Heterosubstituted Derivatives of PtPFPP for O2 Sensing and Cell Analysis: Structure-Activity Relationships.

Biological applications of phosphorescent probes for sensing molecular oxygen (O2) and bioimaging have gained popularity, but their choice is rather limited. We describe a family of new heterosubstituted phosphorescent bioprobes based on the Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye. The probes are produced by simple click modification of its para-fluorine atoms with thiols, such as 1/2-thio-glucose, thio-poly(ethylene glycol) (PEG), or cysteamine. The probes were designed to have one cell-targeting moiety and three polar moieties forming a hydrophilic shell. Their chemical synthesis and purification were optimized to produce high reaction yields and easy scale-up. The ability to perform as cell-permeable or -impermeable probes was tuned by the polarity and molecular charge of the bioconjugate. The new PtPFPP derivatives were characterized for their spectral properties and cell-penetrating ability in the experiments with mammalian cell cultures, using a time-resolved fluorescence reader and PLIM imaging detection. Structure-activity relationships were established. Thus, the tri- and tetra-PEGylated structures showed low cell internalization allowing their use as extracellular probes, while cysteamine derivatives performed as efficient intracellular probes. No significant cytotoxicity was observed for all of the probes under the experimental conditions used.

Total synthesis and biological evaluation of grassypeptolide†A.

Herein, we describe in full our investigations into the synthesis of grassypeptolide A (1) in 17 linear steps with an overall yield of 11.3 %. In particular, this work features the late-stage introduction of sensitive bis(thiazoline) heterocycles and 31-membered macrocyclization conducted at the sterically congested secondary amide site in superb conversion (72 % yield). Biological evaluation indicated that grassypeptolide A significantly inhibited cancer cell proliferation in a dose-dependent manner. It induced cancer cell apoptosis, which was associated with increased cleavage of poly(ADP-ribose) polymerase (PARP) and decreased expression of bcl-2 and bcl-xL. Furthermore, grassypeptolide A also caused cell cycle redistribution by increasing cells in the G1 phase and decreasing cells in the S and G2 phases. In addition, cell cycle arrest was correlated with downregulation of cyclin D and upregulation of p27 and p21.

Anti-inflammatory properties of potato glycoalkaloids in stimulated Jurkat and Raw 264.7 mouse macrophages.

AIMS: The potato glycoalkaloids, α-chaconine, α-solanine and solanidine, along with potato peel extracts were investigated for potential anti-inflammatory effects in vitro. Their potential to reduce two biomarkers of inflammation, cytokine and nitric oxide (NO) productions, were assessed in the stimulated Jurkat and macrophage models, respectively. MAIN METHODS: Cytokine and nitric oxide productions were stimulated in Jurkat and Raw 264.7 macrophages with Concanavalin A (Con A; 25 µg/ml) and lipopolysaccaride (LPS; 1 µg/ml), respectively. Selective concentrations of glycoalkaloids and potato peel extracts were added simultaneously with Con A or LPS for 24h to investigate their potential to reduce inflammatory activity. KEY FINDINGS: α-Chaconine and solanidine significantly reduced interleukin-2 (IL-2) and interleukin-8 (IL-8) productions in Con A-induced Jurkat cells. The potato peel extracts did not influence cytokine production. In LPS-stimulated Raw macrophages, α-solanine, solanidine and two potato peel extracts significantly reduced induced NO production. SIGNIFICANCE: Our findings suggest that sub-cytotoxic concentrations of potato glycoalkaloids and potato peel extracts possess anti-inflammatory effects in vitro and with further investigation may be useful in the prevention of anti-inflammatory diseases.

A novel cyclinE/cyclinA-CDK inhibitor targets p27(Kip1) degradation, cell cycle progression and cell survival: implications in cancer therapy.

p27(Kip1) (p27) binds and inhibits the cyclin E- or cyclin A-associated cyclin-dependent kinases (CDKs)2 and other CDKs, and negatively regulates G1-G2 cell cycle progression. To develop specific CDK inhibitors, we have modeled the interaction between p27 and cyclin A-CDK2, and designed a novel compound that mimics p27 binding to cyclin A-CDK2. The chemically synthesized inhibitor exhibited high potency and selective inhibition towards cyclin E/cyclin A-CDK2 kinase in vitro but not other kinases. To facilitate permeability of the inhibitor, a cell penetrating peptide (CPP) was conjugated to the inhibitor to examine its effect in several cancer cell lines. The CPP-conjugated inhibitor significantly inhibited the proliferation of cancer cells. The treatment of the inhibitor resulted in the increased accumulation of p27 and p21(Cip1/Waf1) (p21) and hypo-phosphorylation of retinoblastoma protein (Rb). The degradation of p27, mediated through the phosphorylation of threonine-187 in p27, was also inhibited. Consequently, exposure of cells to the inhibitor caused cell cycle arrest and apoptosis. We conclude that specific cyclinE/cyclin A-CDK2 inhibitors can be developed based on the interaction between p27 and cyclin/CDK to block cell cycle progression to prevent tumor growth and survival.

