ATP-γ-S-(α,ß-CH2) protects against oxidative stress and amyloid beta toxicity in neuronal culture.

Amyloid beta (Aß) oligomers and oxidative stress, typical of Alzheimer's disease, are highly neurotoxic. Previously we identified ATP-γ-S as a most promising antioxidant and neuroprotectant. To further improve both potency and metabolic stability of ATP-γ-S, we designed a related analogue, ATP-γ-S-(α,ß-CH2). We found that ATP-γ-S-(α,ß-CH2) effectively inhibited ROS formation in PC12 cells subjected to Fe(II)-oxidation, slightly better than ATP-γ-S (IC50 0.18 and 0.20 µM, respectively). Moreover, ATP-γ-S-(α,ß-CH2) rescued primary neurons from Aß42 toxicity, 4-fold more potently than ATP-γ-S, (IC50 0.2 and 0.8 µM, respectively). In addition, the metabolic stability of ATP-γ-S-(α,ß-CH2) in PC12 cells during 4 h of incubation, was up to 20% greater than that of ATP-γ-S and ATP. Previously, we found that ATP-γ-S-(α,ß-CH2) resisted hydrolysis by ecto-nucleotidases such as, NPPs and TNAP, and was found to be ∼7-fold more potent agonist than ATP at P2Y11 receptor. Therefore, we propose ATP-γ-S-(α,ß-CH2) as a promising agent for rescue of neurons from insults typical of Alzheimer's disease.

Inula Viscosa Extract Inhibits Growth of Colorectal Cancer Cells in vitro and in vivo Through Induction of Apoptosis.

Colorectal cancer (CRC) is the second most common cancer in females and the third in males worldwide. Conventional therapy of CRC is limited by severe side effects and by the development of resistance. Therefore, additional therapies are needed in order to combat the problem of selectivity and drug resistance in CRC patients. Inula viscosa (IV) is a well-known medicinal perennial herb in traditional medicine. It is used for different therapeutic purposes, such as; topical anti-inflammatic, diuretic, hemostatic, antiseptic, antiphlogistic, and in the treatment of diabetes. Several studies attempted to reveal the anti-cancer activity of different extracts prepared by different organic solvents from different parts of the IV plant. The aim of the present study is to examine the potential beneficial effects of IV leaf aqueous extract on the growth of colon cancer cells in vitro and in vivo. The results indicated that exposure of colorectal cancer cells to IV extract, significantly reduced cell viability in a dose and time dependent manner. Moreover, treatment of cells with 300 µg/ml of IV extract induced apoptosis, as it was detected by Annexin V/FITC/PI, TUNEL assay, and the activation of caspases. In vivo studies revealed that treatment with 150 or 300 mg/kg IV extract inhibited tumor growth in mice transplanted with MC38 cells. Tumors' weight and volume were significantly (P < 0.001) reduced when compared to untreated-control group. Staining of the paraffin section of tumors revealed that IV treatment inhibited cell proliferation and induced apoptosis. Additionally, no side effects such as; weight loss, behavior changes, ruffled fur or changes in kidney, and liver functions were observed. These results may indicate that active doses of IV extract are not toxic. Further studies are needed in order to identify the structure of the active compounds. Results from this study may contribute to the development of new and efficient strategies for treatment of human colon cancer.

UV irradiation increases ROS production via PKCdelta signaling in primary murine fibroblasts.

Ultraviolet (UV) irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. UVA irradiation represents 90% of the solar UV light reaching the earth's surface, and yet the mechanisms by which it exerts its biological effects are not clear. UVA penetrates into the skin tissue, reaching the basal layers of the active dividing cells and, therefore, the contribution of UVA to skin damage may be significant. The majority of UVA energy is absorbed by unidentified photosensitizers in the cells which are postulated to generate reactive oxygen species (ROS). It has been believed that both chronological aging and photoaging share the same molecular features and, as such, it is very common to utilize UV irradiation for induction of skin aging. To determine the involvement of protein kinase isoforms in chronological aging and photoaging, we utilized in vitro aging model systems of primary murine fibroblasts and primary fibroblasts isolated from PKC null mice. We show for the first time distinct involvement of PKC isoforms PKCdelta and PKCalpha in photoaging versus cellular senescence. While chronological aging is accompanied by overexpression and activation of PKCalpha, UV irradiation and ROS production are associated with photoaging accompanied by PKCdelta downregulation and nuclear translocation.

