Interleukin-6 and Cyclooxygenase-2 downregulation by fatty-acid fractions of Ranunculus constantinopolitanus

<p>Abstract</p> <p>Background</p> <p>Medicinal plants represent alternative means for the treatment of several chronic diseases, including inflammation. The genus <it>Ranunculus</it>, a representative of the Ranunculaceae family, has been reported to possess anti-inflammatory, analgesic, antiviral, antibacterial, antiparasitic and antifungal activities, possibly due to the presence of anemonin and other. Different studies have shown the occurrence of unusual fatty acids (FAs) in Ranunculaceae; however, their therapeutic role has not been investigated. The purpose of this study is to characterize potential anti-inflammatory bioactivities in <it>Ranunculus constantinopolitanus </it>D'Urv., traditionally used in Eastern Mediterranean folk medicine.</p> <p>Methods</p> <p>The aerial part of <it>R. constantinopolitanus </it>was subjected to methanol (MeOH) extraction and solvent fractionation. The bioactive fraction (I.2) was further fractionated using column chromatography, and the biologically active subfraction (Y₂₊₃) was identified using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The effects of I.2 and Y₂₊₃on cell viability were studied in mouse mammary epithelial SCp2 cells using trypan blue exclusion method. To study the anti-inflammatory activities of I.2 and Y₂₊₃, their ability to reduce interleukin (IL)-6 levels was assessed in endotoxin (ET)-stimulated SCp2 cells using enzyme-linked immunosorbent assay (ELISA). In addition, the ability of Y₂₊₃to reduce cyclooxygenase (COX)-2 expression was studied in IL-1-treated mouse intestinal epithelial Mode-K cells via western blotting. Data were analyzed by one-way analysis of variance (ANOVA), Student-Newman-Keuls (SNK), Tukey HSD, two-sample t-test and Dunnett t-tests for multiple comparisons.</p> <p>Results</p> <p>The chloroform fraction (I.2) derived from crude MeOH extract of the plant, in addition to Y₂₊₃, a FA mix isolated from this fraction and containing palmitic acid, C18:2 and C18:1 isomers and stearic acid (1:5:8:1 ratio), reduced ET-induced IL-6 levels in SCp2 cells without affecting cell viability or morphology. When compared to fish oil, conjugated linoleic acid (CLA) and to individual FAs as palmitic, linoleic, oleic and stearic acid or to a mix of these FAs (1:5:8:1 ratio), Y₂₊₃exhibited higher potency in reducing ET-induced IL-6 levels within a shorter period of time. Y₂₊₃ also reduced COX-2 expression in IL-1-treated Mode-K cells.</p> <p>Conclusion</p> <p>Our studies demonstrate the existence of potential anti-inflammatory bioactivities in <it>R. constantinopolitanus </it>and attribute them to a FA mix in this plant.</p

Targeting mitochondria by Zn(II)N-alkylpyridylporphyrins: the impact of compound sub-mitochondrial partition on cell respiration and overall photodynamic efficacy.

Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures

Effect of molecular characteristics on cellular uptake, subcellular localization, and phototoxicity of Zn(2) N-Alkylpyridylporphyrins

Background: Delivering molecules to selected cellular compartments is important for analytical and practical purposes. Results: Varying the length and positions of alkyl substituents results in preferential uptake of zinc porphyrins by particular cellular structures. Conclusion: Uptake, distribution, and phototoxicity of porphyrins depend on charge, lipophilicity, and molecular shape. Significance: Systematic chemical modification provides the basis for rational design of molecules targeting specific cellular compartments. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Lipophilicity of Zn(II) <i>meso</i>-tetrakis(<i>N</i>-alkylpyridinium-2-yl)porphyrins.

<p>Lipophilicity was determined from the partition coefficient between n-octanol and water (log <i>P</i>_ow) and chromatographic retention factor (<i>R</i>_f, compound path/solvent path, determined on silica gel plates using 1∶1:8 KNO_3(sat):H₂O:acetonitrile as a mobile phase).</p><p>Lipophilicity of Zn(II) <i>meso</i>-tetrakis(<i>N</i>-alkylpyridinium-2-yl)porphyrins.</p

Structures of <i>ortho</i> Zn(II) <i>meso</i>-tetrakis(<i>N</i>-alkylpyridinium-2-yl)porphyrins investigated in this study and the <i>para</i> platinum analog (PtTM-4-PyP) [36] of the methyl Zn-porphyrin (ZnTM-2-PyP).

<p>PtTM-4-PyP, Pt(II) <i>meso</i>-tetrakis(<i>N</i>-methylpyridinium-4-yl)porphyrin; Zn porphyrin, Zn(II) <i>meso</i>-tetrakis(<i>N</i>-alkylpyridinium-2-yl)porphyrin, alkyl being methyl (M, ZnTM-2-PyP), n-butyl (nBu, ZnTnBu-2-PyP), n-hexyl (nHex, ZnTnHex-2-PyP).</p

Photoinactivation of cytochrome c oxidase in intact cells.

<p>Cells were washed after 24 hours incubation with 5 and 10 µM of ZnTM-2-PyP, ZnTnBu-2-PyP or ZnTnHex-2-PyP and were illuminated for 30 minutes. Cytochrome c oxidase activity was determined in cell-free lysates. Data are expressed as mean ± SE (n = 4); *p<0.05 compared to control.</p

Effect of phototreatment with Zn porphyrins on MTT reduction and respiration.

<p>Cells were preincubated for 24 h with Zn porphyrins, washed, resuspended in PBS and illuminated for 30 minutes. A: MTT reduction by cells illuminated after preincubation with <i>ortho</i> methyl, butyl or hexyl Zn porphyrins. Concentrations of compounds causing 50% inhibition of MTT reduction (IC₅₀) compared to controls are presented; B: Oxygen consumption by cells preincubated with 5 µM Zn porphyrins and illuminated. Mean ± S.E. is presented (n = 4); * p<0.05 compared to control.</p

Photo-inactivation of cytochrome c oxidase in permeabilized mitochondria incubated with 10 µM ZnTM-2-PyP, ZnTnBu-2-PyP or ZnTnHex-2-PyP for 30 minutes and then illuminated for 30 minutes.

<p>Data are expressed as mean ± SE (n = 4). * p<0.05 compared to control.</p

Fluorescence microscopy images of cells preincubated with Zn-porphyrins.

<p>I: ZnTM-2-PyP; II: ZnTE-2-PyP; III, IV: ZnTnBu-2-PyP; V, VI: ZnTnHex-2-PyP. A: Cells incubated for in the dark with Zn-porphyrin; B: Costaining with DIOC6(3) iodide (II, IV, V), Lysosensor (III), or MitoTracker (VI) C: Overlay.</p

Photoinactivation of cytochrome c oxidase in intact mitochondria.

<p>Mitochondria were incubated with 1 and 5 µM ZnTM-2-PyP, ZnTnBu-2-PyP or ZnTnHex-2-PyP for 15 minutes and were then illuminated for 30 minutes. Data are expressed as mean ± SE (n = 4). *p<0.05 compared to control.</p