Showing papers in "Breast Cancer Research and Treatment in 2004"

Relevance of breast cancer cell lines as models for breast tumours: an update

Abstract: The number of available breast cancer cell (BCC) lines is small, and only a very few of them have been extensively studied. Whether they are representative of the tumours from which they originated remains a matter of debate. Whether their diversity mirrors the well-known inter-tumoural heterogeneity is another essential question. While numerous similarities have long been found between cell lines and tumours, recent technical advances, including the use of micro-arrays and comparative genetic analysis, have brought new data to the discussion. This paper presents most of the BCC lines that have been described in some detail to date. It evaluates the accuracy of the few of them widely used (MCF-7, T-47D, BT-474, SK-BR-3, MDA-MB-231, Hs578T) as tumour models. It is concluded that BCC lines are likely to reflect, to a large extent, the features of cancer cells in vivo. The importance of oestrogen receptor-alpha (gene ESR1) and Her-2/neu (ERBB2) as classifiers for cell lines and tumours is underlined. The recourse to a larger set of cell lines is suggested since the exact origin of some of the widely used lines remains ambiguous. Investigations on additional specific lines are expected to improve our knowledge of BCC and of the dialogue that these maintain with their surrounding normal cells in vivo.

Pharmacological Characterization of 4-hydroxy- N -desmethyl Tamoxifen, a Novel Active Metabolite of Tamoxifen

Abstract: The antiestrogen tamoxifen is extensively metabolized in patients to form a series of compounds with altered affinity for estrogen receptors (ERs), the primary target of this drug. Furthermore, these metabolites exhibit a range of partial agonist and antagonist activities for ER mediated effects that do not depend directly on their absolute affinity for ERs. Thus, clinical response to tamoxifen therapy is likely to depend on the aggregate effect of these different metabolites resulting from their abundance in the patient, their affinity for the receptors, and their agonist/antagonist profile. A recent study has shown that plasma concentrations of the tamoxifen metabolite 4-hydroxy-N-desmethyl tamoxifen (endoxifen), in patents undergoing tamoxifen therapy, are dependent on the cytochrome P450 (CYP) 206 genotype of the patient and that medications commonly prescribed to patients on tamoxifen therapy can also inhibit endoxifen production. In this study we characterized the properties of this metabolite with respect to binding to ERs, ability to inhibit estrogen stimulated breast cancer cell proliferation and the regulation of estrogen responsive genes. We demonstrate that endoxifen has essentially equivalent activity to the potent metabolite 4-hydroxy tamoxifen (4-OH-tam) often described as the active metabolite of this drug. Since plasma levels of endoxifen in patients with functional CYP2D6 frequently exceed the levels of 4-OH-tam, it seems likely that endoxifen is at least as important as 4-OH-tam to the overall activity of this drug and suggests that CYP2D6 status and concomitant administration of drugs that inhibit CYP2D6 activity have the potential to affect response to tamoxifen therapy.

Three-dimensional in vitro tissue culture models of breast cancer - a review

Abstract: Three-dimensional (3D) in vitro breast tumour models have an invaluable role in tumour biology today providing some very important insights into breast cancer. As well as increasing our understanding of homeostasis, cellular differentiation and tissue organization they provide a well defined environment for cancer research in contrast to the complex host environment of an in vivo model. With the recent availability of relevant stromal elements together with the vast array of extracellular matrix constituents available, in vivo like microenvironments can be recreated. These tissue like structures more realistically model the structural architecture and differentiated function of breast cancer than a cellular monolayer providing in vivo like responses to therapeutic agents. Three dimensional in vitro models allow the study of cell-cell and cell-extracellular matrix interactions, in addition to the influence of the microenvironment on cellular differentiation, proliferation, apoptosis and gene expression. Due to their enormous potential 3D cultures are currently being exploited by many other branches of biomedical science with therapeutically orientated studies becoming the major focus of research. In return great progress in 3D culture techniques have been made, largely due to this greater interaction. At present they are being used in studies ranging from investigating the role of adhesion molecules (e.g., E-cadherin) in invasion/metastasis; VEGF and angiogenesis, to tissue modelling and remodelling. Progress in the development of complex 3D culture systems is more productive than ever, however further research is vital.

