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.

Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

Isothiocyanates (ITC) are well-known chemopreventive agents extracted from vegetables. This activity results from the activation of human oxidoreductase. In this letter, the uncompetitive activatory mechanism of ITC was investigated using docking and molecular dynamics simulations. This indicates that NAD(P)H:quinone oxidoreductase can efficiently improve enzyme-substrate recognition within the catalytic site if the ITC activator supports the interaction in the uncompetitive binding site.

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.

NFkappaB activation and drug sensitivity in human neoplastic cells treated with anthracyclines.

NFkappaB (nuclear factor kappaB) is a transcription factor controlling, among others, cell proliferation and apoptosis. The potent activators of NFkappaB are anthracyclines which can activate apoptotic processes. As shown by some authors, NFkappaB activated by these drugs well correlated with their cytotoxic activity. The aim of this study was to assess the effects of doxorubicin (DOX) and its analogs (annamycin, WP903) on the NFkappaB activity in human melanoma cells: a sensitive (ME18) and a resistant to DOX (ME18/R) and its possible correlation with cell sensitivity to these drugs. In the studies, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, ELISA test and confocal microscopy were used. As was shown, DOX, 1.7; 8.6 microM, strongly induced NFkappaB in ME18 cells. Annamycin (ANN), 0.3; 3.0 microM and WP903, 3.0 microM induced NFkappaB in ME18/R cells. PDTC (pyrrolidine dithiocarbamate)--NFkappaB inhibitor made ME18/R cells more sensitive to ANN and WP903 but did not affect cytotoxicity of DOX in ME18 cells. These results suggest that the influence of NFkappaB activation on cytotoxicity of anthracyclines is highly drug- and cell-specific.

The activity of Selol in multidrug-resistant and sensitive human leukemia cells.

Selol is a mixture of selenitetriglycerides synthesized from sunflower oil. As it contains the element selenium in its structure, it is suspected to exhibit chemopreventive and anticancer activity. In this study, the ability of Selol to inhibit cell proliferation and to induce apoptosis was investigated. Three cell lines were used: leukemia HL-60 cell line and multidrug-resistant HL-60/Dox (resistant to doxorubicin) and HL-60/Vinc (resistant to vincristine). Selol was shown to reduce the cell number as a result of treatment with increasing concentrations. For selected concentrations the evidence of apoptosis (changes in mitochondrial potential and caspase activity) was investigated, as well as changes in lysosome distribution. The study has shown that Selol overcame the cell resistance, as doxorubicin-resistant cells were more sensitive towards Selol than sensitive cells.

Differential response of human healthy lymphoblastoid and CCRF-SB leukemia cells to sulforaphane and its two analogues: 2-oxohexyl isothiocyanate and alyssin.

The chemopreventive effect of sulforaphane and two of its analogues on human B-lymphocytes derived cells was evaluated in this study. Two cell lines used in the experiments were: human lymphoblastoid cells and human B-leukemia CCRF-SB. Both cell lines were treated with three structurally related isothiocyanates: sulforaphane, 2-oxohexyl isothiocyanate and alyssin. The viability of cells, induction of a phase II enzyme-quinone reductase, apoptosis induction, GSH content and ROS formation were evaluated. The results indicate the differences between the chemopreventive properties and apoptosis-inducing activity of three isothiocyanates. The significant differences in response to these compounds were observed between healthy lymphoblastoid and leukemia CCRF-SB cells.

Mitochondrial localization of P2Y1, P2Y2 and P2Y12 receptors in rat astrocytes and glioma C6 cells.

We have previously shown that P2Y1, P2Y2 and P2Y12 nucleotide receptors are functionally expressed and active on the cell surface of rat glioma C6 cells. In the present study, we have immunocytochemically shown their sub-cellular colocalization with mitochondria in these cells. The same colocalization of above receptors has been found in rat astrocytes. Additionally, differences in intracellular distribution of examined receptors between both cell lines have been observed. This data indicates that P2Y1, P2Y2 and P2Y12 receptor proteins exist within mitochondria of astrocytes and C6 cells, although their role in these sub-cellular structures remains unclear.

