Chemical Composition and Protective Possibilities of Juglans Nigra Leaves and Green Husks Extracts: DNA Binding and Micronucleus Assay in Human Lymphocytes.

To better understand the mechanism of action of the compounds in the ethanolic extracts of J. nigra leaves and green husks, their binding to CT-DNA was investigated. This study was conducted to elucidate the in vitro protective effect of extracts against chromosomal damage in mitogen-induced human lymphocytes and investigate the possible application of selec+ted extracts as a natural source of polyphenolic compounds. Using HPLC-MS analysis, 103 different compounds were identified as having a higher number of active species, which is consistent with their activity. The frequency of micronuclei (MN) was scored in binucleated cells, and the nuclear proliferation index was calculated. Cyclic voltammetry experiments demonstrate that the nature of the interaction between extracts and CT-DNA is a synergy of electrostatic and intercalative modes, where leaves extracts showed a higher ability to bind to DNA. Extracts showed excellent antioxidant activity. At a concentration of only 4 µg/mL, extract of J. nigra leaves and the green husks reduced the incidence of MN by 58.2% and 64.5%, respectively, compared to control cell cultures.

Optimization of radioprotective dose of Juglans nigra leaf extract using response surface methodology.

J. nigra leaf contains mixture of various pharmacologically active compounds and it is assumed that they may have the potential radioprotective effect. The purpose of this work was to predict radioprotective doses by correlating changes in organ distribution of radiopharmaceuticals with extract dose levels and rat body weight using response surface methodology (RSM) based on a second-order polynomial equation. The extract was administered daily by oral gavage to rats at dose of 6.9, 10.3, or 13.7 mg kg-1 body weight (bw) day-1 for 10 days. On the eleventh day, 0.1 ml of the one radiopharmaceutical (Na99mTcO4, 99mTc-dimercaptosuccinic acid and 99mTc-tin colloid) was injected into the tail vein. The statistical parameters: the coefficient of determination (0.81-0.95), the coefficient of variation (3.05-11.1), the adequate precision (8.82-19.12) and the mean relative percentage deviation (± 2.3-8.2) were indicated the precision and reliability of RSM. Using RSM, the predicted daily dose of leaf extract ranging from 11.19 to 11.99 mg kg-1 bw may be considered as an optimal daily radiopotective dose for protection of organs from radiation in cases of planned radiation exposures.

Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up.

We evaluated urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) prognostic value in postmenopausal, node-negative breast cancer patients bearing tumors with estrogen receptor (ER)/progesterone receptor (PR) expression, treated with locoregional therapy alone, within an early follow-up. We focused our analysis on tumors of histological grade II in order to improve its prognostic value and, consequently, to improve a decision-making process. The cytosol extracts of 73 tumor samples were used for assessing several biomarkers. ER and PR levels were measured by classical biochemical method. Cathepsin D was assayed by a radiometric immunoassay while both uPA and PAI-1 level determinations were performed by enzyme-linked immunosorbent assays. HER-2 gene amplification was determined by chromogenic in situ hybridization (CISH) in primary tumor tissue. Patients bearing tumors smaller than or equal to 2 cm (pT1) or those with low PAI-1 levels (PAI-1 < 6.35 pg/mg) showed favorable outcome compared to patients bearing tumors greater than 2 cm (pT2,3) or those with high PAI-1 levels, respectively. Analyses of 4 phenotypes, defined by tumor size and PAI-1 status, revealed that patients bearing either pT1 tumors, irrespective of PAI-1 levels, or pT2,3 tumors with low PAI-1 levels, had similar disease-free interval probabilities and showed favorable outcome compared to those bearing pT2,3 tumors with high PAI-1 levels. Our findings suggest that tumor size and PAI-1, used in combination as phenotypes are not only prognostic but might also be predictive in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up period.

Trefoil factor 1 in early breast carcinoma: a potential indicator of clinical outcome during the first 3 years of follow-up.

BACKGROUND: A role of an estrogen-regulated, autocrine motogenic factor was assumed to be a major biological role of trefoil factor 1 (TFF1) in breast cancer. TFF1 is regarded as a predictive factor for positive response to endocrine therapy in breast cancer patients. The aim of our study was to examine TFF1 level distribution in breast carcinomas in order to distinguish estrogen-independent from estrogen-dependent TFF1 expression and to evaluate clinical usefulness of TFF1 status in early breast cancer during the first 3 years of follow-up. METHODS: The study included 226 patients with primary operable invasive early breast carcinomas for whom an equal, a 3-year follow-up was conducted. TFF1 levels as well as estrogen receptor (ER) and progesterone receptor (PR) levels were measured in cytosolic extracts of tumor samples by immunoradiometric assay or by use of classical biochemical method, respectively. Non-parametric statistical tests were applied for data analyses. RESULTS: Statistical analysis revealed that TFF1 levels were significantly higher in premenopausal patients (p=0.02), or in tumors with: lower histological grade (p<0.001), positive ER or PR status (p<0.001, in both cases). On the basis of TFF1 level distribution between ER-negative and ER-positive postmenopausal patients with tumors of different histological grade, 14 ng/mg was set as the cut-off value to distinguish estrogen-independent from estrogen-dependent TFF1 expression in breast cancer. Depending on menopausal and PR status, positive TFF1 status identified patients at opposite risk for relapse among ER-positive patients with grade II tumors. Among ER- and PR-positive premenopausal patients with grade II tumors, TFF1 status alone identified patients at opposite risk for relapse. CONCLUSIONS: Determination of TFF1 status might identify patients at different risk for relapse and help in making decision on administering adjuvant therapy for early breast cancer patients during the first 3 years of follow-up.

Cathepsin D as an indicator of clinical outcome in early breast carcinoma during the first 3 years of follow-up.

AIM: The aim of this study was to evaluate clinical usefulness of cathepsin D status in early breast cancer during the first 3 years of follow-up. PATIENTS & METHODS: The study included 226 patients with histologically verified, primary operable invasive early breast carcinomas. Concentrations of estrogen receptor (ER) and progesterone receptor (PR) in breast tumor cytosols were measured by use of the classical biochemical method. The concentration of three cathepsin D forms (52-, 48- and 34-kDa proteins) was determined by a radioimmunoassay RESULTS: On the basis of differences in cathepsin D levels either within an ER(-)/PR(-) phenotype or between this and either ER(+)/PR(+) or ER(+)/PR(-) phenotypes, a concentration of 39 pmol/mg was determined as the cutoff value for distinguishing estrogen-regulated cathepsin D expression. Estrogen-regulated cathepsin D expression was recognized as a high-risk biomarker for low-risk (histological grade I) breast cancer patients and as a low-risk biomarker for high-risk patients (pN(+) pT2,3). CONCLUSION: Determination of cathepsin D status in breast cancer might identify patients at different risk for relapse and might facilitate the selection of more or less aggressive adjuvant therapy for early breast cancer patients during the first 3 years of follow-up.

Potential clinical significance of ERß ON promoter methylation in sporadic breast cancer.

The aim of the study was to assess how hypermethylation of the ON promoter of the estrogen receptor beta (ERß) gene affects its expression (at the mRNA and protein level) and to correlate these with some clinical and histopathological parameters. A total of 131 samples of frozen breast cancer tissue was analyzed. A custom-designed, two-step PCR method was used to measure the methylation index of the ERß gene ON promoter region. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to quantify mRNA of the ERß1 isoform, while ERß1 protein was determined using the Western blot method. There was a significant difference in the methylation index of the ERß gene ON promoter between the groups of patients with negative and positive axillary lymph node status (P = 0.03). In addition, the methylation index of the ON promoter was positively correlated with estrogen receptor alfa (ERα) protein levels (ρ = 0.31, P = 0.02). There was a significant difference in the methylation index of the ON promoter between the progesterone receptor (PR)-negative and PR-positive groups of patients (P = 0.01). ERß1 protein levels were negatively correlated with ERα protein (ρ = -0.27, P < 0.01). The methylation index of the ON promoter could be a more reliable additional parameter for prediction and/or prognosis in breast cancer than ERß1-mRNA and/or protein levels.

Estrogen-regulated cut-off values of pS2 and cathepsin D expression in breast carcinomas.

The purpose of the study was to asses the expression of estrogen-induced pS2 and cathepsin D (CD) that might facilitate biological subgrouping of patients with breast carcinomas (BC) and its potential applicability in clinical oncology. The study included 226 patients with histologically verified BC. Clinico-pathological findings were classified according to age, menopausal status, tumor size, histologic grade, and regional lymph node status. Estrogen and progesterone receptors (ER and PR), as well as CD and pS2 protein concentrations were assayed on the same cytosolic extract in accordance with the recommendation of EORTC. Statistically significant direct correlations were observed between CD expression and axillary node status and between pS2 expression and histologic grade, while the expression of both proteins was related to both ER and PR status. Baseline levels of CD expression were found in patients with SR-negative status and node-negative or tumors less than 2cm. Unfavorable carcinoma subgroups, in relation to pS2 expression, were defined as pre- and postmenopausal carcinomas with histologic grade III. The highest CD level observed in SR-negative unfavorable subgroups (38.7 pmol/mg) and the highest pS2 level observed in ER(-) unfavorable subgroups (14.7 ng/mg) were considered as the cut-off values. These values defined estrogen-regulated expression of CD and pS2 protein that might enable the identification of patients at high risk of disease progression, for whom more aggressive adjuvant approach would be warranted, as well as the identification of patients whose prognosis is so good that adjuvant therapy would not be cost-beneficial.