Induction of G2/M arrest and apoptosis by sesquiterpene lactones in human melanoma cell lines.

Malignant melanoma is a highly aggressive tumor which frequently resists chemotherapy, therefore, the search for new agents for its treatment is of great importance. In this study, we purified the sesquiterpene lactones (SLs), Tomentosin and Inuviscolide from Inula viscosa (Compositae) leaves and studied their anti-cancer potency against human melanoma cell lines in order to develop new agents for melanoma treatment. SLs inhibited the proliferation of three human melanoma cell lines: SK-28, 624 mel and 1363 mel in a dose-dependent manner. We further investigated SLs mechanism of action using SK-28 as a representative cell line model. SLs caused cell-cycle arrest at G(2)/M, accompanied by the appearance of a sub-G0 fraction, indicative of apoptotic cell death. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential (DeltaPsi) and by detection of Caspase-3 activity. Rapid inhibitory phosphorylation of Cdc2 (Thr14 and Tyr15) was seen early after treatment, followed by a later decrease in the expression level of both Cyclin b1 and Cdc2. Induction of p53 and p21(waf1) proteins and phosphorylation of p53 at Ser15 were also detected early after treatment. The anti-apoptotic proteins, p65 subunit of nuclear factor kappaB (NF-kappaB), and Survivin were reduced in a dose-dependent manner. Taken together, these changes partially explain the ability of the SLs to induce G(2)/M arrest and apoptosis. Induction of apoptosis by Tomentosin and Inuviscolide in human aggressive melanoma cell lines has high pharmacological value and implies that SLs might be developed as new agents for melanoma treatment.

Cucurbitacin glucosides: antioxidant and free-radical scavenging activities.

The cucurbitacins are of great interest because of the wide range of biological activities they exhibit in plants and animals. We studied the antioxidant properties of cucurbitacin B + E glucosides (cucurbitacin glucoside combination, CGC) and their direct free-radical scavenging properties, using ESR spectroscopy. Antioxidant activity was measured by the ability of the CGC to reduce preformed ABTS*+ into its native form and to inhibit MDA formation during the oxidation of linoleic acid. In both methods, the CGC exhibited antioxidant activity in a dose-dependent manner as expected from antioxidants. Using ESR spectroscopy, we found that the CGC inhibited *OH-dependent DEPMPO-OH adduct formation, O2*--dependent DEPMPO-OOH adduct formation, and the 1O2-dependent TEMPO adduct generated in the photoradiation-porphin system. The IC50 values were 0.38, 8, and 11 mM, respectively. Together, these data demonstrate that the CGC exhibits antioxidant properties, probably through the involvement of a direct scavenging effect on several free radicals.

Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells.

Our aim was to study the effects of cucurbitacin glucosides extracted from Citrullus colocynthis leaves on human breast cancer cell growth. Leaves were extracted, resulting in the identification of cucurbitacin B/E glucosides. The cucurbitacin glucoside combination (1:1) inhibited growth of ER(+) MCF-7 and ER(-) MDA-MB-231 human breast cancer cell lines. Cell-cycle analysis showed that treatment with isolated cucurbitacin glucoside combination resulted in accumulation of cells at the G(2)/M phase of the cell cycle. Treated cells showed rapid reduction in the level of the key protein complex necessary to the regulation of G(2) exit and initiation of mitosis, namely the p34(CDC2)/cyclin B1 complex. cucurbitacin glucoside treatment also caused changes in the overall cell morphology from an elongated form to a round-shaped cell, which indicates that cucurbitacin treatment caused impairment of actin filament organization. This profound morphological change might also influence intracellular signaling by molecules such as PKB, resulting in inhibition in the transmission of survival signals. Reduction in PKB phosphorylation and inhibition of survivin, an anti-apoptosis family member, was observed. The treatment caused elevation in p-STAT3 and in p21(WAF), proven to be a STAT3 positive target in absence of survival signals. Cucurbitacin glucoside treatment also induced apoptosis, as measured by Annexin V/propidium iodide staining and by changes in mitochondrial membrane potential (DeltaPsi) using a fluorescent dye, JC-1. We suggest that cucurbitacin glucosides exhibit pleiotropic effects on cells, causing both cell cycle arrest and apoptosis. These results suggest that cucurbitacin glucosides might have therapeutic value against breast cancer cells.

Antioxidant activities and anthocyanin content of fresh fruits of common fig (Ficus carica L.).

