Data of oxygen- and pH-dependent oxidation of resveratrol

AbstractWe show here if under physiologically relevant conditions resveratrol (RSV) remains stable or not. We further show under which circumstances various oxidation products of RSV such as ROS can be produced. For example, in addition to the widely known effect of bicarbonate ions, high pH values promote the decay of RSV. Moreover, we analyse the impact of reduction of the oxygen partial pressure on the pH-dependent oxidation of RSV. For further interpretation and discussion of these focused data in a broader context we refer to the article “Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress” (Plauth et al., in press) [1]

Hormetic shifting of redox environment by pro-oxidative resveratrol protects cells against stress

AbstractResveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of this natural product remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to its generation of oxidation products such as reactive oxygen species (ROS). At low nontoxic concentrations (in general <50µM), treatment with resveratrol increased viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, resveratrol treatment led to mild, Nrf2-specific gene expression reprogramming. For example, in primary epidermal keratinocytes derived from human skin this coordinated process resulted in a 1.3-fold increase of endogenously generated glutathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61mVmmol GSH per g protein. After induction of oxidative stress by using 0.78% (v/v) ethanol, endogenous generation of ROS was consequently reduced by 24% in resveratrol pre-treated cells. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that the cellular response to resveratrol treatment is essentially based on oxidative triggering. In physiological microenvironments this molecular training can lead to hormetic shifting of cellular defense towards a more reductive state to improve physiological resilience to oxidative stress

Mechanismen der milden zellulären Stressantwort

Polyphenols, historically known as “vegetable tannins”, feature health- beneficial effects [14]. The phytoalexin trans-resveratrol (RSV) was originally isolated from Veratrum grandiflorum O. Loes [30] and is ascertained to naturally occur in approximately 72 plant species [19]. The “French paradox” [16] and early reports of cancer chemo-preventive properties [19] contributed to the growing popularity of RSV [24, 388, 389]. The human skin is the largest organ of our body [2] and challenged by oxidative and environmental stressors on a daily basis [390–392]. The use of RSV in a skin context is a cutting-edge topic [19, 329] as epidermal keratinocytes are a prime target for RSV-based lotions and emollients. However, the mechanism of action of RSV remains largely elusive and controversially discussed. In this study, neonatal normal human epidermal keratinocyte (NHEK) cells are used as a primary cellular model to investigate the mechanism of action of RSV. We demonstrate that RSV is unstable under physiologically relevant conditions, resulting in the generation of oxidation products and reactive oxygen species (ROS). In addition, RSV increases the cellular viability at “low”, hormetic doses (≤ 50 μM) in representative cell models. The application of ROS scavengers truncates these beneficial effects. Moreover, Nrf2-dependent gene expression is initiated by RSV. A 1.3-fold increase of endogenous glutathione (GSH) is sufficient to cause a quantitative reduction of the cellular redox environment. Consequently, RSV pre-treated cells are more resistant to ethanol-induced oxidative stress and generate 24% less ROS. We propose that the major effect of RSV is to induce a mild oxidative stress resulting in hormetic shifting of cellular metabolism towards a more reductive state.Polyphenole wurden anfänglich als Tannine zur Herstellung von Leder verwendet und sind heutzutage vor allem für ihre gesundheitsfördernden Eigenschaften bekannt [14]. Das Phytoalexin trans-Resveratrol (RSV) wurde ursprünglich aus Veratrum grandiflorum O. Loes isoliert [30] und wurde bisher in ca. 72 verschiedenen Pflanzen nachgewiesen [19]. Besonders das sogenannte französische Paradoxon [16] sowie Berichte über chemo-preventive Eigenschaften [19] rückten RSV in den Fokus der Forschung [24,388,389]. Die Haut ist das größte Organ unseres Körpers [2] und tagtäglich verschiedenen oxidativen und umweltbedingten Stressfaktoren ausgesetzt [390–392]. Die Anwendung von RSV im Hautkontext ist ein hochaktueller Themenbereich, insbesondere, da die epidermalen Keratinozyten ein Hauptziel für RSV-basierte Lotionen darstellen. Ungeachtet dessen ist der Reaktionsmechanismus von RSV weitestgehend unbekannt und wird heftig diskutiert. In dieser Arbeit werden vor allem neonatale normale humane epidermale Keratinozyten (NHEK) verwendet, um den Wirkmechanismus von RSV zu untersuchen. Wir zeigen, dass RSV unter physiologischen Bedingungen instabil ist und dass der Zerfall von RSV die Entstehung von Oxidationsprodukten und reaktiven Sauerstoffspezies (ROS) nach sich zieht. Darüber hinaus erhöht die Anwendung von RSV in kleinen, hormetischen Konzentrationen (≤ 50 μM) die zelluläre Viabilität in diversen Zellmodellen. Eine gleichzeitige Anwendung von Radikalfängern beseitigt diese Viabilitätssteigerung vollständig. Zusätzlich initiiert RSV die Expression von Zielgenen über den Transkriptionsfaktor Nrf2. Eine vergleichsweise kleine, 1,3-fache Steigerung des endogenen Glutathiongehalts (GSH) führt zu einer messbaren, quantitativen Verringerung der zellulären Redoxumgebung. Dementsprechend sind mit RSV vorbehandelte Zellen resistenter gegen bspw. Ethanol-induzierten oxidativen Stress und produzieren 24% weniger ROS. Wir schlagen einen Wirkmechanismus vor, der vor allem auf der Induktion von mildem oxidativen Stress beruht und letzten Endes zu einer hormetischen Verschiebung der zellulären Redoxumgebung führt

