Sulforaphane Protects the Liver against CdSe Quantum Dot-Induced Cytotoxicity

The potential cytotoxicity of cadmium selenide (CdSe) quantum dots (QDs) presents a barrier to their use in biomedical imaging or as diagnostic and therapeutic agents. Sulforaphane (SFN) is a chemoprotective compound derived from cruciferous vegetables which can up-regulate antioxidant enzymes and induce apoptosis and autophagy. This study reports the effects of SFN on CdSe QD-induced cytotoxicity in immortalised human hepatocytes and in the livers of mice. CdSe QDs induced dose-dependent cell death in hepatocytes with an IC₅₀ = 20.4 μM. Pre-treatment with SFN (5 μM) increased cell viability in response to CdSe QDs (20 μM) from 49.5 to 89.3%. SFN induced a pro-oxidant effect characterized by depletion of intracellular reduced glutathione during short term exposure (3–6 h), followed by up-regulation of antioxidant enzymes and glutathione levels at 24 h. SFN also caused Nrf2 translocation into the nucleus, up-regulation of antioxidant enzymes and autophagy. siRNA knockdown of Nrf2 suggests that the Nrf2 pathway plays a role in the protection against CdSe QD-induced cell death. Wortmannin inhibition of SFN-induced autophagy significantly suppressed the protective effect of SFN on CdSe QD-induced cell death. Moreover, the role of autophagy in SFN protection against CdSe QD-induced cell death was confirmed using mouse embryonic fibroblasts lacking ATG5. CdSe QDs caused significant liver damage in mice, and this was decreased by SFN treatment. In conclusion, SFN attenuated the cytotoxicity of CdSe QDs in both human hepatocytes and in the mouse liver, and this protection was associated with the induction of Nrf2 pathway and autophagy.     

Cyclosporine A Induces Apoptotic and Autophagic Cell Death in Rat Pituitary GH3 Cells

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels.     

Role of D-Limonene in Autophagy Induced by Bergamot Essential Oil in SH-SY5Y Neuroblastoma Cells

Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005–0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70^S6K (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125–750 µM) and linalyl acetate (62.5–375 µM), were individually tested at concentrations comparable to those found in 0.005–0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by d-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by d-limonene.     

Cholestane-3β, 5α, 6β-triol Suppresses Proliferation,Migration, and Invasion of Human Prostate Cancer Cells

Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27^Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.     

Allelopathic activities of three carotenoids,neoxanthin, crocin and β-carotene,assayed using protoplast co-culture method with digital image analysis

Allelopathic activities of three carotenoids of a natural pigment group, neoxanthin, crocin and β-carotene, were assayed by the protoplast co-culture method with digital image analysis (DIA-PP method). Effects on three different growth stages of lettuce protoplasts, i.e., cell wall formation, cell division, and yellow pigment accumulation, were investigated using 96-well culture plates. Cell division was inhibited 65–95% by all three carotenoids at 33–100 µM. Inhibition of cell division stage was stronger than at the cell wall formation stage in neoxanthin, and the water-soluble carotenoid, crocin, whose yellow pigment was incorporated into the vacuole of lettuce protoplasts. Neoxanthin at 33 µM and crocin at higher than 100 µM inhibited more than 100% of the yellow pigment accumulation. By contrast, at low concentrations (0.01–1 µM) β-carotene stimulated growth at the cell division stage. At high concentrations of β-carotene (100–500 µM), inhibition was prominent at all three stages, and also in neighboring wells of zero control, which suggested emission of a volatile compound by β-carotene. They were compared with the report of the volatile compound, tulipalin A. Differences in patterns of inhibition of carotenoids on lettuce protoplast growth were compared with reports of another natural pigment, anthocyanin, and anthocyanin-containing red callus cultured in the light, and with that of neoxanthin-containing yellow callus cultured in the dark.     

