Hydroalcoholic extract of Buxus sempervirens shows antiproliferative effect on melanoma, colorectal carcinoma and prostate cancer cells by affecting the autophagic flow.

Buxus sempervirens (European Box, Buxaceae, boxwood) has been used in folk medicine to treat rheumatism, arthritis, fever, malaria and skin ulceration while, in recent years, interest has grown on possible employment of boxwood extracts in cancer therapy. We studied the effect of hydroalcoholic extract from dried leaves of Buxus sempervirens (BSHE) on four human cell lines (BMel melanoma cells, HCT116 colorectal carcinoma cells, PC3 prostate cancer cells, and HS27 skin fibroblasts) to ascertain its possible antineoplastic activity. This extract inhibited proliferation of all cell lines in different degree as shown, after 48 h-exposure and MTS assay, by the values of GR50 (normalized growth rate inhibition50) that were 72, 48, 38, and 32 µg/mL for HS27, HCT116, PC3 and BMel cells, respectively. At the above GR50 concentrations, 99% of all studied cells remained vital showing accumulation of acidic vesicles in the cytoplasm, mainly around nuclei, whereas a higher extract concentration (125 µg/mL) was cytotoxic causing, after 48 h-exposure, death of all BMel and HCT116 cells. Immunofluorescence showed microtubule-associated light chain three protein (LC3, a marker for autophagy) to be localized on the above acidic vesicles when cells were treated for 48 h with BSHE (GR50 concentrations). Western blot analysis revealed, in all treated cells, a significant increase (2.2-3.3 times at 24 h) of LC3II, i.e., the phosphatidylethanolamine conjugate of the cytoplasmic form LC3I that is recruited in autophagosome membranes during autophagy. Such increase was accompanied, in all cell lines treated for 24 h or 48 h with BSHE, by a significant increment (2.5-3.4 times at 24 h) of p62, an autophagic cargo protein undergoing degradation during the autophagic process. Therefore, BSHE appeared to promote autophagic flow with its following blockade and consequent accumulation of autophagosome or autolysosomes. The antiproliferative effects of BSHE also involved cell cycle regulators such as p21 (HS27, BMel and HCT116 cells) and cyclin B1 (HCT116, BMel and PC3 cells) whereas, among apoptosis markers, BSHE only decreased (30%-40% at 48 h) the expression of the antiapoptotic protein survivin. It was concluded that BSHE impairs autophagic flow with arrest of proliferation and death in both fibroblasts and cancer cells, being the latter much more sensitive to these effects.

Inhibitory effects of cadmium on peripheral blood mononuclear cell proliferation and cytokine release are reversed by zinc and selenium salts.

Zinc (Zn) and selenium (Se) exert regulatory activities on immune functions, while cadmium (Cd) is an immunotoxic agent. The object of this study was to detect effects of 10(-4), 10(-5), and 10(-6) M Cd sulphate, Zn sulphate, and sodium selenite, and their combinations on human peripheral blood mononuclear cell (PBMC) proliferation and IFN-gamma and TNF-alpha production. Only 10(-5) M Zn sulphate significantly enhanced spontaneous PBMC proliferation, which was unaffected by the other salts. At 10(-4) and 10(-5) M, Cd sulphate exerted a dose-response inhibitory action on phytohemagglutinin- (PHA-) stimulated PBMC proliferation and cytokine release, while 10(-4) M and 10(-5) M Zn sulphate and 10(-5) M sodium selenite induced a stimulatory effect on both proliferation and cytokine release; 10(-4) M sodium selenite enhanced only the PBMC proliferation; at 10(-6) M, none of the salts changed the PHA-stimulated immune activity. Moreover, 10(-4) and 10(-5) M Zn and 10(-5) M Se strongly upregulated IFN-gamma (a Th1 cytokine) release, even in presence of 10(-5) M Cd, and reduced the inhibitory effects of Cd on PBMC proliferation and TNF-alpha release. This study confirms that Zn and Se both strongly enhance cytokine release induced by mitogenic stimulation, showing also that Zn acts with a broader range of concentrations than Se. This suggests that dietary excess of Se may not have beneficial effects.

Cholesterol- and caveolin-rich membrane domains are essential for phospholipase A2-dependent EDHF formation.