Determination of Impurities in Pharmaceutical Formulations by HPLC

Massively increasing global incidences of colorectal cancer require efficient treatment and prevention strategies. Here, we report unexpected anticancerogenic effects of hydroethanolic Iberis amara extract (IAE), which is known as a widely used phytomedical product for treating gastrointestinal complaints. IAE significantly inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC$_{50}$) of 6 and 9 μg/ml, respectively, and further generated inhibitory effects in PC-3 prostate and MCF7 breast cancer cells. Inhibition of proliferation in HT-29 cells was associated with a G2/M phase cell cycle arrest including reduced expression of various regulatory marker proteins. Notably, in HT-29 cells IAE further induced apoptosis by intracellular formation of reactive oxygen species (ROS). Consistent with predictions derived from our in vitro experiments, bidaily oral gavage of 50 mg/kg of IAE over 4 weeks resulted in significant inhibition of tumor growth in a mouse HT-29 tumor xenograft model. Taken together, Iberis amara extracts could become useful alternatives for preventing and treating the progression of colon cancer

Extra- and intracellular formation of reactive oxygen species (ROS) and of lipid peroxides in HT-29 cells after treatment with IAE.

<p>A, Extracellular formation of ROS in full cell culture medium was kinetically detected using the ROS-sensitive CellROX Orange fluorogenic probe. Data are expressed as mean ± SEM (n = 8). B, Intracellular ROS was detected by flow cytometry of HT-29 cells stained with CellROX Orange after treatment for 24 h. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). C, Intracellular lipid peroxidation was detected by flow cytometry of cells treated for 24 h using the Click-iT technology. Increasing fluorescence intensities are a result of enhanced lipid peroxidation upon treatment. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). D, Intracellular lipid peroxidation was visualized by fluorescence microscopy (green, lipid peroxides; blue, nucleus). Scale bars, 25 μm. n.s. not significant, **p≤0.01, ***p≤0.001 vs. control.</p

Activation of apoptosis signaling pathway in colon carcinoma cells after treatment with IAE.

