A Deadly Liaison between Oxidative Injury and p53 Drives Methyl-Gallate-Induced Autophagy and Apoptosis in HCT116 Colon Cancer Cells.

Methyl gallate (MG), which is a gallotannin widely found in plants, is a polyphenol used in traditional Chinese phytotherapy to alleviate several cancer symptoms. Our studies provided evidence that MG is capable of reducing the viability of HCT116 colon cancer cells, while it was found to be ineffective on differentiated Caco-2 cells, which is a model of polarized colon cells. In the first phase of treatment, MG promoted both early ROS generation and endoplasmic reticulum (ER) stress, sustained by elevated PERK, Grp78 and CHOP expression levels, as well as an upregulation in intracellular calcium content. Such events were accompanied by an autophagic process (16-24 h), where prolonging the time (48 h) of MG exposure led to cellular homeostasis collapse and apoptotic cell death with DNA fragmentation and p53 and γH2Ax activation. Our data demonstrated that a crucial role in the MG-induced mechanism is played by p53. Its level, which increased precociously (4 h) in MG-treated cells, was tightly intertwined with oxidative injury. Indeed, the addition of N-acetylcysteine (NAC), which is a ROS scavenger, counteracted the p53 increase, as well as the MG effect on cell viability. Moreover, MG promoted p53 accumulation into the nucleus and its inhibition by pifithrin-α (PFT-α), which is a negative modulator of p53 transcriptional activity, enhanced autophagy, increased the LC3-II level and inhibited apoptotic cell death. These findings provide new clues to the potential action of MG as a possible anti-tumor phytomolecule for colon cancer treatment.

Anti-Eryptotic Activity of Food-Derived Phytochemicals and Natural Compounds.

Human red blood cells (RBCs), senescent or damaged due to particular stress, can be removed by programmed suicidal death, a process called eryptosis. There are various molecular mechanisms underlying eryptosis. The most frequent is the increase in the cytoplasmic concentration of Ca2+ ions, later exposure of erythrocytes to oxidative stress, hyperosmotic shock, ceramide formation, stimulation of caspases, and energy depletion. Phosphatidylserine (PS) exposed by eryptotic RBCs due to interaction with endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor, causes the RBCs to adhere to vascular wall with consequent damage to the microcirculation. Eryptosis can be triggered by various xenobiotics and endogenous molecules, such as high cholesterol levels. The possible diseases associated with eryptosis are various, including anemia, chronic kidney disease, liver failure, diabetes, hypertension, heart failure, thrombosis, obesity, metabolic syndrome, arthritis, and lupus. This review addresses and collates the existing ex vivo and animal studies on the inhibition of eryptosis by food-derived phytochemicals and natural compounds including phenolic compounds (PC), alkaloids, and other substances that could be a therapeutic and/or co-adjuvant option in eryptotic-driven disorders, especially if they are introduced through the diet.

Inhibitory effects of cynaropicrin on human melanoma progression by targeting MAPK, NF-κB, and Nrf-2 signaling pathways in vitro.

Malignant melanoma is the deadliest skin cancer, due to its propensity to metastasize. MAPKs and NF-κB pathways are constitutively activated in melanoma and promote cell proliferation, cell invasion, metastasis formation, and resistance to therapeutic regimens. Thus, they represent potential targets for melanoma prevention and treatment. Phytochemicals are gaining considerable attention for the management of melanoma because of their several cellular and molecular targets. A screening of a small library of sesquiterpenes lactones selected cynaropicrin, isolated from the aerial parts of Centaurea drabifolia subsp. detonsa, for its potential anticancer effect against melanoma cells. Treatment of human melanoma cells A375 with cynaropicrin resulted in inhibition of cell proliferation and induction of caspase-3-dependent apoptosis. Furthermore, cynaropicrin reduced several cellular malignant features such migration, invasion, and colonies formation through the inhibition of ERK1/2 and NF-κB activity. Cynaropicrin was able to reduce intracellular reactive oxygen species generation, which are involved in all the stages of carcinogenesis. Indeed, cynaropicrin increased the expression of several antioxidant genes, such as glutamate-cysteine ligase and heme oxygenase-1, by promoting the activation of the transcription factor Nrf-2. In conclusion, our results individuate cynaropicrin as a potential adjuvant chemotherapeutic agent for melanoma by targeting several protumorigenic signaling pathways.

Anti-Proliferative Activity of A Hydrophilic Extract of Manna from Fraxinus angustifolia Vahl through Mitochondrial Pathway-Mediated Apoptosis and Cell Cycle Arrest in Human Colon Cancer Cells.

