Giovanna Bartolini

Cytotoxic and antioxidant activity of 4-methylthio-3-butenyl isothiocyanate from Raphanus sativus L. (Kaiware Daikon) sprouts.

There is high current interest in the chemopreventive potential of Brassica vegetables (cruciferae), particularly due to their content in glucosinolates (GL), which upon myrosinase hydrolysis release the corresponding isythiocyanates (ITC). Some ITCs, such as sulforaphane (SFN) from broccoli ( Brassica oleacea italica), have been found to possess anticancer activity through induction of apoptosis in selected cell lines, as well as indirect antioxidant activity through induction of phase II detoxifying enzymes. Japanese daikon ( Raphanus sativus L.) is possibly the vegetable with the highest per capita consumption within the Brassicaceae family. Thanks to a recently improved gram scale production process, it was possible to prepare sufficient amounts of the GL glucoraphasatin (GRH) as well as the corresponding ITC 4-methylthio-3-butenyl isothiocyanate (GRH-ITC) from its sprouts. This paper reports a study on the cytotoxic and apoptotic activities of GRH-ITC compared with the oxidized counterpart 4-methylsulfinyl-3-butenyl isothiocyanate (GRE-ITC) on three human colon carcinoma cell lines (LoVo, HCT-116, and HT-29) together with a detailed kinetic investigation of the direct antioxidant/radical scavenging ability of GRH and GRH-ITC. Both GRH-ITC and GRE-ITC reduced cell proliferation in a dose-dependent manner and induced apoptosis in the three cancer cell lines. The compounds significantly ( p < 0.05) increased Bax and decreased Bcl2 protein expression, as well as producing caspase-9 and PARP-1 cleavage after 3 days of exposure in the three cancer cell lines. GRH-ITC treatment was shown to have no toxicity with regard to normal human lymphocytes (-15 +/- 5%) in comparison with SFN (complete growth inhibition). GRH and GRH-ITC were able to quench the 2,2-diphenyl-1-picrylhydrazyl radical, with second-order rate constants of 14.0 +/- 2.8 and 43.1 +/- 9.5 M(-1) s(-1), respectively (at 298 K in methanol), whereas the corresponding value measured here for the reference antioxidant alpha-tocopherol was 425 +/- 40 M (-1) s (-1). GRH reacted with H2O2 and tert-butyl hydroperoxide in water (pH 7.4) at 37 degrees C, with rate constants of 1.9 +/- 0.3 x 10(-2) and 9.5 +/- 0.3 x 10(-4) M(-1) s (-1) (paralleling recently developed synthetic antioxidants) being quantitatively (>97%) converted to GRE. It is demonstrated that GRH-ITC has interesting antioxidant/radical scavenging properties, associated with a selective cytotoxic/apoptotic activity toward three human colon carcinoma cell lines, and very limited toxicity on normal human T-lymphocytes.

(-)-Epigallocatechin-3-gallate downregulates Pg-P and BCRP in a tamoxifen resistant MCF-7 cell line.

We investigated the anticancer effect of EGCG treatment on a breast carcinoma cell line resistant to tamoxifen (MCF-7Tam cells). As there are no reports about the molecular mechanisms implicated in EGCG treatment of tamoxifen resistant breast carcinoma cells, we studied the effects of EGCG treatment on three plasma membrane proteins that are involved in the mechanism of drug-resistance: Multidrug Resistance Protein (MRP1), P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). EGCG treatment (10-100 microg/ml for 24-72 hours) caused cell growth inhibition and dose-dependent apoptosis: after 100 microg/ml EGCG treatment for 24 hours, Bax expression increased and Bcl2 expression decreased (p<0.05). Coherently, Annexin V-FITC apoptosis assay detected a significant increase in labelled cells (p<0.05). EGCG did not affect MRP1: in contrast, 100 microg/ml EGCG administration caused P-gp decrease to 53% of control cells (p<0.001) and this effect was not due to downregulation of P-gp gene expression. EGCG induced P-gp decrease even when MG132, a strong proteasome inhibitor, was given together with EGCG to MCF-7Tam cells. EGCG treatment also inhibited BCRP activity: mRNA transcription and protein level did not change after treatment, but mitoxantrone test demonstrated a strong inhibition of BCRP activity (p<0.001). In conclusion, the present results showed that EGCG could down-regulate the activity of two molecules that play a key role in drug metabolism and transport and that are highly expressed in tamoxifen resistant breast carcinoma cells. The interaction of EGCG and drugs used in the therapy of estrogen sensitive breast carcinoma ought to be subject of studies and the potential use of EGCG in drug-resistant diseases ought to be better considered.