Fig fruit has been a typical component in the health-promoting Mediterranean diet for millennia. To study the potential health-promoting constituents of fig fruits, six commercial fig varieties differing in color (black, red, yellow, and green) were analyzed for total polyphenols, total flavonoids, antioxidant capacity, and amount and profile of anthocyanins. Using reversed-phase liquid chromatography (RP-LC), various concentrations of anthocyanins but a similar profile was found in all varieties studied. Hydrolysis revealed cyanidin as the major aglycon. Proton and carbon NMR confirmed cyanidin-3-O-rhamnoglucoside (cyanidin-3-O-rutinoside; C3R) as the main anthocyanin in all fruits. Color appearance of fig extract correlated well with total polyphenols, flavonoids, anthocyanins, and antioxidant capacity. Extracts of darker varieties showed higher contents of phytochemicals compared to lighter colored varieties. Fruit skins contributed most of the above phytochemicals and antioxidant activity compared to the fruit pulp. Antioxidant capacity correlated well with the amounts of polyphenols and anthocyanins (R2 = 0.985 and 0.992, respectively). In the dark-colored Mission and the red Brown-Turkey varieties, the anthocyanin fraction contributed 36 and 28% of the total antioxidant capacity, respectively. C3R contributed 92% of the total antioxidant capacity of the anthocyanin fraction. Fruits of the Mission variety contained the highest levels of polyphenols, flavonoids, and anthocyanins and exhibited the highest antioxidant capacity.

Unique natural antioxidants (NAOs) and derived purified components inhibit cell cycle progression by downregulation of ppRb and E2F in human PC3 prostate cancer cells.

Prostate cancer (PCA) is the leading cause of cancer mortality among older men in Western countries. Epidemiological studies have shown correlation between a lower risk of PCA and a higher consumption of antioxidants. However, the mechanism by which antioxidants exert their effects is still unknown. In the present study, we explored the signaling mechanism through which unique natural antioxidant derived from spinach extract (NAO) exerts their beneficial effects in the chemoprevention of PCA using human PC3 cells. Probing into the effect of NAO and its derived polyphenols on cell-cycle G1 arrest, we found that they cause cell-cycle prolongation. NAO and its two derived purified components exhibited a significant increase in the level of p21cip1 expression after 36 h of starvation, followed by 18 h of treatment with NAO in the presence of serum. In addition, under similar conditions, the expressed level of Cyclin A and CDK-2 in the PC3 cells was significantly reduced after treatment with NAO or its purified components. Immunoblot analysis demonstrated a significant increase in the hypophosphorylated form of pRb and a decrease in ppRb. NAO and its purified derived components were found to downregulate the protein expression of another member of the pRb family, p107, as well as that of E2F-1. These results suggest that NAO-induced G1 delay and cell cycle prolongation are caused by downregulation of the protein expression of ppRb and E2F in the human PCA cell line PC3.

Scavenging of reactive oxygen species by a novel glucurinated flavonoid antioxidant isolated and purified from spinach.

NAO is a natural water soluble antioxidant that was isolated and purified from spinach leaves. Using HPLC, NMR, and CMR spectroscopy, the main components were identified as flavonoids and p-coumaric acid derivatives. The NAO was found to be a very effective antioxidant in several in vivo and in vitro biological systems. In the present study, the antioxidant activity of the novel antioxidant glucurinated flavonoid (GF) isolated and characterized from NAO, is compared to well-known antioxidants. In addition, the direct free radical scavenging properties of the purified component GF were studied using the electron spin resonance (ESR) technique. GF and NAO were found to be superior to EGCG and NAC and to the Vitamin E homologue Trolox in inhibiting reactive oxygen species (ROS) formation in the autooxidation system of linoleic acid and in fibroblasts exposed to metal oxidation. GF and NAO were found to inhibit the ESR signal intensity of DMPO-O(2) radical formation during the riboflavin photodynamic reaction. 10 mM GF caused approximately 90% inhibition in the intensity of the ESR signal, while NAO at a concentration of 60 microg/ml caused an inhibition of about 50%. Using the Fenton reaction, GF and NAO were found to inhibit DMPO-OH radical formation. A concentration of 2 mM GF caused a 70% inhibition in the intensity of the DMPO-OH radical ESR signal, while propyl gallate at the same concentration caused only 50% inhibition. Furthermore, both GF and NAO also inhibited the (1)O(2) dependent TEMPO radical generated in the photoradiation TPPS4 system. About 80% inhibition was obtained by 4 mM GF. The results obtained indicate that the natural antioxidants derived from spinach may directly affect the scavenging of ROS and, as a consequence, may be considered as effective sources for combating oxidative damage.

