

Three pairs of enantiomers of the unknown sulforaphane analogs bearing organofluorine substituents bonded to the sulfinyl sulfur atom and having different number of methylene groups in the central carbon chain were synthesized and fully characterized, including determination of their absolute configurations. All the new compounds were tested in vitro for their cytotoxicity against melanoma cells to show increased activity in comparison with the natural sulforaphane. The influence of the particular structural changes in the molecule on the cytotoxicity is discussed.

Interactions between drugs and sulforaphane modulate the drug metabolism enzymatic system.

BACKGROUND: Sulforaphane (SFN) is a potent chemopreventive agent, which is widely consumed in diet or as a diet supplement. It modulates the enzymes of II and III metabolism phase. In this paper, the influence of SFN and three commonly consumed drugs: furosemide, verapamil and ketoprofen on II and III metabolisms phase enzymes was studied. We have also investigated if the interactions between SFN and the drugs occur resulting in enzymatic system disturbances. METHODS: The Caco-2 cells were incubated with SFN and drugs separately or in a mixture simultaneously or subsequently. The impact of the compounds on the cell viability and NADPH:quinine reductase (QR) activity was determined. The expression of glutathione-S-transferase (GST) isoenzymes GSTA3, GSTM1, P-glycoprotein (PgP) and multidrug resistance protein 1 (MRP1) genes was measured by qPCR method. Since these enzymes are regulated by Nrf2 pathway, the localization of Nrf2 (Nuclear erythroid 2-related factor) after exposure to the mixtures of SFN and the drugs was evaluated by confocal microscopy. RESULTS: SFN was shown to interact antagonistically with the studied drugs. At most cases an increase in enzymatic activity and expression was observed. The most significant changes were observed in case of enzymes regulated by Nrf2: QR, GSTA1 and GSTA3 and also MRP1. PgP was shown to be not altered by the studied compounds. COCNCLUSION: The interaction between SFN and furosemide, verapamil and ketoprofen modify the activity of enzymatic system involved in drug metabolism and transport. This may lead to drug effectiveness alteration and also to multidrug resistance (MDR) development.

Cytotoxicity of PP(Arg)2- and PP(Ala)2(Arg)2-based photodynamic therapy and early stage of apoptosis induction in human breast cancers in vitro.

Mitochondria are cell energetic centers where ATP is produced. They also play a very important role in the PDT as intracellular sites of photosensitizer localization. Photosensitizers gathering in mitochondria (like porphyrin derivatives used in this work) are more effective in tumor cell destruction. Moreover, it was assumed that di-amino acid substituents attached to porphyrin ring will strengthen the effectivity of interaction with membrane receptors of examined cells. MTT assay was performed to investigate the influence of PP(Arg)(2) and PP(Ala)(2)(Arg)(2)-based PDT on breast cancer cell viability for 24 h, 48 h and 120 h after cell irradiation. Then the influence of PP(Ala)(2)(Arg)(2)- and PP(Arg)(2)-mediated PDT on early mitochondrial apoptosis induction via measurements of the transmembrane mitochondrial potential changes was examined. Results showed that lower energy doses and maximal nontoxic photosensitizer doses of PP(Ala)(2)(Arg)(2) and PP(Arg)(2) applied in PDT can imply apoptotic cell death. It was confirmed that modification of the protoporphyrin IX by attaching two alanine substituents raised the efficiency of photodynamic therapy.

Isothiocyanate-drug interactions in the human adenocarcinoma cell line Caco-2.

Isothiocyanates, among which alyssin is counted, are the compounds that have proved chemopreventive properties and the ability to induce the 2 and the 3 detoxification phase by affecting the transcription factor nuclear erythroid 2-related factor (Nrf2). Having a positive effect on the human body, these compounds are used as dietary supplements. Because of the observed increase in the consumption of dietary supplements taken along with the drugs routinely used in medical practice, this study examined the possibility of interactions between alyssin and drugs, which could have an impact on cell metabolism. We have determined the effects of the tested substances and their interactions on the expression and activity of the phase 2 genes, as well as on the drug transport, which could be influenced by affecting the expression of transport proteins that belong to the 3 phase of metabolism. It was also studied whether the transcription factor Nrf2 is responsible for the interactions that occurred. The results showed that the interactions between alyssin and the tested drugs strengthen or weaken the effect of the drugs given separately depending on the concentration of alyssin and the type of drug. Even though Nrf2 is involved in the interaction, it seems that it is not the only factor regulating the interactions between the tested medications.

Combination treatment with 5-fluorouracil and isothiocyanates shows an antagonistic effect in Chinese hamster fibroblast cells line-V79.