Constitutive activation of transcription factor NFkappaB as a possible marker of sensitivity of neoplastic cells to anthracyclines.

NFkappaB, a member of the Rel family of transcription factors has been found to be critically important for control of cell proliferation and apoptosis. Although rare, there are systems in which NFkappaB has occurred as pro-apoptotic factor. The potent activators of NFkappaB are anthracycline anticancer drugs which induce the events of apoptosis. These results could point to the pro-apoptotic role of NFkappaB. Recent studies have shown that activation of that transcription factor well correlated with cytotoxic activity of anthracyclines. Potential mechanism through which NFkappaB could play a role in tumorigenesis involves its constitutive activation which was shown in a wide variety of tumour types. The aim of this study was to define the level of activity of NFkappaB in the native human tumour cells differing in sensitivity to anthracycline analogs to study the mechanism responsible for cytotoxic action of these drugs. Constitutive activation of NFkappaB was determined with use of ELISA test and confocal microscopy. As was shown, both methods have occurred as quite comparable. Constitutive activation of NFkappaB was observed in all neoplastic cells tested independently on sensitivity to anthracyclines. However, these results do not exclude the supportive role of NFkappaB in apoptotic activity of these drugs. It is necessary to study the influence of the compounds tested here on NFkappaB activation and on the induction and intensity of apoptotic processes.

Inhibition of cell cycle and induction of apoptosis by sulforaphane in cell lines carrying various inherited BRCA1 mutations.

The important aspect of sulforaphane (SFN) chemopreventive activity is its ability to induce cell growth inhibition and apoptosis. In this study, the effect of SFN on lymphoblastoid cells derived from people carrying four different germ-line mutations in BRCA1 gene was tested and compared to the effect of SFN on wild-type cells. The mutations studied were C61G, 3819del5, 4153delA and 5382INSC. Changes in cell viability and density after SFN treatment were evaluated, as well as cell cycle progression, changes in mitochondrial membrane potential, and phosphatidylserine externalization. SFN was shown to reduce cell viability and density in all cell lines tested. Cell cycle block in S-phase and the occurence of simultaneous apoptosis were observed. The concentration of SFN needed to elicit a comparable effect on each cell line was varied. We found that the effect of SFN on cells carrying different inherited mutations depended on mutation type.

Polycyclic aromatic hydrocarbons: physicochemical properties, environmental appearance and impact on living organisms.

Human exposure to environmental pollution is of great interest nowadays. Many substances present in the environment are considered as carcinogenic to humans. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. In this paper, PAHs are described: their physicochemical properties, natural and anthropogenic sources, environmental, dietary and occupational exposure and impact on human health. Metabolical carcinogenic activation of PAHs and the role of metabolism products as biomarkers of PAH exposure have been discussed.

Sulforaphane-mediated induction of a phase 2 detoxifying enzyme NAD(P)H:quinone reductase and apoptosis in human lymphoblastoid cells.

The effect of sulforaphane on human lymphoblastoid cells originating from a patient of a high cancer risk was studied. Sulforaphane (SFN) is a naturally occurring substance of chemopreventive activity. In our study, changes in cell growth, induction of apoptosis and phase 2 enzymes as well as glutathione level were examined. Apoptosis was tested by confocal microscopy at three stages: change in mitochondrial membrane potential, caspase activation and phosphatidylserine externalization. We show that SFN increases the activity of the detoxification system: it increases quinone reductase activity at low concentration (0.5-1 microM) and raises glutathione level in a dose-dependent manner. At higher doses (2.5-10 microM) sulforaphane is a cell growth modulator, as it caused cell growth cessation (IC50 = 3.875 microM), and apoptosis inducer. The results obtained suggest that sulforaphane acts as a chemopreventive agent in human lymphoblastoid cells.