<p>A, HT-29 and T84 cells were treated for 24 h. Enzymatic activation of caspases 2, 3/7, 6, 8 and 9 was determined by use of luminescence-based assays. Data are normalized to control treatment and are expressed as mean ± SEM (n = 4). B, Whole cell lysates from HT-29 cells treated for 24 h were analyzed for the expression of total and cleaved proteins of caspase 3, caspase 9 and PARP by immunoblotting. Numbers indicate densitometric ratios of the cleaved to total proteins normalized to control treatments. C, Fluorescence microscopy of HT-29 cells treated for 24 h. Cleaved caspase 3 was labeled green, F-actin red and the nucleus blue. Scale bars, 25 μm. D, HT-29 cells were treated for 6 h. DNA fragmentation was detected through accumulation of cytoplasmic BrdU-labeled DNA by ELISA. Data are normalized to control treatment and are expressed as mean ± SEM (n = 5). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control.</p

Cell cycle analysis of HT-29 colon carcinoma cells after treatment with 30 μg/ml IAE for 24 h.

<p>A, Cell cycle was analyzed by flow cytometry of propidium iodide stained cells. Histograms (top) show one representative experiment for each treatment condition. Bar plots (bottom) show percent of cell population in apoptotic SubG1, G0/G1, S and G2/M phases of the cell cycle and are expressed as mean ± SEM (n = 3). *p≤0.05, ***p≤0.001 vs. control. B, Whole cell lysates were analyzed for the expression of cyclin A2, cyclin D3, CDK2, CDK4, CDK6 and GAPDH proteins by immunoblotting using specific antibodies.</p

Cytotoxicity test and evaluation of inhibition of tumor growth by treating HT-29 tumor xenograft mouse models with IAE.

<p>Athymic nude mice were ectopically implanted with 5 million HT-29 cells in the flank and orally gavaged bidaily by 50 mg/kg IAE or vehicle for 4 weeks. A, Cytotoxicity was tested for colon cancer and primary colon cells (IC₅₀ = 25.82 μg/mL (HT-29); IC₅₀ = 36.12 μg/mL (CCD 841 CoN)). Data are expressed as mean ± SD (n = 4). B, Body weight during entire experiment. C, Tumor volume during entire experiment and images of exemplary xenografts of untreated and IAE-treated mice at end point. D, Tumor weight at the end of the study. Data are expressed as mean ± SEM (n = 18). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control (one-tailed t test).</p

Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.

<p>Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.</p

Amorfrutin C Induces Apoptosis and Inhibits Proliferation in Colon Cancer Cells through Targeting Mitochondria

A known (<b>1</b>) and a structurally related new natural product (<b>2</b>), both belonging to the amorfrutin benzoic acid class, were isolated from the roots of <i>Glycyrrhiza foetida</i>. Compound <b>1</b> (amorfrutin B) is an efficient agonist of the nuclear peroxisome proliferator activated receptor (PPAR) gamma and of other PPAR subtypes. Compound <b>2</b> (amorfrutin C) showed comparably lower PPAR activation potential. Amorfrutin C exhibited striking antiproliferative effects for human colorectal cancer cells (HT-29 and T84), prostate cancer (PC-3), and breast cancer (MCF7) cells (IC₅₀ values ranging from 8 to 16 μM in these cancer cell lines). Notably, amorfrutin C (<b>2</b>) showed less potent antiproliferative effects in primary colon cells. For HT-29 cells, compound <b>2</b> induced G0/G1 cell cycle arrest and modulated protein expression of key cell cycle modulators. Amorfrutin C further induced apoptotic events in HT-29 cells, including caspase activation, DNA fragmentation, PARP cleavage, phosphatidylserine externalization, and formation of reactive oxygen species. Mechanistic studies revealed that <b>2</b> disrupts the mitochondrial integrity by depolarization of the mitochondrial membrane (IC₅₀ 0.6 μM) and permanent opening of the mitochondrial permeability transition pore, leading to increased mitochondrial oxygen consumption and extracellular acidification. Structure–activity-relationship experiments revealed the carboxylic acid and the hydroxy group residues of <b>2</b> as fundamental structural requirements for inducing these apoptotic effects. Synergy analyses demonstrated stimulation of the death receptor signaling pathway. Taken together, amorfrutin C (<b>2</b>) represents a promising lead for the development of anticancer drugs