Optical coherence tomography: feasibility for basic research and image-guided surgery of breast cancer.

Abstract: Diagnostic trends in medicine are being directed toward cellular and molecular processes, where treatment regimens are more amenable for cure. Optical imaging is capable of performing cellular and molecular imaging using the short wavelengths and spectroscopic properties of light. Diffuse optical tomography is an optical imaging technique that has been pursued as an alternative to X-ray mammography. While this technique permits non-invasive optical imaging of the whole breast, to date it is incapable of resolving features at the cellular level. Optical coherence tomography (OCT) is an emerging high-resolution biomedical imaging technology that for larger and undifferentiated cells can perform cellular-level imaging at the expense of imaging depth. OCT performs optical ranging in tissue and is analogous to ultrasound except reflections of near-infrared light are detected rather than sound. In this paper, an overview of the OCT technology is provided, followed by images demonstrating the feasibility of using OCT to image cellular features indicative of breast cancer. OCT images of a well-established carcinogen-induced rat mammary tumor model were acquired. Images from this common experimental model show strong correlation with corresponding histopathology. These results illustrate the potential of OCT for a wide range of basic research studies and for intra-operative image-guidance to identify foci of tumor cells within surgical margins during the surgical treatment of breast cancer.

Is Male Breast Cancer Similar or Different than Female Breast Cancer

Abstract: Objective. To determine if male breast carcinogenesis was similar to its more common female counterpart, we compared incidence patterns among men and women with breast cancer.

Monitoring response to primary chemotherapy in breast cancer using dynamic contrast-enhanced magnetic resonance imaging.

Abstract: Purpose. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) allows analysis of both tumor volume and contrast enhancement pattern using a single tool. We sought to investigate whether DCE-MRI could be used to predict histological response in patients undergoing primary chemotherapy (PCT) for breast cancer. Patients and methods. Thirty patients with breast cancer, clinical diameter >3 cm or stage III A/B, received anthracycline and taxane based PCT. DCE-MRI was performed at the baseline, after two cycles and after four cycles of PCT, before surgery. Histological response was assessed using a five-point scheme. Grade 4 (small cluster of dispersed residual cancer cells) and grade 5 (no residual viable cancer cell) were defined as a major histopathological response (MHR). Results. Univariate analysis showed that a >65% reduction in the tumor volume and a reduction in the early enhancement ratio (ECU) after two cycles of PCT were associated with a MHR. Multivariate analysis revealed that tumor volume reduction after two cycles of PCT was independently associated with a MHR (odds ratio [OR] 39.968, 95% confidence interval [CI] 3.438–464.962, p < 0.01). ECU reduction was still associated with a MHR (OR 2.50, 95% CI 0.263–23.775), but it did not retain statistical significance (p = 0.42). Combining tumor volume and ECU reduction after two cycles of PCT yielded a 93% diagnostic accuracy in identifying tumors achieving a pathological complete response (pCR) (histopathological grade 5). Conclusions. DCE-MRI allows prediction of the effect of neoadjuvant chemotherapy in breast cancer. Although in our study tumor volume reduction after two cycles had the strongest predictive value, DCE-MRI has the potential to provide functional parameters that could be integrated to optimize neoadjuvant chemotherapy strategies.

High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer.