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents.

Drugs that inhibit DNA topoisomerase I and DNA topoisomerase II have been widely used in cancer chemotherapy. We report herein the results of a focused medicinal chemistry effort around novel ellipticinium salts which target topoisomerase I and II enzymes with improved solubility. The salts were prepared by reaction of ellipticine with the required alkyl halide and evaluated for DNA intercalation, topoisomerase inhibition and growth inhibition against 12 cancer cell lines. Results from the topoisomerase I relaxation assay indicated that all novel ellipticine derivatives behaved as intercalating agents. At a concentration of 100 µM, specific topoisomerase I inhibition was not observed. Two of the derivatives under investigation were found to fully inhibit the DNA decatenation reaction at a concentration of 100 µM, indicative of topoisomerase II inhibition. N-Alkylation of ellipticine was found to enhance the observed growth inhibition across all cell lines and induce growth inhibition comparable to that of Irinotecan (CPT-11; GI(50) 1-18 µM) and in some cell lines better than Etoposide (VP-16; GI(50) = 0.04-5.2 µM). 6-Methylellipticine was the most potent growth inhibitory compound assessed (GI(50) = 0.47-0.9 µM). N-Alkylation of 6-methylellipticine was found to reduce this response with GI(50) values in the range of 1.3-28 µM.

Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells.

The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and ß-sitosterol, have previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2 cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1 (2CMP:1DHB) and a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The 2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation products was found to be similar to that previously observed for cholesterol, ß-sitosterol and stigmasterol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells incubated with the 7-keto and 7ß-OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the presence of 3DHB:1CMP-3ß,5α,6ß-triol and 2CMP:1DHB-5ß,6ß-epoxide. An additional oxidation product synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of phytosterol oxide cytotoxicity.

Oxidized derivatives of dihydrobrassicasterol: cytotoxic and apoptotic potential in U937 and HepG2 cells.

The ability of phytosterol compounds to reduce plasma serum cholesterol levels in humans is well investigated. However, phytosterols are structurally similar to cholesterol with a double bond at the C5-6 position and are therefore susceptible to oxidation. Much research has been carried out on the biological effects of cholesterol oxidation products (COPs) in vitro. In contrast, there is less known about phytosterol oxidation products (POPs). From previous studies, it is apparent that oxidized derivatives of the phytosterols, ß-sitosterol and stigmasterol, are cytotoxic in vitro but are less potent than their COP counterparts. In the present study, the cytotoxic and apoptotic potential of oxidized derivatives of dihydrobrassicasterol (DHB) including 5α,6α-epoxyergostan-3ß-ol (α-epoxide), 5ß,6ß-epoxyergostan-3ß-ol (ß-epoxide), ergost-5-en-7-on-3ß-ol (7-keto), ergost-5-ene-3ß,7ß-diol (7-ß-OH), and ergostane-3ß,5α,6ß-triol (triol) were evaluated in the U937 and HepG2 cell lines. In general, 7-keto, 7-ß-OH, and triol derivatives had a significant cytotoxic impact on U937 and HepG2 cells. The oxides appear to be more toxic toward U937 cells. In line with previous findings, the POPs investigated in this study were less potent than the equivalent COPs. The results add to the body of data on the toxicity of individual POPs.

Pleiotropic role for monocyte C-fms protein in response to vascular injury: potential therapeutic target.

OBJECTIVES: We examined the role of C-fms+ cells in response to vascular injury with a focus on the temporal and spatial platelet interactions, monocyte survival and proliferation within the evolving neointimal lesion and monocyte proliferation within the circulation and specified monocyte reservoir sites. Finally, we investigated the therapeutic effect of C-fms kinase inhibition (CFKI) on neointimal hyperplasia post vessel injury. METHODS AND RESULTS: We utilized murine carotid-wire injury, a transgenic C-fms reporting mouse model, confocal microscopy, shear-flow studies, specific C-fms signalling inhibition to determine the activation, mobilization and recruitment of C-fms+ monocytes in the context of early and late vessel remodelling. C-fms+ cells were recruited as early as 4h and accumulated over time in the neointima following injury. Monocyte interaction with platelet thrombus under flow and in vivo, in addition to monocyte mobilisation into the circulation post-injury was impaired by CFKI administration. Sustained inhibition of C-fms over 1-2 weeks abrogated the neointimal response but preserved re-endothelialisation post-injury. CONCLUSION: These data establish C-fms as a key regulator of the vascular response to injury and a potentially attractive therapeutic target in disease states where neointimal hyperplasia, monocyte activation and pathologic remodelling are prominent and endothelial homeostasis is desirable.