Structure and Mechanics of Supporting Cells in the Guinea Pig Organ of Corti

The mechanical properties of the mammalian organ of Corti determine its sensitivity to sound frequency and intensity, and the structure of supporting cells changes progressively with frequency along the cochlea. From the apex (low frequency) to the base (high frequency) of the guinea pig cochlea inner pillar cells decrease in length incrementally from 75–55 µm whilst the number of axial microtubules increases from 1,300–2,100. The respective values for outer pillar cells are 120–65 µm and 1,500–3,000. This correlates with a progressive decrease in the length of the outer hair cells from >100 µm to 20 µm. Deiters''cell bodies vary from 60–50 µm long with relatively little change in microtubule number. Their phalangeal processes reflect the lengths of outer hair cells but their microtubule numbers do not change systematically. Correlations between cell length, microtubule number and cochlear location are poor below 1 kHz. Cell stiffness was estimated from direct mechanical measurements made previously from isolated inner and outer pillar cells. We estimate that between 200 Hz and 20 kHz axial stiffness, bending stiffness and buckling limits increase, respectively,∼3, 6 and 4 fold for outer pillar cells, ∼2, 3 and 2.5 fold for inner pillar cells and ∼7, 20 and 24 fold for the phalangeal processes of Deiters''cells. There was little change in the Deiters''cell bodies for any parameter. Compensating for effective cell length the pillar cells are likely to be considerably stiffer than Deiters''cells with buckling limits 10–40 times greater. These data show a clear relationship between cell mechanics and frequency. However, measurements from single cells alone are insufficient and they must be combined with more accurate details of how the multicellular architecture influences the mechanical properties of the whole organ.     

The Role of Heat Shock Protein 70 in the Protective Effect of YC-1 on β-Amyloid-Induced Toxicity in Differentiated PC12 Cells

Neurodegenerative brain disorders such as Alzheimer’s disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aβ_25–35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ_25–35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ_25–35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5–10 µM) prevented the cell death induced by Aβ_25–35. In addition, YC-1 (1, 10 µM) significantly blocked Aβ_25–35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5–20 µM) alone or combined with Aβ_25–35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aβ_25–35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ_25–35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.     

Covalent binding to tubulin by isothiocyanates. A mechanism of cell growth arrest and apoptosis

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.     

Mitochondria Are the Target Organelle of Differentiation-Inducing Factor-3, an Anti-Tumor Agent Isolated from Dictyostelium Discoideum

Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY), and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 5–20 µ M suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY-DIF-3 at 20 µM, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 1–3 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler) induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells.     

Design,synthesis, anticancer evaluation,and molecular modelling studies of novel tolmetin derivatives as potential VEGFR-2 inhibitors and apoptosis inducers

Novel tolmetin derivatives 5a–f to 8a–c were designed, synthesised, and evaluated for antiproliferative activity by NCI (USA) against a panel of 60 tumour cell lines. The cytotoxic activity of the most active tolmetin derivatives 5b and 5c was examined against HL-60, HCT-15, and UO-31 tumour cell lines. Compound 5b was found to be the most potent derivative against HL-60, HCT-15, and UO-31 cell lines with IC₅₀ values of 10.32 ± 0.55, 6.62 ± 0.35, and 7.69 ± 0.41 µM, respectively. Molecular modelling studies of derivative 5b towards the VEGFR-2 active site were performed. Compound 5b displayed high inhibitory activity against VEGFR-2 (IC₅₀ = 0.20 µM). It extremely reduced the HUVECs migration potential exhibiting deeply reduced wound healing patterns after 72 h. It induced apoptosis in HCT-15 cells (52.72-fold). This evidence was supported by an increase in the level of apoptotic caspases-3, -8, and -9 by 7.808-, 1.867-, and 7.622-fold, respectively. Compound 5b arrested the cell cycle in the G0/G1 phase. Furthermore, the ADME studies showed that compound 5b possessed promising pharmacokinetic properties.     