Application of sulforaphane--does it lead to improvement of islet graft survival after warm and/or cold ischemia.

OBJECTIVES: Brain stem death results in ischemic damage of organs. To prevent many agents are being tested this damage. The aim of our study was to determine effects of sulforaphane (SF) on recovery, viability, lipid peroxidation, metabolic and endocrine function of islets isolated from rat pancreata and treated with warm and/or cold ischemia and then transplanted syngeneically. METHODS: Rat pancreata were recovered from non-heart beating rats after intraducatal injection of collagenase solution after 15 or 30 minutes of warm ischemia time (WT). Cold ischemia (CT) was obtained by storage of distended and harvested glands in tubes with UW solution in 4 degrees C for 120 minutes. Sulforaphane was administered 24 hours before isolation islets in concentration 24mg/kg b.w. Diabetes was achieved by intravenouse injection of streptozotocin (STZ 65mg/kg b.w.). Islets were transplanted into the liver through the portal vein. Experimental protocol included four groups: Group I: fresh pancreata not treated with SF, WT=0, CT=0; Group II: 15 or 30 min. of WT, with or without SF; Group III: 15 or 30 min. of WT and 120 min. of CT, with or without SF; Group IV: 120 min. of CT, with or without SF RESULTS: Stimulation index in all groups with sulforaphane was larger from 1.0 (normal response of islets on high concentration glucose in medium). Metabolic activity (MTT) in group II (WT15, SF), gr. IV (CT, SF) and in gr. III (WT15, CT, SF; p>0.05) was lower compared to control group (I). The concentration of MDA in groups with SF increased as compared to controls. The highest recovery and cell viability was observed in group IV (CT, SF) and in gr. III (WT15, CT, SF; p<0.05). In groups exposed to 30 min. of warm ischemia and/or 120 min. of cold preservation was observed higher % of dead islets cells. In vivo study shows that islets graft isolated from rat pancreata treated with sulforaphane reverse diabetes. CONCLUSIONS: Based on results we can conclude that SF in concentration 24mg/kg b.w. reveals protective effect on preserved pancreas and may have a potential clinical implication to improve hemodynamically unstable pancreas donor condition.

Sulforaphane and 2-oxohexyl isothiocyanate induce cell growth arrest and apoptosis in L-1210 leukemia and ME-18 melanoma cells.

Flow cytometry and laser-scanning confocal microscopy were used to study the effect of sulforaphane (SFN) and 2-oxohexyl isothiocyanate on the growth and viability of mouse leukemia L-1210 and human melanoma ME-18 cells during their exponential growth. Sulforaphane belongs to a group of compounds known as isothiocyanates. Isothiocyanates mainly occur in Cruciferous family. In particular, they occur in many vegetables such as broccoli and their sprouts. SFN and 2-oxohexyl isothiocyanate are potent inducers of detoxication phase 2 enzymes in mouse tissues and murine hepatoma cells in culture. Sulforaphane was shown to induce cell growth arrest in a dose dependent manner, followed by cell death. Sulforaphane induced the cell death via an apoptotic process. Two markers of apoptosis were investigated: phosphatidylserine externalization, which occurs in the early stages of apoptosis, and DNA strand breaks. Our results strongly suggest of chemopreventive activity toward cancer by the induction of apoptosis by SFN and 2-oxohexyl isothiocyanate.

Chemoprevention of cancerogenesis--the role of sulforaphane.

Isothiocyanates are a group of naturally occurring compounds with interesting medical properties, such as antimicrobial, antioxidant and antitumor activities. In our work we were trying to present the tumoricidal activity of new synthesized derivatives of one isothiocyanate: 1-isothio-cyanato-(4R)-(methylsulfinyl) butane [sulforaphane]. Many chemical substances derived from plants, undoubtedly have protective properties. The effectiveness of sulforaphane is based on induction of hepatic detoxifying enzymes.