Design and synthesis of α-carboxy phosphononucleosides.

Rhodium catalyzed O-H insertion reactions employing α-diazophosphonate 20 with appropriately protected thymidine, uridine, cytosine, adenosine and guanosine derivatives leads to novel 5'-phosphononucleoside derivatives. Deprotection led to a novel series of phosphono derivatives bearing a carboxylic acid moiety adjacent to the phosphonate group with potential antiviral and/or anticancer activity. The phosphononucleosides bearing an α-carboxylic acid group are envisaged as potential diphosphate mimics. Conversion to mono- and diphosphorylated phosphononucleosides has been effected for evaluation as nucleoside triphosphate mimics. Most of the novel phosphononucleosides proved to be inactive against a variety of DNA and RNA viruses. Only the phosphono AZT derivatives 56-59 showed weak activity against HIV-1 and HIV-2.

Qualitative and quantitative comparison of the cytotoxic and apoptotic potential of phytosterol oxidation products with their corresponding cholesterol oxidation products.

Phytosterols contain an unsaturated ring structure and therefore are susceptible to oxidation under certain conditions. Whilst the cytotoxicity of the analogous cholesterol oxidation products (COP) has been well documented, the biological effects of phytosterol oxidation products (POP) have not yet been fully ascertained. The objective of the present study was to examine the cytotoxicity of beta-sitosterol oxides and their corresponding COP in a human monocytic cell line (U937), a colonic adenocarcinoma cell line (CaCo-2) and a hepatoma liver cell line (HepG2). 7beta-Hydroxysitosterol, 7-ketositosterol, sitosterol-3beta,5alpha,6beta-triol and a sitosterol-5alpha,6alpha-epoxide-sitosterol-5beta,6beta-epoxide (6:1) mixture were found to be cytotoxic to all three cell lines employed; the mode of cell death was by apoptosis in the U937 cell line and necrosis in the CaCo-2 and HepG2 cells. 7beta-Hydroxysitosterol was the only beta-sitosterol oxide to cause depletion in glutathione, indicating that POP-induced apoptosis may not be dependent on the generation of an oxidative stress. A further objective of this study was to assess the ability of the antioxidants alpha-tocopherol, gamma-tocopherol and beta-carotene to modulate POP-induced cytotoxicity in U937 cells. Whilst alpha/gamma-tocopherol protected against 7beta-hydroxycholesterol-induced apoptosis, they did not confer protection against 7beta-hydroxysitosterol- or 7-ketositosterol-induced toxicity, indicating that perhaps COP provoke different apoptotic pathways than POP. beta-Carotene did not protect against COP- or POP-induced toxicity. In general, results indicate that POP have qualitatively similar toxic effects to COP. However, higher concentrations of POP are required to elicit comparable levels of toxicity.

Comparison of the cytotoxic effects of beta-sitosterol oxides and a cholesterol oxide, 7beta-hydroxycholesterol, in cultured mammalian cells.

Phytosterols are plant sterols found in foods such as oils, nuts and vegetables. Phytosterols, in the same way as cholesterol, contain a double bond and are susceptible to oxidation. The objective of the present study was to assess the potential toxic effects of beta-sitosterol oxides on U937 cells. The effects of increasing concentrations (0-120 microm) of beta-sitosterol oxides on cellular cytotoxicity, apoptosis, antioxidant status and genotoxicity was assessed over 12, 24 and 48 h exposure periods. Following 12 h, the viability of cells treated with 120 microm-beta-sitosterol oxides was reduced to 51.7 % relative to control. At 24 and 48 h, both 60 and 120 microm-beta-sitosterol oxides caused a significant decrease in cell viability. For comparison, a decrease in viability of cells treated with a cholesterol oxide, 7beta-hydroxycholesterol (7beta-OH, 30 microm), was evident at 24 h. An increase in apoptotic cells, assessed using Hoechst 33342, indicates that the mode of cell death in U937 cells following exposure to 7beta-OH (30 microm) and beta-sitosterol oxides (60 and 120 microm) was by apoptosis. The increase in apoptotic cells after 12 h following treatment with 120 microm-beta-sitosterol oxides was accompanied by a decrease in cellular glutathione. Similarly, 7beta-OH (30 microm) treatment resulted in decreased glutathione at 12 h. Catalase activity was not affected by any of the treatments. beta-Sitosterol oxides had no genotoxic effects on U937 and V79 cells as assessed by the comet and sister chromatid exchange assays respectively. In general, the results indicate that thermally oxidised derivatives of beta-sitosterol demonstrate similar biological effects as 7beta-OH in U937 cells, but at higher concentrations.