Prostaglandin and thromboxane biosynthesis in isolated platelet-free human monocytes: I. A modified procedure for the characterization of the prostaglandin spectrum produced by resting and activated monocytes

We have developed a technique to isolate monocytes from human peripheral blood. The technique takes special care of completely eliminating platelets which are usually present in other preparations. Monocytes obtained in good yields (2.5-5.0 X 10(5) cells/ml blood), were found to be 70-80% pure on the basis of morphological and histochemical criteria. Contamination was largely due to the presence of lymphocytes. Monocytes were incubated in the presence or absence of arachidonic acid and TXB2, PGE2 and 6-keto-PGF1 alpha were measured by specific and sensitive radio-immunoassays. It was found that when cells were incubated for up to 1 hr, the production of PGs was low or absent even in the presence of 10 microM arachidonic acid in the incubation medium. However, when incubations were carried out for 24 hrs in the presence of at least 1% fetal calf serum a dramatic increase in TXB2 production occurred, with levels as high as 150 ng X 10(6) cells. The ratio TXB2/PGE2 was around 3, while 6-keto-PGF1 alpha was produced at a much lower level. In the same conditions, when care was taken to evaluate PGs already present in fetal serum and/or cross reactivity due to media generally employed, purified human lymphocytes appeared unable to produce detectable levels of the three PGs tested.

Prostaglandin and thromboxane biosynthesis in isolated platelet-free human monocytes. III: The induction of cycloxygenase by colony stimulating factor. 1

Previous observations showing the presence in the serum of a component capable of regulating prostanoid biosynthesis in human cultured monocytes, have led us to suspect its presence in human platelets. We have purified this serum monocytotropic factor (SMF) and have shown its identity with a component of platelet membranes. Surprisingly its structure appeared to be very similar to that of a polypeptide growth factor never before identified in platelets: the colony stimulating factor-1 (CSF-1 or M-CSF). Here we show that SMF and CSF-1 have very similar biological properties. Thus, CSF-1 when released from human platelets is capable of triggering the differentiation pathway leading from blood monocytes to resident macrophages. It is likely that cycloxygenase induction plays a pivotal role in these events.

Biosynthesis of prostaglandins in parenchymal and nonparenchymal rat liver cells

Rat hepatocyte suspensions were separated into parenchymal and nonparenchymal (endothelial-rich) cells and the conversion of arachidonate into prostaglandin E was studied. Most of the intracellular labeled arachidonate was incorporated into phospholipids. Fractionation of parenchymal cell phospholipids by thin-layer chromatography showed the highest specific activity in the phosphatidylserine plus phosphatidylinositol zone followed by phosphatidylcholine and phosphatidylethanolamine; sphingomyelin was not labeled. The conversion of arachidonate into prostaglandins was 1% and 4.3% in parenchymal and nonparenchymal cells respectively. Nonparenchymal cells synthesized 5-fold more prostaglandin E-like material than parenchymal cells. Quantitation of prostaglandin E biosynthesis by radioimmunoassay revealed that in the presence of cold arachidonate sinusoidal cells synthesized 1200 pg of immunogenic prostaglandin E per mg protein, in parenchymal cells the figure was 175 pg per mg protein. Parenchymal cells had little effect on platelet aggregation, whereas nonparenchymal cells inhibited aggregation in a dose-dependent fashion. This suggests that sinusoidal liver cells synthesize a prostacyclin-like compound in addition to prostaglandin E2.

Regulation of thromboxane A2 biosynthesis in platelet-free human monocytes and the possible role of polypeptide growth factor(s) in the induction of cyclooxygenase system.

It has previously been shown that platelet-free human monocytes, when properly incubated in the presence of animal and human sera, became capable of producing large amounts of thromboxane A2 and prostaglandin E2. The characteristics of these processes are reported here. Prostaglandin biosynthesis was time and cell concentration dependent; 24 h of incubation at 37 degrees C and 0.5 X 10(6) cells per ml medium were found to give the most reproducible results. Human monocytes produced thromboxane A2 and prostaglandin E2 in a typical ratio which ranged from 2.0 to 5.0 (28 experiments). Animal and human sera were similarly effective, while serum obtained from platelet-free blood was much less active. The activity of all sera tested was stable to heating (100 degrees C for 2-10 min) and extreme pH values (pH 2 and 11). It was unstable when the serum was heated at pH 11 and after 2-mercaptoethanol treatment. These observations prompted us to check the effect of polypeptide growth factors having properties similar to those reported above, such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor as well as insulin and transferrin. None of these, alone or in various combinations, was capable of eliciting a stimulation comparable with that of serum. Stimulation due to sera was, as expected, dose dependently inhibited by acetylsalicylic acid and more efficiently by indomethacin; unexpectedly it was also inhibited by protein synthesis inhibitors such as actinomycin D and cycloheximide in conditions under which no toxic effect of the drugs was evident. On the basis of these results we conclude that: (a) polypeptide growth factor(s) with a molecular weight at least 30 000 (as judged by Amicon ultrafiltration) is involved in the regulation of prostaglandin biosynthesis); (b) such a factor(s) acts by inducing rather than by activating the cyclooxygenase system.