OBJECTIVE: Cholesterol-rich membrane domains, which contain the scaffold protein caveolin-1 (Cav-1) (caveolae), represent an important structural element involved in endothelial signal transduction. The present study was designed to investigate the role of these signaling platforms in the generation of endothelial-derived hyperpolarizing factor (EDHF). METHODS: Caveolae were disrupted by cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD 10 mM). MbetaCD-induced modulation of non-nitric oxide-/non-prostanoid-dependent (EDHF)-mediated vasorelaxation was studied in pig coronary arteries. Effects of MbetaCD on endothelial Ca(2+) signaling and phospholipase A(2) (cPLA(2)) activity were determined using fura-2 imaging and measurement of [(3)H]-arachidonate mobilization in cultured pig aortic endothelial cells (PAEC). Cellular localization of caveolin-1 and phospholipase A(2) was investigated by cell fractionation, and interaction of cPLA(2) with caveolin-1 was tested by immunoprecipitation experiments. RESULTS: MbetaCD inhibited EDHF-mediated relaxations of pig coronary arteries induced by bradykinin (100 nM) or ionomycin (300 nM) but not relaxations induced by the NO donor DEA/NO (1 microM). Exposure of arteries to cholesterol-saturated MbetaCD failed to affect EDHF-mediated relaxations. Cholesterol depletion with MbetaCD did not affect bradykinin or ionomycin-induced Ca(2+) signaling in pig aortic endothelial cells, but was associated with enhanced basal and reduced Ca(2+)-dependent release of arachidonic acid (AA). Cell fractionation experiments indicated targeting of cPLA(2) to low density, caveolin-1 rich membranes and immunoprecipitation experiments demonstrated association of phospholipase A(2) with the scaffold protein of caveolae, caveolin-1. Cholesterol depletion with MbetaCD did not disrupt the interaction between cPLA(2) and caveolin-1 but prevented targeting of cPLA(2) to low density membranes. Exogenous supplementation of arachidonic acid after cholesterol depletion partially restored EHDF responses in pig coronary arteries. CONCLUSION: The integrity of caveolin-1-containing membrane microdomains is prerequisite for arachidonic acid recruitment and EDHF signaling in porcine arteries.

Novel hypotensive agents from Verbesina caracasana: structure, synthesis and pharmacology.

The number and the pharmacological activities of drugs featuring a guanidine group is actually amazing. Many synthetic guanidine derivatives have attracted pharmacologists in search of new antihypertensive drugs for their ability to block adrenergic nerve activity through central and/or peripheral mechanisms. As a result, compounds such as guanethidine, guanabenz, guanfacine, and pinacidil have been introduced in antihypertensive drug therapy. A crude methanol extract of the Venezuelan plant Verbesina caracasana Fries (Compositae), intravenously administered to mice, was found to induce biological effects such as erection of hair, initial stimulation and subsequent blockade of breathing. Biologically controlled purification yielded a series of active guanidine derivatives, namely G1-G7, which were extensively studied with the focus on the following items: (1) The structure determination of the active compounds by spectral data and a set of reactions; (2) The confirmation of the structures by a biogenetically oriented synthesis; (3) The study of the pharmacological profiles of the isolated drugs; (4) The synthesis of analogous and homologous products in the effort to shed some light on the structure-activity relationship. The metabolites of V. caracasana were characterized, in anesthetized rats, as hypotensive drugs of high (G2), mild (G1, G7) and low (G3,G5,G6) potency, devoid of consistent actions in heart rate, and provided with moderate stimulatory effects on cardiac inotropism and breathing (at selected non-toxic intravenous doses). Autonomic neurogenic components and/or peripheral adrenergic and cholinergic receptor-related pathways were involved in the cardiovascular effects. Synthetic analogs and homologs of G1 and G5 were all shown to be hypotensive drugs of low-mild potency, not affecting appreciably cardiac inotropism and/or breathing. The pharmacodynamic differences among the studied compounds were likely to depend on their ability to cross the blood-brain barrier, lipophilicity and pharmacokinetics. Since most of the compounds did not induce reflex tachycardia and depression of myocardial contractility as the majority of the antihypertensive drugs, they might be useful in the treatment of arterial hypertension of various genesis.

Different effects of platinum, palladium, and rhodium salts on lymphocyte proliferation and cytokine release.

The effects of graded concentrations of Pt, Pd, and Rh salts on spontaneous and PHA-stimulated peripheral blood mononuclear cell (PBMC) proliferation and IFN-gamma, TNF-alpha, and IL-5 release were the focus of this study. Spontaneous PBMC proliferation was inhibited by all 10(-4) M salts (with the exception of PtCl2), while it was enhanced by 10(-5) M PtCl2 as well as by 10(-5) and 10(-6) M (NH4)2[RhCl6] and RhCl3 (but not by 10(-7) M salts). Pt, Pd, and Rh compounds showed similar effects on PHA-stimulated PBMC proliferation and cytokine release; however, the effects on IFN-gamma release were stronger. Thus, 10(-4) and 10(-5) M (NH4)2[PtCl6] and 10(-4) M (NH4)2[PtCl4] inhibited the PHA-stimulated immune activity; 10(-4) M PtCl2 did not exert activity, while 10(-6) M (NH4)2[PtCl6] and 10(-5) and 10(-6) M (NH4)2[PtCl4] and PtCl2 enhanced PBMC proliferation and/or cytokine release. (NH4)2[PdCl6] showed stronger dose-related inhibitory effects (present also at 10(-7) M concentration) on PHA-stimulated proliferation and cytokine release than (NH4)2[PdCl4], PdCl2, or Rh salts; the inhibitory activity of (NH4)2[RhCl6] was slightly higher than that of RhCl3. In conclusion, this study shows that: (a) the immune capacity of Pt, Pd, and Rh depends on speciation; (b) low concentrations of Pt salts stimulate spontaneous and PHA-stimulated immune responses; (c) the in vitro activity of Pd compounds (which are only inhibitory) is higher than that of Pt and Rh salts. These findings are consistent with the observations that sensitization and allergic contact dermatitis in response to Pd are increased in the general population, although the roles of cross-sensitization to Pd and Ni are difficult to determine.