15-Lipoxygenase Metabolites of Docosahexaenoic Acid Inhibit Prostate Cancer Cell Proliferation and Survival

A 15-LOX, it is proposed, suppresses the growth of prostate cancer in part by converting arachidonic, eicosatrienoic, and/or eicosapentaenoic acids to n-6 hydroxy metabolites. These metabolites inhibit the proliferation of PC3, LNCaP, and DU145 prostate cancer cells but only at ≥1–10 µM. We show here that the 15-LOX metabolites of docosahexaenoic acid (DHA), 17-hydroperoxy-, 17-hydroxy-, 10,17-dihydroxy-, and 7,17-dihydroxy-DHA inhibit the proliferation of these cells at ≥0.001, 0.01, 1, and 1 µM, respectively. By comparison, the corresponding 15-hydroperoxy, 15-hydroxy, 8,15-dihydroxy, and 5,15-dihydroxy metabolites of arachidonic acid as well as DHA itself require ≥10–100 µM to do this. Like DHA, the DHA metabolites a) induce PC3 cells to activate a peroxisome proliferator-activated receptor-γ (PPARγ) reporter, express syndecan-1, and become apoptotic and b) are blocked from slowing cell proliferation by pharmacological inhibition or knockdown of PPARγ or syndecan-1. The DHA metabolites thus slow prostate cancer cell proliferation by engaging the PPARγ/syndecan-1 pathway of apoptosis and thereby may contribute to the prostate cancer-suppressing effects of not only 15-LOX but also dietary DHA.     

EXPRESSION OF THE MITOCHONDRIAL GENOME IN HELA CELLS : XV. Effect of Inhibition of Mitochondrial Protein Synthesis on Mitochondrial Formation

The effect of selective inhibition of mitochondrial protein synthesis by chloramphenicol at 40 or 200 µg/ml on the formation of mitochondria in HeLa cells was investigated. HeLa cells, under the conditions used in the present work, grow at a decreasing rate for at least four cell generations in the presence of 40 µg/ml chloramphenicol, and for two generations in the presence of 200 µg/ml chloramphenicol. The progressive cell growth inhibition which begins after 2 days of exposure of the cells to 40 µg/ml chloramphenicol is immediately or gradually reversible, upon removal of the drug, for periods up to at least 8 days of treatment, though there is a progressive loss of cloning efficiency. In cells which have been treated for 6–7 days with 40 or 200 µg/ml of chloramphenicol, mitochondrial protein synthesis occurs at a normal or near-normal rate 1 h after removal of the drug. Mitochondria increase normally in number and show a normal size and amount of cristae in the presence of either concentration of drug. However, in 4–5% of the mitochondrial profiles the cristae appear to be arranged in unusual, circular, looped or whorled configuration.     

Synthesis,computational study and biological evaluation of 9-acridinyl and 1-coumarinyl-1,2,3-triazole-4-yl derivatives as topoisomerase II inhibitors

Topoisomerase (IIB) inhibitors have been involved in the therapies of tumour progression and have become a major focus for the development of anticancer agents. New three-component hybridised ligands, 1,4-disubstituted-1,2,3-triazoles (8–17), were synthesised via a 1,3-dipolar cycloaddition reaction of 9-azidoacridine/3-azidocoumarin with N/O-propargyl small molecules under click reaction conditions. Cancer cell growth inhibition of the synthesised triazoles was tested against human cell-lines in the NCI-60-cell-panel, and the most active compounds tested against topoisomerase (IIB)-enzymes. The acridinyl ligands (8–10) revealed 60–97% cell growth inhibition in six cancer cell-panels. Cell-cycle analysis of MCF7 and DU-145 cells treated with the active acridinyl ligands exhibited cell-cycle arrest at G2/M phase and proapoptotic activity. In addition, compound 8 displayed greater inhibitory activity against topoisomerase (IIB) (IC₅₀ 0.52 µM) compared with doxorubicin (IC₅₀ 0.83 µM). Molecular dynamics simulation studies showed the acridine–triazole–pyrimidine hybrid pharmacophore was optimal with respect to protein–ligand interaction and fit within the binding site, with optimal orientation to allow for intercalation with the DNA bases (DG13, DC14, and DT9).     

Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells

Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer’s disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1–42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10–30 μM) or resveratrol (20 μM) prevented these Aβ1–42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet⁺ for 48 h and then placed in Tet^- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet^- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.     

Activation of Methanogenesis by Cadmium in the Marine Archaeon Methanosarcina acetivorans

Methanosarcina acetivorans was cultured in the presence of CdCl₂ to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41–69% of the heavy metal from the culture and accumulated 231–539 nmol Cd/mg cell protein. This is the first report showing that (i) Cd²⁺ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii) a methanogenic archaea is able to remove a heavy metal from aquatic environments.     