Manna is produced from the spontaneous solidification of the sap of some Fraxinus species, and, owing its content in mannitol, is used in medicine as a mild laxative. Manna is also a rich source of characteristic bio-phenols with reducing, antioxidant and anti-inflammatory properties. This study assesses the activity of a hydrophilic extract of manna (HME) on cellular and molecular events in human colon-rectal cancer cells. HME showed a time- and concentration-dependent anti-proliferative activity, measured by MTT assay, in all the cell lines examined, namely Caco-2, HCT-116 and HT-29. The amounts of HME that caused 50% of cell death after a 24 h treatment were 8.51 ± 0.77, 10.73 ± 1.22 and 28.92 ± 1.99 mg manna equivalents/mL, respectively; no toxicity was observed in normally differentiated Caco-2 intestinal cells. Hydroxytyrosol, a component of HME known for its cytotoxic effects on colon cancer cells, was ineffective, at least at the concentration occurring in the extract. Through flow-cytometric techniques and Western blot analysis, we show that HME treatment causes apoptosis, assessed by phosphatidylserine exposure, as well as a loss of mitochondrial membrane potential, an intracellular formation of reactive oxygen species (ROS), increases in the levels of cleaved PARP-1, caspase 3 and Bax, and a decrease in Bcl-2 expression. Moreover, HME interferes with cell cycle progression, with a block at the G1/S transition. In conclusion, the phytocomplex extracted from manna exerts an anti-proliferative activity on human colon cancer cells through the activation of mitochondrial pathway-mediated apoptosis and cell cycle arrest. Our data may suggest that manna could have the potential to exert chemo-preventive effects for the intestine.

7-Keto-Cholesterol and Cholestan-3beta, 5alpha, 6beta-Triol Induce Eryptosis through Distinct Pathways Leading to NADPH Oxidase and Nitric Oxide Synthase Activation.

BACKGROUND/AIMS: We showed that patho-physiological concentrations of either 7-keto-cholesterol (7-KC), or cholestane-3beta, 5alpha, 6beta-triol (TRIOL) caused the eryptotic death of human red blood cells (RBC), strictly dependent on the early production of reactive oxygen species (ROS). The goal of the current study was to assess the contribution of the erythrocyte ROS-generating enzymes, NADPH oxidase (RBC-NOX), nitric oxide synthase (RBC-NOS) and xanthine oxido-reductase (XOR) to the oxysterol-dependent eryptosis and pertinent activation pathways. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, reactive oxygen/nitrogen species (RONS) and nitric oxide formation from 2',7'-dichloro-dihydrofluorescein (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) -dependent fluorescence, respectively; Akt1, phospho-NOS3 Ser1177, and PKCζ from Western blot analysis. The activity of individual 7-KC (7 µM) and TRIOL (2, µM) on ROS-generating enzymes and relevant activation pathways was assayed in the presence of Diphenylene iodonium chloride (DPI), N-nitro-L-arginine methyl ester (L-NAME), allopurinol, NSC23766 and LY294002, inhibitors in this order of RBC-NOX, RBC-NOS, XOR and upstream regulatory proteins Rac GTPase and phosphoinositide3 Kinase (PI3K); hemoglobin oxidation from spectrophotometric analysis. RESULTS: RBC-NOX was the target of 7-KC, through a signaling including Rac GTPase and PKCζ, whereas TRIOL caused activation of RBC-NOS according to the pathway PI3K/Akt, with the concurrent activity of a Rac-GTPase. In concomitance with the TRIOL-induced .NO production, formation of methemoglobin with global loss of heme were observed, ascribable to nitrosative stress. XOR, activated after modification of the redox environment by either RBC-NOX or RBC-NOS activity, concurred to the overall oxidative/nitrosative stress by either oxysterols. When 7-KC and TRIOL were combined, they acted independently and their effect on ROS/RONS production and PS exposure appeared the result of the effects of the oxysterols on RBC-NOX and RBC-NOS. CONCLUSION: Eryptosis of human RBCs may be caused by either 7-KC or TRIOL by oxidative/nitrosative stress through distinct signaling cascades activating RBC-NOX and RBC-NOS, respectively, with the complementary activity of XOR; when combined, the oxysterols act independently and both concur to the final eryptotic effect.

Quality, functional and sensory evaluation of pasta fortified with extracts from Opuntia ficus-indica cladodes.

BACKGROUND: The stems of Opuntia ficus-indica, known as cladodes, are a rich source of soluble fibers, which makes them an important candidate for the production of functional foods. Tagliatelle of durum wheat fortified with Opuntia cladode extract (OCE) at different levels of addition (10-30%, v/w) was prepared on a laboratory scale and quality characteristics and sensory acceptability were assessed. RESULTS: The main quality parameters (optimal cooking time, swelling index, cooking loss, dry matter) and sensory analysis on a nine-point hedonic scale were comparable with those of the control pasta sample (no added OCE) when durum wheat was supplemented with OCE at up to 20% (v/w). An in vitro human simulated gastrointestinal digestion in the presence of cholesterol or its main dietary oxidized derivative, 7-ketocholesterol, showed that OCE-fortified pasta strongly reduced the bioaccessibility of both the sterols (the higher the incorporated OCE level, the higher the effect). Moreover the extent of starch digestion decreased with increasing levels of OCE. CONCLUSION: The results of the present study indicate that OCE-fortified pasta comprises a food with healthy properties, such as blood cholesterol- and glucose-lowering capabilities. © 2019 Society of Chemical Industry.

Indicaxanthin from Opuntia Ficus Indica (L. Mill) impairs melanoma cell proliferation, invasiveness, and tumor progression.

BACKGROUND: A strong, reciprocal crosstalk between inflammation and melanoma has rigorously been demonstrated in recent years, showing how crucial is a pro-inflammatory microenvironment to drive therapy resistance and metastasis. PURPOSE: We investigated on the effects of Indicaxanthin, a novel, anti-inflammatory and bioavailable phytochemical from Opuntia Ficus Indica fruits, against human melanoma both in vitro and in vivo. STUDY DESIGN AND METHODS: The effects of indicaxanthin were evaluated against the proliferation of A375 human melanoma cell line and in a mice model of cutaneous melanoma. Cell proliferation was assessed by MTT assay, apoptosis by Annexin V-Fluorescein Isothiocyanate/Propidium Iodide staining, protein expression by western blotting, melanoma lesions were subcutaneously injected in mice with B16/F10 cells, chemokine release was quantified by ELISA. RESULTS: Data herein presented demonstrate that indicaxanthin effectively inhibits the proliferation of the highly metastatic and invasive A375 cells as shown by growth inhibition, apoptosis induction and cell invasiveness reduction. More interestingly, in vitro data were paralleled by those in vivo showing that indicaxanthin significantly reduced tumor development when orally administered to mice. The results of our study also clarify the molecular mechanisms underlying the antiproliferative effect of indicaxanthin, individuating the inhibition of NF-κB pathway as predominant. CONCLUSION: In conclusion, we demonstrated that indicaxanthin represents a novel phytochemical able to significantly inhibit human melanoma cell proliferation in vitro and to impair tumor progression in vivo. When considering the resistance of melanoma to the current therapeutical approach and the very limited number of phytochemicals able to partially counteract it, our findings may be of interest to explore indicaxanthin potential in further and more complex melanoma studies in combo therapy, i.e. where different check points of melanoma development are targeted.

Short-term cactus pear [Opuntia ficus-indica (L.) Mill] fruit supplementation ameliorates the inflammatory profile and is associated with improved antioxidant status among healthy humans.

BACKGROUND: Dietary ingredients and food components are major modifiable factors protecting immune system and preventing the progression of a low-grade chronic inflammation responsible for age-related diseases. OBJECTIVE: Our study explored whether cactus pear (Opuntia ficus-indica, Surfarina cultivar) fruit supplementation modulates plasma inflammatory biomarkers in healthy adults. Correlations between inflammatory parameters and antioxidant status were also assessed in parallel. DESIGN: In a randomised, 2-period (2 weeks/period), crossover, controlled-feeding study, conducted in 28 healthy volunteers [mean age 39.96 (±9.15) years, BMI 23.1 (±1.5) kg/m2], the effects of a diet supplemented with cactus pear fruit pulp (200 g, twice a day) were compared with those of an equivalent diet with isocaloric fresh fruit substitution. RESULTS: With respect to control, cactus pear diet decreased ( p < 0.05) the pro-inflammatory markers such as tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, interferon-γ (INF)-γ, IL-8, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), whereas it increased ( p < 0.05) the anti-inflammatory marker IL-10. Moreover, the diet decreased ratios between pro-inflammatory biomarkers (CRP, IL-6, IL-8, TNF-α) and anti-inflammatory biomarker (IL-10) ( p < 0.05). Cactus pear supplementation caused an increase ( p < 0.05) in dermal carotenoids (skin carotenoid score, SCS), a biomarker of the body antioxidant status, with correlations between SCS and CRP (r = -0.905, p < 0.0001), IL-8 (r = -0.835, p < 0.0001) and IL-10 (r = 0.889, p < 0.0001). CONCLUSIONS: The presently observed modulation of both inflammatory markers and antioxidant balance suggests cactus pear fruit as a novel and beneficial component to be incorporated into current healthy dietary habits.

Indicaxanthin from cactus pear fruit exerts anti-inflammatory effects in carrageenin-induced rat pleurisy.

Nutritional research has shifted recently from alleviating nutrient deficiencies to chronic disease prevention. We investigated the activity of indicaxanthin, a bioavailable phytochemical of the betalain class from the edible fruit of Opuntia ficus-indica (L. Miller) in a rat model of acute inflammation. Rat pleurisy was achieved by injection of 0.2 mL of λ-carrageenin in the pleural cavity, and rats were killed 4, 24, and 48 h later; exudates were collected to analyze inflammatory parameters, such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α); cells recruited in pleura were analyzed for cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) expression, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation. Indicaxanthin (0.5, 1, or 2 µmol/kg), given orally before carrageenin, time- and dose-dependently, reduced the exudate volume (up to 70%) and the number of leukocytes recruited in the pleural cavity (up to 95%) at 24 h. Pretreatment with indicaxanthin at 2 µmol/kg inhibited the carrageenin-induced release of PGE(2) (91.4%), NO (67.7%), IL-1ß (53.6%), and TNF-α (71.1%), and caused a decrease of IL-1ß (34.5%), TNF-α (81.6%), iNOS (75.2%), and COX2 (87.7%) mRNA, as well as iNOS (71.9%) and COX-2 (65.9%) protein expression, in the recruited leukocytes. Indicaxanthin inhibited time- and dose- dependently the activation of NF-κB, a key transcription factor in the whole inflammatory cascade. A pharmacokinetic study with a single 2 µmol/kg oral administration showed a maximum 0.22 ± 0.02 µmol/L (n = 15) plasma concentration of indicaxanthin, with a half-life of 1.15 ± 0.11 h. When considering the high bioavailability of indicaxanthin in humans, our findings suggest that this dietary pigment has the potential to improve health and prevent inflammation-based disorders.

Phytochemical indicaxanthin suppresses 7-ketocholesterol-induced THP-1 cell apoptosis by preventing cytosolic Ca(2+) increase and oxidative stress.

7-Ketocholesterol (7-KC)-induced apoptosis of macrophages is considered a key event in the development of human atheromas. In the present study, the effect of indicaxanthin (Ind), a bioactive pigment from cactus pear fruit, on 7-KC-induced apoptosis of human monocyte/macrophage THP-1 cells was investigated. A pathophysiological condition was simulated by using amounts of 7-KC that can be reached in human atheromatous plaque. Ind was assayed within a micromolar concentration range, consistent with its plasma level after dietary supplementation with cactus pear fruit. Pro-apoptotic effects of 7-KC were assessed by cell cycle arrest, exposure of phosphatidylserine at the plasma membrane, variation of nuclear morphology, decrease of mitochondrial trans-membrane potential, activation of Bcl-2 antagonist of cell death and poly(ADP-ribose) polymerase-1 cleavage. Kinetic measurements within 24 h showed early formation of intracellular reactive oxygen species over basal levels, preceding NADPH oxidase-4 (NOX-4) over-expression and elevation of cytosolic Ca²âº, with progressive depletion of total thiols. 7-KC-dependent activation of the redox-sensitive NF-κB was observed. Co-incubation of 2·5 µm of Ind completely prevented 7-KC-induced pro-apoptotic events. The effects of Ind may be ascribed to inhibition of NOX-4 basal activity and over-expression, inhibition of NF-κB activation, maintaining cell redox balance and Ca homeostasis, with prevention of mitochondrial damage and consequently apoptosis. The findings suggest that Ind, a highly bioavailable dietary phytochemical, may exert protective effects against atherogenetic toxicity of 7-KC at a concentration of nutritional interest.

Antioxidant activity of Sicilian pistachio (Pistacia vera L. var. Bronte) nut extract and its bioactive components.

Pistacia vera L. is the only species of Pistacia genus producing edible nuts. This paper investigates the antioxidant potential of a Sicilian variety of pistachio nut by chemical as well as biological assays and measured antioxidant vitamins and a number of antioxidant polyphenols in either the hydrophilic and/or the lipophilic nut extract. In accordance with the majority of foods, the total antioxidant activity, measured as a TAA test, was much higher (50-fold) in the hydrophilic than in the lipophilic extract. Substantial amounts of total phenols were measured. The hydrophilic extract inhibited dose-dependently both the metal-dependent and -independent lipid oxidation of bovine liver microsomes, and the Cu+2-induced oxidation of human low-density lipoprotein (LDL). Peroxyl radical-scavenging as well as chelating activity of nut components may be suggested to explain the observed inhibition patterns. Among tocopherols, gamma-tocopherol was the only vitamin E isomer found in the lipophilic extract that did not contain any carotenoid. Vitamin C was found only in a modest amount. The hydrophilic extract was a source of polyphenol compounds among which trans-resveratrol, proanthocyanidins, and a remarkable amount of the isoflavones daidzein and genistein, 3.68 and 3.40 mg per 100 g of edible nut, respectively, were evaluated. With the exception of isoflavones that appeared unmodified, the amounts of other bioactive molecules were remarkably reduced in the pistachio nut after roasting, and the total antioxidant activity decreased by about 60%. Collectively, our findings provide evidence that the Sicilian pistachio nut may be considered for its bioactive components and can effectively contribute to a healthy status.

Supplementation with cactus pear (Opuntia ficus-indica) fruit decreases oxidative stress in healthy humans: a comparative study with vitamin C.

BACKGROUND: Cactus pear (Opuntia ficus-indica) fruit contains vitamin C and characteristic betalain pigments, the radical-scavenging properties and antioxidant activities of which have been shown in vitro. OBJECTIVE: We investigated the effects of short-term supplementation with cactus pear fruit compared with vitamin C alone on total-body oxidative status in healthy humans. DESIGN: In a randomized, crossover, double-treatment study, 18 healthy volunteers received either 250 g fresh fruit pulp or 75 mg vitamin C twice daily for 2 wk, with a 6-wk washout period between the treatments. Before (baseline) and after each treatment, 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)) and malondialdehyde in plasma, the ratio of reduced to oxidized glutathione (GSH:GSSG) in erythrocytes, and lipid hydroperoxides in LDL were measured as biomarkers of oxidative stress; plasma Trolox-equivalent antioxidant activity (TEAC) and vitamins A, E, and C were evaluated as indexes of antioxidant status. RESULTS: Both treatments caused comparable increases compared with baseline in plasma concentrations of vitamin E and vitamin C (P < 0.05); vitamin A and TEAC did not change significantly. After supplementation with cactus pear fruit, 8-epi-PGF(2)alpha and malondialdehyde decreased by approximately 30% and 75%, respectively; GSH:GSSG shifted toward a higher value (P < 0.05); and LDL hydroperoxides were reduced by almost one-half. Supplementation with vitamin C did not significantly affect any marker of oxidative stress. CONCLUSIONS: Consumption of cactus pear fruit positively affects the body's redox balance, decreases oxidative damage to lipids, and improves antioxidant status in healthy humans. Supplementation with vitamin C at a comparable dosage enhances overall antioxidant defense but does not significantly affect body oxidative stress. Components of cactus pear fruit other than antioxidant vitamins may play a role in the observed effects.

Antioxidant activities of sicilian prickly pear (Opuntia ficus indica) fruit extracts and reducing properties of its betalains: betanin and indicaxanthin.

Sicilian cultivars of prickly pear (Opuntia ficus indica) produce yellow, red, and white fruits, due to the combination of two betalain pigments, the purple-red betanin and the yellow-orange indicaxanthin. The betalain distribution in the three cultivars and the antioxidant activities of methanolic extracts from edible pulp were investigated. In addition, the reducing capacity of purified betanin and indicaxanthin was measured. According to a spectrophotometric analysis, the yellow cultivar exhibited the highest amount of betalains, followed by the red and white ones. Indicaxanthin accounted for about 99% of betalains in the white fruit, while the ratio of betanin to indicaxanthin varied from 1:8 (w:w) in the yellow fruit to 2:1 (w:w) in the red one. Polyphenol pigments were negligible components only in the red fruit. When measured as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalents per gram of pulp, the methanolic fruit extracts showed a marked antioxidant activity. Vitamin C did not account for more than 40% of the measured activity. In addition, the extracts dose-dependently inhibited the organic hydroperoxide-stimulated red cell membrane lipid oxidation, as well as the metal-dependent and -independent low-density lipoprotein oxidation. The extract from the white fruit showed the highest protection in all models of lipid oxidation. Purified betanin and indicaxanthin were more effective than Trolox at scavenging the [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] diammonium salt cation radical. Cyclic voltammetric measurements show two anodic waves for betanin and indicaxanthin, and differential pulse voltammetry shows three anodic waves for betanin, with calculated peak potentials of 404, 616, and 998 mV, and two anodic waves for indicaxanthin, with peak potentials of 611 and 895 mV. Betanin underwent complex formation through chelation with Cu(2+), whereas indicaxanthin was not modified. These findings suggest that the above betalains contribute to the antioxidant activity of prickly pear fruits.