Antioxidative effect of dietary flavonoid isoquercitrin on human ovarian granulosa cells HGL5 in vitro.

This study aimed to examine the effect of dietary flavonoid isoquercitrin on ovarian granulosa cells using the immortalized human cell line HGL5. Cell viability, survival, apoptosis, release of steroid hormones 17beta-estradiol and progesterone, and human transforming growth factor-beta2 (TGF-beta2) and TGF-beta2 receptor as well as intracellular reactive oxygen species (ROS) generation were investigated after isoquercitrin treatment at the concentration range of 5-100 microg.ml-1. It did not cause any significant change (p>0.05) in cell viability as studied by AlamarBlue assay in comparison to control. No significant change was observed (p>0.05) in the proportion of live, dead and apoptotic cells as revealed by apoptotic assay using flow cytometry. Similarly, the release of 17beta-estradiol, progesterone, TGF-beta2 and its receptor were not affected significantly (p>0.05) by isoquercitrin as detected by ELISA, in comparison to control. Except for the highest concentration of 100 microg.ml-1, which led to oxidative stress, isoquercitrin exhibited antioxidative activity at lower concentration used in the study (5, 10, 25, and 50 microg.ml-1) by hampering the production of intracellular ROS, in comparison to control, as detected by chemiluminescence assay (p<0.05). Findings of the present study indicate an existence of the antioxidative pathway that involves inhibition of intracellular ROS generation by isoquercitrin in human ovarian granulosa cells.

Detection of circulating tumor cells in metastatic breast cancer patients.

OBJECTIVE: The objective of this study was the detection of circulating tumor cells (CTCs) in metastatic breast cancer patients. METHODS: Since only small numbers of circulating tumor cells (CTCs) are found in peripheral blood, at first we performed immunomagnetic separation as a concentration method suitable for selecting circulating tumor cells in peripheral blood. This was followed by analysis of isolated cells with the aid of laser scanning cytometry (LSC). Twenty eight patients with metastatic breast cancer were enrolled in the study and the control group consisted of 19 clinically healthy women. Six milliliters of peripheral blood was drawn for the analyses, but only in two patients the blood has been drawn twice. Blood samples were taken when no chemotherapy was administered, but hormonal therapy has been allowed. RESULTS: The positivity for CTCs was found in 20 (50.0 %) patients with metastatic breast cancer patients, while in 6 (31.6 %) healthy controls false positive circulating epithelial-like cells were detected. Because we did not use CD45 staining, we could not distinguish these circulating epithelial-like cells from CTCs. In a majority of metastatic breast cancer patients we found a mixed population of HER-2 gene expressing CTCs. We found that HER2+ CTCs in high numbers are CK19 + CTCs, while almost all HER2-CTCs are CK19- CTCs. CONCLUSION: The described method was found promising for estimating HER2 status on CTCs from peripheral blood in metastatic breast cancer patients.

Circulating tumour cells in breast cancer--review.

Disseminated malignancies are responsible for the majority of cancer-related deaths. During the metastatic process, circulating tumour cells (CTCs) are generated. The presence of CTCs, epithelial cells found in the peripheral blood, is an essential step in establishing distant metastases. Circulating epithelial cells have the morphology of malignant cells and their number in the blood correlates with tumour burden. To identify CTCs in peripheral blood, two major approaches are used involving additional antibodies and nucleic acid-based techniques. Tumour cells with HER-2 overexpression are frequently resistant to cytotoxic drugs and radiotherapy. Wider clinical application of the detection of minimal residual disease is partly limited by the lack of standardized methods for detection. Recent studies suggest that in addition to the prognostic significance of tumour cells, determination of CTCs may be important in therapy monitoring or as potential targets for targeted therapy. Persistence of minimal residual disease after primary treatment may be an indication for extensive adjuvant treatment in order to prevent relapse of the disease. Detection of CTCs and the use of prognostic markers such as HER-2 overexpression may help us to better understand the biology and clinical significance of the presence of CTCs in breast cancer patients.

Chromatin texture, DNA index, and S-phase fraction in primary breast carcinoma cells analysed by laserscanning cytometry.

OBJECTIVES: Laser scanning cytometry (LSC) is a slide-based technique capable of measuring a number of biological parameters both in immobilised cell suspensions and in formalin-fixed paraffin-embedded tissue sections. BACKGROUND: High proliferation rate in surgically removed breast tumours is an unfavourable prognostic factor. In node negative cases it can help distinguish patients with higher risk for distant metastases from those with a lower risk. PATIENTS AND METHODS: In a prospective study we investigated 140 breast tumours, of which 113 were invasive ductal carcinomas, 11 were invasive lobular carcinomas, and 16 tumours were of other histological types. Cells for LSC investigations were prepared from fresh, surgically removed tumours by mechanical disintegration. After fixation the cells were stained with FITC-conjugated anti-cytokeratin (CK-FITC) to distinguish CK+ tumour cells from CK- stroma, and with propidium iodide to stain DNA. RESULTS: We identified three S-phase fraction (SPF) groups, with low (30 patients), moderate (54 patients), and high SPF (51 patients). Thirty-seven tumours were diploid, 83 were aneuploid, while 5 tumours had a bimodal distribution of DNA content. Chromatin texture values were increasing in the respective subclasses from the hypodiploid group to the tetraploid/hypertetraploid group. CONCLUSION: The measurement of DNA content and SPF of tumours by LSC completed by and correlated with other biological properties of the tumour cells may be a useful tool in assessing prognosis and clinical outcome of patients with breast cancer. (Tab. 5, Fig. 4, Ref. 18). Full Text (Free, PDF) www.bmj.sk.

Immunity profile in breast cancer patients.

OBJECTIVES: Despite the multifactorial pathogenesis of malignant transformation, it is assumed that deficiency in some immune mechanisms plays a considerable role in its development. BACKGROUND: Chronically activated immune cells exert tumour-promoting effects directly by influencing the proliferation and survival of neoplastic cells, as well as by indirect modulation of neoplastic microenvironments in favour of tumour progression. PATIENTS AND METHODS: We refer to results of two separate investigations that aim to monitor the immune functions in patients with breast cancer. In the first investigation, we compare the picture of basic cellular immunity profile of patients in early stage of breast cancer with those suffering from advanced disease; in the second one, we compare the production of Th1-cytokines in patients in different stages of breast cancer and atopic healthy controls. RESULTS: We recognized that the totals of T-lymphocytes and T-helpers were lower and the expression of HLADR on T-lymphocytes were higher in patients with advanced disease; the expression of IL-2 and LFN-gamma by T-lymphocytes was decreased in metastatic breast cancer patients, however IL-2 production was increased in patients in early stage of disease. CONCLUSION: We conclude that the role of immune system in cancer development is ambivalent as it may be not only protective, but also harmful (Tab. 1, Fig. 3, Ref. 22). Full Text (Free, PDF) www.bmj.sk.

Advanced detection and measurement of cells on membrane from peripheral blood by laser scanning cytometry (LSC) in early stage breast cancer patients.

BACKGROUND: The aim of our study was the potential detection of circulating tumour cells (CTCs) in early stage breast cancer patients. Our approach was cell microfiltration through polycarbonate membrane as a concentration method suitable for CTC selection in peripheral blood. The isolated cells on membrane were further analysed by laser scanning cytometry. METHODS: Sixteen patients were enrolled in the study, of which 13 had early stage breast carcinoma and 3 patients had metastatic breast carcinoma. The analyses were performed from 9 ml of peripheral blood, in one patient blood was drawn twice. Blood samples were taken after adjuvant chemotherapy but prior to adjuvant radiotherapy. The control group consisted of 12 clinically healthy subjects. CONCLUSIONS: In the control group 3 subjects out of 12 had 1 CTC, the mean CTC numbers being 0.25 +/- 0.45. In the early stage breast cancer patients 0-36 CTCs were detected (mean 13.9 +/- 12.9 CTCs. 10 patients out of 13 had more than 2 CTCs (62%). The detection and measurement of cells on membrane is a simple and reproducible method of detection of CTCs in peripheral blood. Sensitivity of the method is 88.5%. Detection of CTCs seems to be a promising method for the monitoring of adjuvant therapy in early stage breast cancer patients and for the identification of high risk patients in whom elevated numbers of CTCs are persisting following the termination of adjuvant therapy (Tab. 1, Fig. 4, Ref. 35). Full Text (Free, PDF) www.bmj.sk.

In vivo fluorescence diagnostics and photodynamic therapy of gastrointestinal superficial polyps with aminolevulinic acid. A clinical and spectroscopic study.

In the present study initial results of clinical study related to the treatment of patients having different types of precancerous lesions in the area of esophagus, stomach and intestine by photodynamic therapy (PDT) based on aminolevulinic acid (ALA-PDT) are reported. The procedure was performed by laser fibre system with the light guides introduced through biopsy channel of an endoscope. In addition, in vivo fluorescent diagnostics and spectral analyses of biopsies were performed. Each patient had a positive response to therapy. In two cases there was a total response and in other five cases more than sixty percent of suspected area was removed. Additionally, sigilocellular carcinoma of stomach was revealed in one case. It appears from the results of this study, that the treatment of precancerous lesions with ALA-PDT could be successful treatment modality.