Anthony J. Leathem

Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay

Antioxidant species may act in vivo to decrease oxidative damage to DNA, protein and lipids thus reducing the risk of coronary heart disease and cancer. Phytoestrogens are plant compounds which are a major component of traditional Asian diets and which may be protective against certain hormone-dependent cancers (breast and prostate) and against coronary heart disease. They may also be able to function as antioxidants, scavenging potentially harmful free radicals. In this study, the effects of the isoflavonoids (a class of phytoestrogen) genistein and equol on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide significantly increased the levels of DNA strand breaks. Pre-treatment of the cells with both genistein and equol offered protection against this damage at concentrations within the physiological range. This protection was greater than that offered by addition of the known antioxidant vitamins ascorbic acid and alpha-tocopherol, or the compounds 17beta-oestradiol and Tamoxifen which have similar structures to isoflavonoids and are known to have weak antioxidant properties. These findings are consistent with the hypothesis that phytoestrogens can, under certain conditions, function as antioxidants and protect against oxidatively-induced DNA damage.

PREDICTIVE VALUE OF LECTIN BINDING ON BREAST-CANCER RECURRENCE AND SURVIVAL

A new approach for predicting long-term survival of breast-cancer patients is the detection of carbohydrate expression in paraffin-embedded sections of the primary tumour. The binding of a lectin (HPA), derived from the albumin gland of the Roman snail, Helix pomatia, to N-acetyl-galactosaminyl oligosaccharides appears valuable in assessing long-term prognosis. The clinical progress of 179 patients, followed-up for 15-20 years, was related to staining of paraffin sections of their primary breast cancers by HPA. All patients had had mastectomy but were not stratified by pathology or treatment. There were significant differences, in premenopausal patients, between groups with and without HPA staining in both time to first recurrence and survival time. HPA binding provides an extra tool for staging to aid decisions in early adjuvant treatment, with the advantage of being applicable to routinely fixed paraffin-embedded material.

Proteome and glycosylation mapping identifies post-translational modifications associated with aggressive breast cancer

Changes in glycosylation of glycoproteins and glycolipids is a common feature of cancer and may influence cancer cell behaviour, perhaps by enabling cell‐cell interactions which favour metastasis or by allowing cancer cells to evade immuno‐surveillance. Studies to identify glycosylation changes in human cancer have often used immuno‐histochemical techniques with lectins or antibodies and human tissue sections. Whilst some detailed biochemical studies have been performed there are few clinically relevant studies since the numbers of specimens evaluated are often very small. Using an immuno‐histochemical approach, we have found that the lectin HPA from the albumen gland of Helix pomatia detects aggressive metastatic breast cancers. We have sought to identify the breast cancer associated oligosaccharides and the proteins to which they are attached via 2‐DE for proteome analysis and HPLC separations for glycosylation mapping. Sixty‐nine breast cancer specimens were studied. The HPA staining pattern, clinical treatment and follow‐up data were known. Oligosaccharides that related to HPA staining were identified and found in elevated levels in metastatic breast cancer specimens. Human breast and colorectal cancer cells grown in vitro and in a clinically relevant animal model of metastasis also show increased levels of the oligosaccharides. We are now structurally characterising the oligosaccharides and aim to use them as diagnostic tools and targets for immuno‐therapy of breast and other solid tumours.

Do phytoestrogens reduce the risk of breast cancer and breast cancer recurrence? What clinicians need to know

Oestrogen is an important determinant of breast cancer risk. Oestrogen-mimicking plant compounds called phytoestrogens can bind to oestrogen receptors and exert weak oestrogenic effects. Despite this activity, epidemiological studies suggest that the incidence of breast cancer is lower in countries where the intake of phytoestrogens is high, implying that these compounds may reduce breast cancer risk, and possibly have an impact on survival. Isoflavones and lignans are the most common phytoestrogens in the diet. In this article, we present findings from human observational and intervention studies related to both isoflavone and lignan exposure and breast cancer risk and survival. In addition, the clinical implications of these findings are examined in the light of a growing dietary supplement market. An increasing number of breast cancer patients seek to take supplements together with their standard treatment in the hope that these will either prevent recurrence or treat their menopausal symptoms. Observational studies suggest a protective effect of isoflavones on breast cancer risk and the case may be similar for increasing lignan consumption although evidence so far is inconsistent. In contrast, short-term intervention studies suggest a possible stimulatory effect on breast tissue raising concerns of possible adverse effects in breast cancer patients. However, owing to the dearth of human studies investigating effects on breast cancer recurrence and survival the role of phytoestrogens remains unclear. So far, not enough clear evidence exists on which to base guidelines for clinical use, although raising patient awareness of the uncertain effect of phytoestrogens is recommended.