The isothiocyanates present in the cruciferous plants were proved to have the antiproliferative and cytotoxic effect on cancer cell lines. Natural compounds in combination with chemotherapy agents enhance anticancer activities of drugs and reduce their toxicity. The aim of the presented study was to determine an effect of isothiocyanates and 5-fluorouracil used alone or in combination (in sequential or co-administrative treatments) on normal cell lines-V79. There were compared abilities of three isothiocyanates to interact with 5-fluorouracil. There was also investigated the mechanism of interaction and influence of isothiocyanates on 5-fluorouracil. The cell survival was evaluated with MTT assay. Combination effects between isothiocyanates and 5-fluorouracil were estimated in the way described by Chou and Talalay. The cycle progression and the living cells number were determined with flow cytometry. The type of cell death was detected with a confocal microscope. There was observed an antagonistic effect which was mainly dependent on the cell cycle distribution e.g., sulforaphane increased the cell number in the G2/M phase, whereas 5-fluorouracil and combination of these two compounds increased the cell number in the S phase. If each compound blocked the S phase of the cell cycle, their combination increased the cell number in the S phase, but the increase was not statistically significant when compared with single substance treatments. The highest antagonistic effect in normal cells was obtained for co-administrated 5-fluorouracil and 2-oxoheptyl isothiocyanate at the fraction affected at 0.5 and 0.75. Isothiocyanates did not affect 5-fluorouracil cytotoxicity in normal cell lines-V79.

Selenitetriglicerydes affect CYP1A1 and QR activity by involvement of reactive oxygen species and Nrf2 transcription factor.

Selenitetriglycerides are a group of compounds that contain selenium (Se) (IV). In this paper, we present the results of examinations of three structurally-related selenitetriglicerydes that contain various Se concentrations: 2%, 5% and 7% Selol. The present study concentrates on the effect of Selol on phase 1 and 2 enzyme activity and the implications of free radicals and the nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway in the activity of this compound. The cytotoxic and cytostatic activities of the three kinds of Selol were evaluated; however, the cytotoxic effect was observed only for 7% Selol. Our results show that 2% Selol acts as a monofunctional inducer of phase 2 enzyme activity, and the induction is mediated by the Nrf2 transcription factor. Selol 7% acts in an opposite manner and induces phase 1 with simultaneous inhibition of phase 2 enzyme activity. The differential effect can be associated with the increase in Se content, leading to a change in the structure of the compound.

The effect of isothiocyanates on CYP1A1 and CYP1A2 activities induced by polycyclic aromatic hydrocarbons in Mcf7 cells.

Polycyclic aromatic hydrocarbons (PAHs)--environmental carcinogens--are metabolized by CYP1A1 and CYP1A2 enzymes to oxy-derivatives, which are able to bind to DNA and initiate carcinogenesis. PAHs induce CYP1A1 and CYP1A2 activity, which increases the risk of development of carcinogenesis. Isothiocyanates (ITCs), naturally occurring in Brassica vegetables, possess chemopreventive properties and are able to reduce the CYP1A enzyme activity. In this paper we report our study of the ability of ITCs: sulforaphane and its analogues: isothiocyanate-2-oxohexyl and alyssin, to inhibit CYP1A1 and CYP1A2 enzyme activity induced by the PAHs, anthracene (ANT) and dibenzo[a,h]anthracene (DBA) in human breast cancer cell line Mcf7. The aim was to determine whether the differences in structure of ITCs change their inhibitory properties, and whether these properties depend on the type of inducer. The results indicate that the properties of ITCs depend on the type of PAH: ITCs are more potent in inhibiting activity induced by the weaker inducer. It was also found that the change in ITCs' structure influences their activities. ITC 2-oxohexyl was the weakest inhibitor, whereas sulforaphane and alyssin exhibited similar potency. The study revealed that inhibition of CYP1A1 activity is direct whereas inhibition of CYP1A2 activity is not only direct but is also caused by the level of protein disturbance.

Sulforaphane and its analogues inhibit CYP1A1 and CYP1A2 activity induced by benzo[a]pyrene.

CYP1A1 and CYP1A2 enzymes metabolize polycyclic aromatic hydrocarbons (PAHs) to the reactive oxyderivatives. PAHs can induce the activity of both enzymes, which increases its conversion and enhances risk of carcinogenesis. Thus, the inhibition of CYP enzymes is recognized as a cancer chemoprevention strategy. A well-known group of chemopreventive agents is isothiocyanates, which occur naturally in Brassica vegetables. In this paper, a naturally occurring sulforaphane and its two synthetic analogues isothiocyanate-2-oxohexyl and alyssin were investigated. The aim of the study was to determine whether the differences in the isothiocyanate structure change its ability to inhibit CYP1A1 and CYP1A2 activity induced by benzo[a]pyrene in HepG2 and Mcf7 cells. Also a mechanistic study was performed including isothiocyanates' influence on CYP1A1 and CYP1A2 catalytic activity, enzymatic protein level, and AhR translocation. It was shown that both enzymes were significantly induced by benzo[a]pyrene, and isothiocyanates were capable of decreasing the induced activity. The inhibitory properties depend on the types of isothiocyanate and enzyme. In general, CYP1A2 was altered in the more meaningful way than CYP1A1 by isothiocyanates. Sulforaphane exhibited weak inhibitory properties, whereas both analogues were capable of inhibiting BaP-induced activity with the similar efficacy. The mechanistic study revealed that analogues decreased the CYP1A2 activity via the protein-level reduction and CYP1A1 directly. The results indicate that isothiocyanates can be considered as potent chemopreventive substances and the change in the sulforaphane structure increases its chemopreventive potency.