Some aspects of the ecology of the desert snail Sphincterochila prophetarum in relation to energy and water flow.

Between 1976-1978, we studied the ecology of the Sphincterochila prophetarum, the most common snail, on stony slopes in Northern Negev. Energy and water flow determinations were made at the individual and population levels on a field observation basis and metabolic data were derived from laboratory simulations.The snails are active during 5-7% of the time during the year, while the remaining 95% of the time, the snails are in aestivation under stones. The energy and water inputs during the short feeding periods are very fast. Only 8% of the total energy in the eaten food are assimilated. Eighty-nine percent of the total assimilated energy are utilized in respiration during the various periods of activity and dormancy.The water loss during the feeding and activity period is about 37% of the total annual input. The snails lose twice as much the amount of water during the winter dormancy periods than in the summer dormancy period.

Nucleoside 5'-phosphorothioate derivatives as oxidative stress protectants in PC12 cells.

Iron-induced oxidative damage of mitochondria contributes to cellular death seen in neurodegenerative diseases, therefore, there is a demand for nontoxic, biocompatible, and effective Fe-ion chelators. We evaluated the chelation of Fe(II) by phosphate derivatives using ferrozine as an indicator. We studied the effect of phosphate derivatives on inhibiting Fe(II)-induced oxidative stress in PC12 cells, and metabolic stability in PC12 cells was evaluated. Nucleotides containing phosphorothioate moieties inhibited ROS formation better than natural nucleotides and were more metabolically stable in PC12 cells. Finally, we elucidated that these nucleotides activate the MAP-kinase pathway that contributes to protection of PC12 cells under oxidative stress.

EPR studies of O(2)(*-), OH, and (1)O(2) scavenging and prevention of glutathione depletion in fibroblast cells by cyanidin-3-rhamnoglucoside isolated from fig (Ficus carica L.) fruits.

Cyanidin-3-rhamnoglucoside (C3R) is the major anthocyanin in fresh fig fruits. In this study, the free radical scavenging potential of C3R was evaluated in vitro using several free radical generators. This naturally occurring anthocyanin was superior to other tested natural antioxidants in scavenging ABTS(*+). Electron paramagnetic resonance served to determine the scavenging properties of C3R toward superoxide radical anion (O(2)(*-)), hydroxyl radical ((*)OH), and singlet radical ((1)O(2)). The protection of NIH-3T3 fibroblast cells was then tested as the inhibition of reactive oxygen species (ROS) formation in a dose-dependent manner. It was further demonstrated that treatment with C3R elevates the reduced glutathione (GSH) concentration and the redox ratio (GSH/GSSG) in fibroblast cells in a dose-dependent manner. Moreover, C3R reduced the induction of ROS by butathionine sulfoximine (BSO) and elevated the redox ratio. Thus, it is suggested that C3R in fresh fig fruits is a potent scavenger and may influence endogenous antioxidant systems of consumers.

Protection of fibroblasts (NIH-3T3) against oxidative damage by cyanidin-3-rhamnoglucoside isolated from fig fruits (Ficus carica L.).

Anthocyanins, plant secondary metabolites, have been recognized for their health-promoting properties when consumed by humans. In this study, the antioxidant properties of a major anthocyanin in fresh fig fruits, cyanidin-3-rhamnoglucoside (C3R), were evaluated by various assays in vitro and correlated with the protection afforded by C3R to cultured NIH-3T3 fibroblast cells. C3R inhibited lipid peroxidation from producing peroxy radicals (ROO(*)) and MDA in a dose-dependent manner, and a high calculated stoichiometric coefficient [n] for peroxy radicals was demonstrated. In addition to its scavenging of reactive oxygen species (ROS), C3R showed a strong chelating activity toward the Fe(2+) ion. Finally, pretreatment with C3R inhibited proapoptotic processes that were initiated by the oxidation of lysosome membranes in fibroblast cells. The high antioxidant potential, with several modes of action of purified C3R, may contribute to health benefits gained by the consumption of fresh fig fruits.

Differential attenuation of oxidative/nitrosative injuries in early prostatic neoplastic lesions in TRAMP mice by dietary antioxidants.