Abstract: Previous studies in small series of patients with invasive breast cancer suggested a prognostic value of Ep-CAM overexpression in primary tumor tissue. To corroborate these findings, we performed a retrospective analysis of Ep-CAM expression using a tissue microarray containing tissue specimens from a large patient set. Ep-CAM expression was evaluated by immunohistochemistry in breast cancer tissue from 1715 patients with documented raw survival data. High level Ep-CAM expression (overexpression) was found in 41.7% of tumor samples, low level expression was found in 48.0% and no expression in 10.3% of tumor samples. Ep-CAM expression predicted poor overall survival in this patient cohort (p < 0.0001). Overall survival decreased significantly with increasing Ep-CAM expression. However, in this patient sample Ep-CAM expression was not an independent prognostic marker by multivariate analysis. Subgroup analysis revealed that Ep-CAM expression was a prognostic marker in node-positive (p < 0.0001) but not in node-negative (p= 0.58) breast cancer patients. Intriguingly, Ep-CAM expression was predictive for a dismal prognosis in patients receiving adjuvant cytotoxic (p= 0.03) or hormonal therapy (p < 0.0001) but not in untreated patients (p= 0.41). In summary, this study provides strong evidence that expression of Ep-CAM is a powerful marker of poor prognosis in node-positive invasive breast carcinoma and a potential predictive marker of sensitivity to adjuvant hormonal and/or cytotoxic treatment modalities.

The Addition of Manual Lymph Drainage to Compression Therapy For Breast Cancer Related Lymphedema: a Randomized Controlled Trial

Abstract: Purpose. The purpose of this investigation was to compare the reduction in arm lymphedema volume achieved from manual lymph drainage massage (MLD) in combination with multi-layered compression bandaging (CB) to that achieved by CB alone.

Rho proteins and cancer.

Abstract: The Rho family of GTPases has been intensively studied for their roles in signal transduction processes leading to cytoskeletal-dependent responses, including cell migration and phagocytosis In addition, they are important regulators of cell cycle progression and affect the expression of a number of genes, including those for matrix-degrading proteases implicated in cancer invasion So far, the expression of some Rho family members has been found to be increased in some human cancers, and some cancer-associated mutations in Rho family regulators have been characterized This makes Rho protein signalling pathways attractive targets for cancer therapy However, there is little evidence so far from animal studies to define if and how Rho proteins contribute to cancer cell proliferation, survival, invasion and metastasis

Use of complementary and alternative medicine by chinese women with breast cancer.

Abstract: The use of complementary and alternative medicine (CAM) has been rapidly increasing among cancer patients. The aim of this study is to evaluate the prevalence and patterns of CAM use, particularly patients' intentions and their perceived effectiveness of using Chinese herbal medicine (CHM), as well as the relations between the herbal medicine use and demographic and clinical factors among Chinese women with breast cancer. We analyzed the data from a population-based sample of 1065 breast cancer women in urban Shanghai. Patients' average age at diagnosis was 48.1 years and the median time from the initial diagnosis to the follow-up survey was 4.3 years. Overall, 98% of patients had used at least one form of CAM therapy after diagnosis of breast cancer. The most popular CAM modality was traditional Chinese medicine (86.7%), followed by the use of supplements (84.8%), physical exercises (65.5%), and support group attendance (16.6%). CHM was used by 86.4% of patients, while acupuncture was used only by 4.9% of patients. Treating cancer (81.5%) was the most common intentions of using CHM. Other cited intentions included enhancing the immune system (12%), preventing metastasis of cancer or managing other discomforts (7.9%), and lessening menopausal symptoms (4.7%). The majority of patients reported that they had benefited from the use of CHM. Patients who were younger, married, had higher education or income, received chemotherapy or radiotherapy, or had recurrence/metastasis of cancer tended to use CHM more frequently than other patients. The relations between patient characteristics and use of CHMs varied with users' intentions. Given the high prevalence of CAM use among breast cancer patients, research is urgently needed to systematically evaluate the efficacy and safety of CAM use, particularly use of herbal medicines.

Prognostic value of matrix metalloproteinases (MMP-2 and MMP-9) in patients with lymph node-negative breast carcinoma.