Lignans and breast cancer risk in pre- and post-menopausal women: meta-analyses of observational studies

Phyto-oestrogens are plant compounds structurally similar to oestradiol, which have been proposed to have protective effects against breast cancer. The main class of phyto-oestrogens in the Western diet is lignans. Literature reports on the effect of lignans in breast cancer risk have been conflicting. We performed three separate meta-analyses to examine the relationships between (i) plant lignan intake, (ii) enterolignan exposure and (iii) blood enterolactone levels and breast cancer risk. Medline, BIOSIS and EMBASE databases were searched for publications up to 30 September 2008, and 23 studies were included in the random effects meta-analyses. Overall, there was little association between high plant lignan intake and breast cancer risk (11 studies, combined odds ratio (OR): 0.93, 95% confidence interval (95% CI): 0.83–1.03, P=0.15), but this association was subjected to marked heterogeneity (I2=44%). Restricting the analysis to post-menopausal women, high levels of plant lignan intake were associated with reduced breast cancer risk (7 studies, combined OR: 0.85, 95% CI: 0.78, 0.93, P<0.001) and heterogeneity was markedly reduced (I2=0%). High enterolignan exposure was also associated with breast cancer (5 studies, combined OR: 0.73, 95% CI: 0.57, 0.92, P=0.009) but, again, there was marked heterogeneity (I2=63%). No association was found with blood enterolactone levels (combined OR: 0.82, 95% CI: 0.59–1.14, P=0.24). In conclusion, plant lignans may be associated with a small reduction in post-menopausal breast cancer risk, but further studies are required to confirm these results.

Lectin microarray profiling of metastatic breast cancers

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 µm section of a primary breast tumor or <1 µL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.

A sensitive assay to measure biomarker glycosylation demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate cancer compared with benign prostatic hyperplasia.

BACKGROUND Prostate specific antigen (PSA) measurement is used for the diagnosis of prostate cancer (PCa) but the test lacks specificity due to the number of false positive readings. The glycosylation of PSA is altered in PCa but studies in this area have been limited to few clinical samples and/or require advanced laboratory facilities. An assay to assess PSA glycosylation was established using equipment available in most routine biomedical testing laboratories. METHODS Serum samples from patients with PCa or benign prostatic hyperplasia (BPH) were used. PSA (range 4-10 ng/ml) was affinity purified, separated and probed with the lectin Ulex europaeus (UEA-1; specific for α1,2 linked fucose). An enzyme-linked immunosorbent lectin assay (ELLA) with colorimetric detection was devised and PSA fucosylation assessed in a further independent set of 26 samples. RESULTS Free PSA (fPSA) from PCa patients showed a significant increase in fucosylation compared with fPSA from patients with BPH. The ELLA was 92% specific and 69% sensitive for PCa over BPH. In comparison, fPSA measurement was 70% specific and 56% sensitive (threshold set to 25% tPSA) for PCa over BPH. CONCLUSIONS Changes in glycosylation of PSA were identified using 50 μl of serum with PSA in the range of 4-10 ng/ml, this represents a more specific and sensitive test for PCa based on fucosylation changes of fPSA.

Lectin-binding properties of human breast cancer cell lines and human milk with particular reference to Helix pomatia agglutinin.

Several studies have shown binding of a variety of lectins to breast cancer cells in tissue sections. In particular, binding of the lectin from the Roman snail, Helix pomatia agglutinin (HPA), to breast cancer cells is linked with a poor prognosis. The molecular basis for lectin binding to metastatic breast cancers is not known. To elucidate this in a model system, lectin-binding patterns of seven human breast cancer cell lines were investigated, their cell membranes were isolated, and HPA binding was assessed. In addition, the influence of fixation and processing on lectin-binding sites was also investigated. Binding of lectins to the tumor cells was very heterogeneous between and within the different cell lines and was influenced by fixation and processing. However, some cell lines showed HPA-binding sites both in vivo and in tissue sections. Analysis of the isolated cell membrane glycoproteins from these cell lines on Western blots revealed that HPA can bind to several membrane glycoproteins. In contrast, human milk shows only one major milk glycoprotein that is HPA-positive. Therefore, a switch in glycosylation appears to be taking place during the transformation to a metastatic phenotype.

Effect of red clover-derived isoflavone supplementation on insulin-like growth factor, lipid and antioxidant status in healthy female volunteers: a pilot study

Background: Isoflavones are estrogen-like plant compounds that may protect against cardiovascular disease and endocrine-responsive cancer. Isoflavones may, because of their ability to act as selective estrogen receptor modulators, alter insulin-like growth factor (IGF) status.Objective: The aim of this study was to assess the effect of 1-month isoflavone supplementation (86 mg/day red clover-derived isoflavones) on IGF status.Design and subjects: Healthy pre- (n=16) and postmenopausal (n=7) women were invited to take part in a randomised, placebo-controlled crossover study with a minimum 2-month washout period.Results: For premenopausal subjects, the change in IGF-1, IGF-BP1 and IGF-BP3 assessed at different points of the menstrual cycle did not differ between isoflavone and placebo phase. However, the change in IGF-1, when examined pre- and post-supplementation, was nonsignificantly reduced (P=0.06) on the isoflavone supplement compared to placebo. For postmenopausal subjects, the change in IGF-1, IGF-BP1 and IGFBP-3 concentrations over the supplementation period did not differ between isoflavone or placebo phase. Isoflavones increased HDL in postmenopausal women compared to placebo (P=0.02) but did not alter either cholesterol or triacylglycerol concentrations, and had no effect on antioxidant status.Conclusions: This study shows that 1-month supplementation with red clover isoflavones has a positive effect on HDL cholesterol, but at most a small effect on IGF status in premenopausal and no effect in postmenopausal subjects. Further studies are required to ascertain the role these dietary compounds may have to play in breast cancer prevention.Sponsorship: The isoflavone and placebo supplements were kindly supplied by Novogen (North Ryde, Sydney, Australia). This study was supported by Action Against Breast Cancer registered charity number 1020967.

Histochemistry to detect Helix pomatia lectin binding in breast cancer: methodology makes a difference.

A number of studies have shown that altered cellular glycosylation, as detected by binding of Helix pomatia lectin to paraffin sections, is associated with metastatic disease and consequent poor patient prognosis in breast and other cancers. In a 24-year retrospective study, sections of 373 primary breast cancers were stained for binding of the lectin using two different histochemical techniques: a direct method (using peroxidase-conjugated lectin) and an indirect method (using native, unconjugated lectin). Similar percentages of cases were positive (79%) and negative (21%) for lectin binding with either technique, but there was enormous inconsistency when individual cases were examined. A total of 38/373 (10.2%) cases that were negative by the indirect method were positive by the direct method, and 37/373 (9.9%) cases that were negative by the direct method were positive by the indirect method. Life tables calculated for lectin staining vs nonstaining cases showed a very strong correlation between lectin binding and long-term survival (p < 0.0001) when staining was performed by the indirect method, but only very weak correlation with prognosis (p < 0.03, borderline significance) when the direct technique was employed. SDS-PAGE revealed that there were differences in breast cancer glycoproteins recognized by native lectin and peroxidase-conjugated lectin immobilized on Sepharose 4B affinity beads. Helix pomatia lectin binding appears to be an intriguing and potentially valuable marker of biological behavior in breast cancer. This study emphasizes the importance of selecting an appropriate immunohistochemical technique for its visualization.