Simple and Dendritic Cyclam Derivatives. Photophysical Properties, Effect of Protonation and Zn2+ Coordination, Preliminary Screening as Inhibitors of Tumour Cell Growth

We have synthesized two novel dendrimers (BG1 and BG2) consisting of a 1,4,8,11-tetraazacyclotetradecane (cyclam, 1) core with appended four dimethoxybenzene and eight benzyl units (BG1) and twelve dimethoxybenzene and sixteen benzyl units (BG2). The absorption and luminescence spectra of these compounds and the changes taking place upon protonation and Zn2+ coordination of their cyclam core have been investigated in acetonitrile-dichloromethane 1:1 v/v solution. For comparison purposes, the absorption and luminescence spectra of 1,4,8,11-tetrabenzyl-cyclam (2), and dendrons BD1 and BD2, model compounds of the branches of BG1 and BG2 respectively, have also been studied. BD1, BD2, BG1, and BG2 exhibit the absorption and emission spectra of their 1,3-dimethoxybenzene unit, but in the two dendrimers the emission intensity is quenched by the cyclam amine groups and increases upon protonation and metal coordination. In order to test if these cyclam derivatives have an antitumour effect, we have studied their action on proliferation in the human neuroblastoma TS12 cell line. Screening experiments have shown that cell proliferation was (i) strongly reduced by the tetrabenzyl substituted cyclam 2, and (ii) unaffected by cyclam and the benzo dendrimers BG1 and BG2. Antitumour screening experiments have also been performed on the tetranaphthyl substituted cyclam 3 and the naphtho-dendrimer NG2, whose photophysical properties have been previously studied. Cell proliferation came out to be moderately reduced by 3, whereas dendrimer NG2 had no effect, similar to dendrimers BG1 and BG2.

Prostanoids as second messengers of polypeptide growth factors

Prostaglandin H synthase (PGHs), also known as cyclooxygenase, is an unstable enzyme whose mRNA has an half life of 10 minutes. Some polypeptide factors have been reported to induce the enzyme in target cells. We have purified and characterized a component of animal sera which behaves as a potent inducer of human monocyte PGHs. This factor. called serum monocytotropic factor, has been identified in human platelets and it appcars to be structurally and biochemically different from identified platelet factors, such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), while showing strong similarities to colony stimulating factor 1 (CSF-1), so far undetected in platclets. Morcover, we have shown, by immunoblot analysis, that CSF-1 behaves as a potent and specific induccr of monocyte PGHs.The hypothesis that prostanoids may be considered as second messengers of platelet CSF-1 like factor, as well as of other growth factors and that PGHs induction plays a pivotal role in this process, will be illustrated.

Immune dysfunction in primary biliary cirrhosis. II. Increased production of prostaglandin E

In a previous study we observed that after in vitro treatment with indomethacin, lymphocyte response to phytohaemagglutinin (PHA) in primary biliar) cirrhosis (PBC) patients was higher than that of controls. We know that indomethacin also inhibits prostanoid production, and thus in the present work we directly measured prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production by mononuclear cells and monocytes from 12PBC patients. 11 control subjects, and three control disease patients (alcoholic cirrhosis. AC). PHA‐stimulated enriched monocytes from PBC patients produced approximately threefold more PGE2 (after 48 h of culture) than did normal and AC monocytes (P<0.05). TXB2 production was similar in all groups studied. We also made cultures in which PBC‐purified lymphocytes proliferated better than PBC mononuclear cells (i.e. lymphocytes plus monocytes). Thus, a monocyte population producing PGE2 could be responsible, at least in part, for the hyporesponsiveness to PHA observed in PBC patients.

Thromboxane A2 synthase activity in platelet free human monocytes

Soon after platelets, the highest amounts of thromboxane A2 (TXA2) can be detected in human monocytes activated by serum. Using platelet-free human monocytes, we have shown that foetal calf serum (FCS) induces prostaglandin H synthase (PGH synthase) after 16 h of incubation, as shown by the use of transcriptional inhibitors and Western blotting. The effect of serum can be in part mimicked by recombinant colony stimulating factor-1 (hr CSF-1). It is not known whether the limiting step leading from arachidonate to TXA2 is represented solely by the level of PGH synthase or also by the level of TXA2 synthase. We approached this problem by using a Western blot specific for the enzyme, as well as by using PGH2 as substrate. The results show that TXA2 synthase is constitutively expressed in monocytes, i.e., its levels were high soon after their isolation, and similar to those observed after 24 h of incubation with serum. However TXA2 failed to be synthesized until at least 3 h of incubation, and the pattern of synthesis was dependent on the kinetics of PGH synthase induction. In any condition in which TXA2 synthase was immunodetectable, using PGH2 as substrate a high rate of conversion to TXB2 could be detected. Experiments with actinomycin D and cycloheximide indicate that the half-life of TXA2 synthase was longer than 16 h, therefore much longer than that of PGH synthase, that the gene coding for it is fully active in resting monocytes, and that the conversion of arachidonate to TXA2 induced by serum or CSF-1 is dependent solely on the de novo synthesis of PGH synthase.