Design,synthesis, and analysis of antiproliferative and apoptosis-inducing activities of nitrile derivatives containing a benzofuran scaffold: EGFR inhibition assay and molecular modelling study

New cyanobenzofurans derivatives 2–12 were synthesised, and their antiproliferative activity was examined compared to doxorubicin and Afatinib (IC₅₀ = 4.17–8.87 and 5.5–11.2 µM, respectively). Compounds 2 and 8 exhibited broad-spectrum activity against HePG2 (IC₅₀ = 16.08–23.67 µM), HCT-116 (IC₅₀ = 8.81–13.85 µM), and MCF-7 (IC₅₀ = 8.36–17.28 µM) cell lines. Compounds 2, 3, 8, 10, and 11 were tested as EGFR-TK inhibitors to demonstrate their possible anti-tumour mechanism compared to gefitinib (IC₅₀ = 0.90 µM). Compounds 2, 3, 10, and 11 displayed significant EGFR TK inhibitory activity with IC₅₀ of 0.81–1.12 µM. Compounds 3 and 11 induced apoptosis at the Pre-G phase and cell cycle arrest at the G2/M phase. They also increased the level of caspase-3 by 5.7- and 7.3-fold, respectively. The molecular docking analysis of compounds 2, 3, 10, and 11 indicated that they could bind to the active site of EGFR TK.     

Zoledronate Attenuates Angiogenic Effects of Angiotensin II-Stimulated Endothelial Progenitor Cells via RhoA and MAPK Signaling

Background

New vessel formation plays a pivotal role in the pathogenesis of neovascular-related diseases. Endothelial progenitor cells (EPCs) were found to contribute to neovascular-related diseases and interference with EPC neovascularization may be a novel target for these diseases. Zoledronate (Zol) was reported to exhibit anti-angiogenic effect. Basing on these evidences, we proposed that Zol may affect EPC function to exert novel anti-angiogenic effect. In this study, we therefore investigated the effects of Zol on multiple aspects of EPC function and explored the underlying mechanisms involved.

Methodology/Principal Findings

EPCs were cultured from bone marrow derived mononuclear cells. The potential effects of Zol on Angiotensin II (Ang II)-stimulated EPC proliferation, migration, adhesion, in vitro tube formation were investigated. The results showed that Ang II (1 µM) enhanced EPC migration, adhesion, in vitro tube formation but had no effect on cell proliferation. Zol (75 and 100 µM) inhibited proliferation of EPCs and 50 µM geranylgeranyol (GGOH) could reverse the decrease of EPC proliferation. We found for the first time that Zol (50–100 µM) dose dependently attenuated migration, adhesion, and in vitro tube formation of EPCs stimulated by Ang II. GGOH could reverse the attenuation of EPC function induced by Zol. However, Zol did not induce EPC apoptosis. In addition, the underlying mechanisms were determined. The results revealed that Zol markedly down-regulated active RhoA stimulated by Ang II and inhibited the phosphorylation of Erk1/2 and JNK. Moreover, RhoA silencing resulted in a notable inhibition of EPC in vitro tube formation, suggesting that RhoA suppression played a pivotal role in Zol antiangiogenic effect.

Conclusions/Significance

These findings suggested that Zol attenuated the promotion of EPC function stimulated by Ang II and exhibited novel antiangiogenic effect via RhoA and MAPK signaling. Thus, Zol may be served as a novel therapeutic agent for neovascular-related diseases treatment.     

Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy

Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP^–/– fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP^–/– fibroblasts were exposed for 3 weeks to 20 µM GPI 6150 (1,11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (K_i = 60 nM) and one of the most potent PARP inhibitors to date (IC₅₀ = 0.15 µM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by ~91%, to about the residual levels in PARP^–/– cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.     

Nivalenol and Deoxynivalenol Affect Rat Intestinal Epithelial Cells: A Concentration Related Study

The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5–80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G₀/G₁ interphase and in the S phase, with a concomitant reduction in the fraction of cells in G₂. Interestingly, when administered at lower concentrations (0.1–2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins.     

Frondoside A Suppressive Effects on Lung Cancer Survival,Tumor Growth,Angiogenesis, Invasion,and Metastasis

A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer.