Magdalena Kedzierska

An Extract from Berries of Aronia melanocarpa Modulates the Generation of Superoxide Anion Radicals in Blood Platelets from Breast Cancer Patients

Plant antioxidants protect cells against oxidative stress. Because oxidative stress (measured by different biomarkers) is observed in breast cancer patients, the aim of this study was to establish the effects of a polyphenol-rich extract of Aronia melanocarpa (final concentration of 50 microg/mL, 5 min, 37 degrees C) on superoxide anion radicals (O(2)(-*)) and glutathione (GSH) in platelets from patients with breast cancer and in a healthy group in vitro. Generation of O(2)(-*) in platelets before and after incubation with the extract was measured by cytochrome C reduction. Using HPLC, we determined the level of glutathione in blood platelets. We observed a statistically significant increase of biomarkers of oxidative stress such as O(2)(-*) and a decrease in GSH in platelets from patients with breast cancer compared with the healthy group. We showed that the extract from A. melanocarpa added to blood platelets significantly reduced the production of O(2)(-*) in platelets not only from the healthy group but also from patients with breast cancer. Considering the data presented in this study, we have demonstrated the protective role of the extract from A. melanocarpa in patients with breast cancer in vitro.

Comparative studies on homocysteine and its metabolite–homocysteine thiolactone action in blood platelets in vitro

Homocysteine (Hcy), an intermediate formed during the catabolism of the essential dietary amino acid methionine, and its cyclic thioester, homocysteine thiolactone (TL) formed from Hcy in plasma, may be implicated in pathological haemostasis and atherosclerosis. The mechanism by which TL exerts the prothrombotic effect and influences blood platelets remains unclear. Activation of blood platelets plays an important role in prothrombotic events. The aim of our study was to establish and compare the influence of a reduced form of homocysteine (at final doses of 10–100 µM) and its cyclic thioester, homocysteine thiolactone (0.1–1 µM), on platelet activation induced by thrombin (platelet aggregation), on platelet protein modifications (determined by parameters such as level of protein carbonyl groups, 3-nitrotyrosine residues in proteins) and on superoxide anion radicals () generation using the model system in vitro. We have observed that TL, like its precursor, Hcy, stimulates the generation of in platelets and causes an augmentation of platelet aggregation induced by thrombin. Our present results in vitro also demonstrate that Hcy (10–100 µM) and TL at lower doses than Hcy (0.1–1 µM) cause modification of platelet proteins: diminished formation of carbonyl groups and distinctly decreased tyrosine nitration in platelet proteins after thrombin stimulation, but increased platelet aggregation induced by thrombin. TL like Hcy (at concentrations corresponding to concentrations in blood during hyperhomocysteinemia) modifies platelet responses to an important physiological agonist–thrombin.

Effects of the commercial extract of aronia on oxidative stress in blood platelets isolated from breast cancer patients after the surgery and various phases of the chemotherapy.

Since the extract from berries of Aronia melanocarpa presents antioxidative properties in plasma and in blood platelets, not only from healthy group, but also from patients with benign breast diseases and in patients with invasive breast cancer before surgery, the aim of our present study was to evaluate the oxidative stress by measuring the level of various biomarkers of this process such as the generation of superoxide anion radicals (O(2)(-·)), the amount of carbonyl groups and 3-nitrotyrosine in proteins or the amount of glutathione in blood platelets isolated from breast cancer patients after the surgery and after various phases of the chemotherapy in the presence of A. melanocarpa extract (Aronox) in vitro. We demonstrated in platelet proteins from patients with invasive breast cancer (after the surgery and after various phases of the chemotherapy) higher level of carbonyl groups than in control healthy group. The level of 3-nitrotyrosine in platelet proteins from patients with invasive breast cancer was also significantly higher than in healthy subject group. We observed an increase of other biomarkers of oxidative stress such as O(2)(-·) and a decrease of GSH in platelets from patients with breast cancer (after the surgery and after various phases of the chemotherapy) compared to the healthy group. In model system in vitro our results showed that the commercial extract from berries of A. melanocarpa due to antioxidant action, significantly reduced the oxidative/nitrative stress in platelets from patients with invasive breast cancer caused by the surgery and various phases of the chemotherapy.

The nitrative and oxidative stress in blood platelets isolated from breast cancer patients: The protectory action of aronia melanocarpa extract

Since mechanisms involved in the relationship between oxidative stress and breast cancer are still unclear, the aim of our present study was to evaluate oxidative/nitrative modifications of blood platelet proteins by measuring the level of biomarkers of oxidative/nitrative stress such as carbonyl groups, thiol groups and 3-nitrotyrosine in proteins in patients with benign breast diseases and in patients with invasive breast cancer, and compare with the control group. Levels of carbonyl groups and 3-nitrotyrosine residues in platelet proteins were measured by ELISA and a competition ELISA, respectively. The method with 5,5′-dithio-bis(2-nitro-benzoic acid) has been used to analyse free thiol groups in platelet proteins. Patients were hospitalized in the Department of Oncological Surgery, Medical University of Lodz, Poland. Exogenous antioxidants reduce oxidative stress, therefore we also investigated in a model system in vitro the effects of a polyphenol rich extract of Aronia melanocarpa (Rosaceae, final concentration of 50 µg/ml, 5 min, 37°C) on modified blood platelet proteins as well from patients with breast cancer and from the healthy group. We demonstrated in platelet proteins from patients with invasive breast cancer a higher level of carbonyl groups than in the control healthy group (p < 0.02). The level of 3-nitrotyrosine in platelet proteins from patients with invasive breast cancer was also significantly higher than in the healthy subject group (p < 0.001). In contrast, the amount of thiol groups in platelet proteins from patients was significantly lower (about < 50%) than in control blood platelets. In a model system in vitro we also observed that the extract from berries of A. melanocarpa (50 µg/ml, 5 min, 37°C) due to antioxidant action, significantly reduced the oxidative/nitrative stress (measured by thiol groups and 3-nitrotyrosine) in platelets, not only from the healthy group, but also from patients with benign breast diseases and in patients with invasive breast cancer.

Effect of aronia on thiol levels in plasma of breast cancer patients

The various specific biomarkers of oxidative stress in plasma from patients with breast cancer, as well as biomarkers (the level of lipid hydroperoxides, conjugated dienes, thiobarbituric acid reactive substances) have been described. The aim of our present study was to evaluate the amount of low-molecular-weight thiols (which are physiological free radical scavengers) and establish the effects of the extract from A. melanocarpa on the amount of these thiols in plasma obtained from patients with invasive breast cancer, patients with benign breast diseases and from healthy volunteers. We observed in patients the higher amounts of homocysteine in plasma from patients in comparison to plasma from the control group; however the total level of glutathione, cysteine, cysteinylglycine, and the amount of thiols in reduced and oxidized forms was changed (e.g., in patients, the decrease of glutathione and cysteine reached about 50% of total values). Moreover, we showed that in the presence of the extract of A. melanocarpa (50 µg/mL, 5 min, 37°C), changes in amount of thiols in plasma from patients with invasive breast cancer and patients with benign breast diseases were significantly reduced in vitro. Considering the data presented in this study, we suggest that the extract from A. melanocarpa has an effect on thiol metabolism and the levels of all tested thiols observed in plasma obtained from breast cancer patients.

The changes of blood platelet activation in breast cancer patients before surgery, after surgery, and in various phases of the chemotherapy

Blood platelets from patients with cancer (before or after the surgery) exhibit a variety of qualitative abnormalities. Different anti-cancer drugs may also induce the oxidative/nitrative stress in blood platelets and change their hemostatic properties. The aim of our study was to explain the effect of superoxide anion radicals () production on hemostatic properties of blood platelets (activated by a strong physiological agonist – thrombin) from breast cancer patients before the surgery, after the surgery, and after various phases (I–IV) of chemotherapy (doxorubicin and cyclophosphamide). Patients were hospitalized in the Department of Oncological Surgery and at the Department of Chemotherapy, Medical University of Lodz, Poland. We measured the platelet aggregation as the marker of hemostatic activity of blood platelets. We observed an increase of in thrombin-activated blood platelets from patients with breast cancer (before or after the surgery and after various phases of the chemotherapy) compared to the healthy group. Our other experiments demonstrated that aggregation (induced by thrombin) of blood platelets from patients with breast cancer before the surgery, after the surgery, and after various phases of the chemotherapy differs from aggregation of platelets obtained from healthy volunteers. Moreover, our results showed the correlation between the generation and changes of platelet aggregation in breast cancer patients before the surgery, after the surgery, and after the chemotherapy (I and IV phases). Considering the data presented in this study, we suggest that the production of in blood platelets (activated by thrombin) obtained from breast cancer patients may induce the changes of platelet aggregation, which may contribute in thrombosis in these patients.

Effects of polyphenol-rich extract from berries of Aronia melanocarpa on the markers of oxidative stress and blood platelet activation

Bioactive substances found in numerous foods can be successfully and safely used to modify various cellular functions and affect the oxidative stress. Aronia melanocarpa fruits (Rosaceae) are one of the richest plant sources of phenolic substances shown to have anti-inflammatory, antitumor, antioxidative and antiplatelet activities. We investigated antioxidant properties of the extract from berries of A. melanocarpa by the estimation of the selected and other biomarkers of oxidative stress, i.e. the level of 8-epi-prostaglandin F2 (8-EPI) (by immunoassay kit) and the amount of glutathione (by HPLC method) in control platelets and platelets treated with H2O2. The expression of αIIbβ3 (a marker of platelet activation) was measured by flow cytometer. The antioxidative and antiplatelet properties of the tested extract were also compared with the action of a well characterized antioxidative and antiplatelet commercial monomeric polyphenol–resveratrol. The extract from berries of A. melanocarpa (at the highest tested concentration −100 µg/ml) decreased the production of 8-EPI (a marker of lipid peroxidation) in control blood platelets and platelets treated with H2O2 (2 mM). A combined action of the tested plant extract and H2O2 evoked a significant increase of reduced form of glutathione in platelets compared with cells treated with H2O2 only. Moreover, the tested plant extract (at the highest used concentration −100 µg/ml) reduced the expression of αIIbβ3 on blood platelets. Comparative studies indicate that the tested plant extract was found to be more reactive in blood platelets than the solution of pure resveratrol.

Changes of platelet antioxidative enzymes during oxidative stress: The protective effect of polyphenol-rich extract from berries of Aronia melanocarpa and grape seeds

Aronia melanocarpa fruits (Rosaceae) and grape seeds (seeds of Vitis vinifera, Vitaceae) are two of the richest plant sources of phenolic substances, and they have been shown to have various biological activities. The aim of the present study was to investigate and compare the action of phenolic extracts (at concentrations 5–100 µg/mL) of two different plants, berries of A. melanocarpa (chokebbery) and grape seeds, on the activities of various antioxidative enzymes, the amount of glutathione (as an important component of redox status) in control the platelets and platelets treated with H2O2 (the strong physiological oxidant) in vitro. The properties of these two tested extracts were also compared with the action of a well characterized antioxidative and antiplatelet commercial monomeric polyphenol – resveratrol. The extract from berries of A. melanocarpa, like the extract from grape seeds, reduced the changes in activities of different antioxidative enzymes (glutathione peroxidase, superoxide dismutase, and catalase) in platelets treated with H2O2. The action of the two tested plant extracts and H2O2 evoked a significant increase of reduced glutathione in platelets compared with platelets treated with H2O2 only. Comparative studies indicate that the two tested plant extracts had similar antioxidative properties, and were found to be more reactive in blood platelets than the solution of resveratrol.