In vitro effects of platinum compounds on lymphocyte proliferation and cytokine release.

In vitro immune effects of Pt compounds of occupational and/or environmental importance, or those used in cancer treatment were studied. Spontaneous and PHA-stimulated proliferation of peripheral blood mononuclear cells (PBMC) and in vitro release of TNF-alpha, IFN-gamma, and IL-5 were assessed in presence of high and very low concentrations of Pt salts: 10(-4) and 10(-7) M (NH4)2[PtCl6], (NH4)2[PtCl4], PtCl4, PtCl2, Na2PtI6, and cis-diaminedichloroPt (CisPt). Spontaneous and PHA-stimulated PBMC proliferation were both inhibited by 10(-4) M (NH4)2[PtCl6] and (NH4)2[PtCl4], while only PHA-stimulated proliferation was inhibited by 10(-4) M CisPt, without significant effects of the other Pt salts. TNF-alpha release from PBMC was reduced by 10(-4) M (NH4)2[PtCl6] and INF-gamma release was reduced by 10(-4) and 10(-7) M hexa- and tetrachloroplatinate and 10(-4) M Na2PtI6, but not by other Pt salts. IL-5 release (related to the Th2 immune response) was inhibited by 10(-4) M (NH4)2[PtCl6], (NH4)2[PtCl4] and Na2PtI6, but it was enhanced by both 10(-4) and 10(-7) M PtCl4. PtCl2 did not influence the immune effects. The study shows Pt salts have immune effects and their potency is ranked in the following order: (NH4)2[PtCl6] > (NH4)2[PtCl4] > Na2PtI6 and CisPt > PtCl4 > PtCl2. These results indicate that certain Pt salts affect lymphocyte proliferation and cytokine release. The intracellular mechanisms responsible for such effects have not been identified.

Cadmium induces p53-dependent apoptosis in human prostate epithelial cells.

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.

Variability of biological effects of silicas: different degrees of activation of the fifth component of complement by amorphous silicas.

A biogenic and a pyrogenic amorphous silica were incubated in normal human plasma and compared on a per unit surface basis for their ability to split C5 molecules and yield small C5a peptides. Since C5a peptides induce selective chemotactic attraction of polymorphonuclear leukocytes (PMN), measurement of PMN-induced chemotaxis was used as an index of C5 activation. Though to a lesser extent than the crystalline forms, amorphous silicas can promote the cleavage of C5 protein and generation of C5a-like fragment. The biogenic silica, which differs from the pyrogenic variety in particle shape, level of contaminants, and degree of surface hydrophilicity, besides specific surface, induced a greater response. Both silicas activated C5 through a process which seems to involve multiple events similar to those induced by crystalline silica. C5 molecules are adsorbed and hydroxyl radicals are generated through Haber Weiss cycles catalyzed by the redox-active iron present at the particle surface either as trace impurities or chelated from plasma by silanol groups. In turn, these radicals convert native C5 to an oxidized C5-like form C5(H2O2). Finally, C5(H2O2) is cleaved by protease enzymatic action of plasma kallikrein activated by the same silica dusts, yielding a product, C5a(H2O2), having the same functional characteristic as C5a.

Cleavage of the fifth component of human complement and release of a split product with C5a-like activity by crystalline silica through free radical generation and kallikrein activation.

The effects of the same form of crystalline silica variously modified were compared to investigate the mechanisms by which silica activates C5 molecules. After incubation in human plasma, silica generated C5a-type fragments that stimulated polymorphonuclear leukocyte chemotaxis. This activity was totally abolished when plasma, adsorbed with antiserum against C5a or thermally inactivated, was used. Pretreatment of plasma with deferoxamine, 1,3 dimethyl-2-thiourea, or aprotinin markedly inhibited or totally abolished C5 activation. Finally, a significant increase in kallikrein activity was detected after incubation of silica particles in plasma. The results seem to indicate that the activation of C5 by crystalline silica occurs through a complex mechanism: the redox-active iron possibly present at the silica surface catalyzes, via Haber-Weiss cycles, the production of hydroxyl radicals, which in turn convert native C5 to an oxidized C5-like form. This product is then cleaved by kallikrein, activated by the same silica particles, yielding oxidized C5a with the same functional properties as C5a. The different types of the same form of silica exhibited different reactivity. Two separate properties of the dusts seem to contribute to C5 activation: the potential to release hydroxyl radicals and the extent of C5 adsorption at the surface. The degree of surface hydrophobicity/hydrophilicity appeared sufficient to explain the different responses.