BACKGROUND: Dietary antioxidants with yet unproven efficacies in averting prostate cancer (PCa) are widely used in the United States as preventives. Experimental evidence establishing a causal relationship between oxidative and nitrosative stress (OS/NS) and PCa development and showing its modulation by dietary antioxidants would help justify their usage. METHODS: The TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) mouse model was used to demonstrate the OS/NS-associated damage, as evident by 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE)-protein-adducts and nitrotyrosine (Ntyr), in prostatic premalignant lesions, and to evaluate the antioxidant efficacy of various dietary supplements [natural antioxidant (NAO) from spinach extracts, (-) epigallocatechin-3-gallate (EGCG), or N-acetylcystein (NAC)] during the early PCa development. RESULT: We show, for the first time, that oxidative/nitrosative damages of genomic DNA and cellular proteins were discretely localized in premalignant lesions, but not in adjacent morphologically normal epithelia, of TRAMP prostates; these injuries were absent in age-matched nontransgenic littermates. The extent of OS/NS-related injuries correlated well with the tempo of development and prevalence of premalignant lesions in various prostatic lobes and exhibited a clear trend of increase from 12 to 20 weeks of age. Treatment of TRAMP mice with various antioxidants as dietary supplements resulted in differential alleviation of OS/NS-related prostatic injuries. The antioxidant potencies of the dietary supplements did not fully correlate with their documented antiPCa actions, suggest that they may exert additional "nonantioxidant," antitumor effects in this model. CONCLUSIONS: Our data indicate that in TRAMP mice, OS/NS injuries are likely involved in early prostatic tumorigenesis and can be modulated by various antioxidants.

Composition, efficacy, and safety of spinach extracts.

Spinach leaves, containing several active components, including flavonoids, exhibit antioxidative, antiproliferative, and antiinflammatory properties in biological systems. Spinach extracts have been demonstrated to exert numerous beneficial effects, such as chemo- and central nervous system protection and anticancer and antiaging functions. In this review article, we present a compilation of data generated in our laboratories and those of other investigators describing the chemical composition of spinach, its beneficial effects, relative safety information, and its recommended inclusion in the human diet. A powerful, water-soluble, natural antioxidant mixture (NAO), which specifically inhibits the lipoxygenase enzyme, was isolated from spinach leaves. The antioxidative activity of NAO has been compared to that of other known antioxidants and found to be superior in vitro and in vivo to that of green tea, N-acetylcysteine (NAC), butylated hydroxytoluene (BHT), and vitamin E. NAO has been tested for safety and is well tolerated in several species, such as mouse, rat, and rabbit. NAO has been found to be nonmutagenic and has shown promising anticarcinogenic effects in a few experimental models, such as skin and prostate cancer; it has not shown any target-organ toxicity or side effects. The current review provides epidemiological and preclinical data supporting the efficacy of extracts of spinach and the safety of its consumption.

A natural antioxidant mixture from spinach does not have estrogenic or antiestrogenic activity in immature CD-1 mice.

The use of natural antioxidants and flavonoids in nutritional and pharmaceutical applications is increasing. Because some phytochemicals such as genistein, found in soy products, have estrogenic activity, we investigated the estrogenic potential of a natural antioxidant mixture (NAO) isolated from spinach leaves, using an in vivo uterotrophic bioassay and an in vitro transcriptional activation assay for the estrogen receptor (ER). Outbred female CD-1 mice (17 d old) were given subcutaneous injections of 17beta-estradiol or genistein [500 and 500,000 microg /(kg x d), respectively] as positive controls or NAO [1000 to 1,000,000 microg/(kg x d)] for 3 d. Uterine wet weight/body weight ratios were determined. Both 17beta-estradiol and genistein significantly increased uterine wet weight ratios compared with untreated controls, but NAO did not. Histological examination of the uterus showed that 17beta-estradiol and genistein increased epithelial cell height, number and gland development, but NAO did not. Estrogenic activity of NAO was investigated in vitro using the ER transcriptional activation assay. BG1Luc4E2 cells expressing ER were stably transfected with a luciferase reporter gene responsive to estrogens. 17beta-estradiol dose dependently increased luciferase activity; NAO had no effect. When NAO was tested for antiestrogenic activity, it did not lessen the effects of 17beta-estradiol. These data suggest that NAO does not have estrogenic or antiestrogenic activity. Thus, an antioxidant mixture has been identified that does not have potentially adverse estrogenic activity.