Abstract: Twenty-five to thirty percent of patients with node-negative breast cancer are expected to relapse following surgery, therefore great efforts have been made to identify new prognostic markers that could be useful in defining patients for additional therapy. The expression of MMP-2 and MMP-9 has been associated with high potential of metastasis in several human carcinomas including breast cancer. In the present study we examined the prognostic value of immunoreactive MMP-2/MMP-9 protein in 270 consecutive lymph node negative cases who received radical mastectomy or modified radical mastectomy. Among the patients, 211 cases received adjuvant endocrine therapy and/or adjuvant chemotherapy. Using immunohistochemical assay, we found that 56.7% of the resected tumors were positive for MMP-2 whereas 59.6% of the samples were positive for MMP-9. Chi2 test demonstrated a significant direct association between MMP-2 and MMP-9 (p < 0.001); positive immunostaining of MMP-2 was significantly related to higher tumor grade (p < 0.001) and larger tumor size (p = 0.012); positive immunostaining of MMP-9 was significantly related to higher tumor grade (p = 0.002). In univariate analysis, using Cox-proportional hazard model we found MMP-2, MMP-9 and the co-expression of MMPs (MMP2/MMP9) were significantly associated with patients' relapse free survival (p = 0.016, 0.015 and 0.013 respectively) but not overall survival (p = 0.122, 0.320 and 0.091 respectively). Log-rank test also showed that MMP-2, MMP-9 or the co-expression of MMP2/MMP9 was unfavorable prognostic factor for relapse free survival but not overall survival. In subgroup analysis, we found MMPs were more prognostic for patients with no adjuvant treatment than for patients with adjuvant therapy. In multivariate analysis, using Cox-proportional hazard model we found co-expression of MMPs, larger tumor size and higher tumor grade were unfavorable for relapse free survival (p = 0.038, 0.007 and 0.015 for each). We concluded that MMP-2 and MMP-2 are unfavorable prognostic factors in breast cancer patients. They might be potential predictive factor for adjuvant systemic therapy. The co-expression of MMP-2 and MMP-9 has significantly prognostic value in node-negative patients.

The role of the guanine nucleotide exchange factor Tiam1 in cellular migration, invasion, adhesion and tumor progression.

Abstract: While advances in molecular genetics have provided new insights into molecular alterations that lead to the development of many tumors, including breast carcinoma, the genetic and epigenetic alterations that result in metastatic spread of the disease, from which afflicted patients ultimately succumb, are much more poorly understood. Important biologic processes in the development of metastasis include increased migration and invasion of tumor cells. While the regulation of these processes is complex, they are controlled in part by small G proteins of the Rho family, including Rho, Rac, and Cdc42, that are involved in cytoskeletal organization. These proteins, active when bound to GTP, are, in turn, regulated by guanine nucleotide exchange factors (GNEFs) and guanine nucleotide activating proteins. The GNEF Tiam1 catalyzes nucleotide exchange for Rac in vivo, and Rac, Cdc42 and Rho in vitro. Tiam1 was identified first in 1994 by in vitro selection for invasiveness in T-lymphoma cells. Accordingly, Tiam1 has been shown to increase invasion in T-lymphoma cells, as well as to increase cellular migration in fibroblasts, and to promote motility in some neuronal cells. In contrast, Tiam1 has been demonstrated to increase cellular adhesion in some epithelial cell populations. Thus, Tiam1 has multiple roles in regulating cellular functions, likely dependent on the cell type, the substratum, transformation status of the cells, and the activation state of small G proteins in a given cell. Increasing evidence has focused on Tiam1's regulation, as well as Tiam1's role in cancer progression and metastasis. Recent results from other laboratories and ours have demonstrated that increased Tiam1 expression correlates with grade of breast cancer in humans and metastatic potential of human breast carcinoma cell lines in nude mice. This review will discuss Tiam1's cellular functions and methods of regulation, and will highlight Tiam1's contribution to cancer progression and metastasis.

Evidence for an association of human papillomavirus and breast carcinomas.

Abstract: Human papillomavirus (HPV) DNA has been detected in breast carcinoma by different laboratorial techniques, suggesting the virus could play a role in the pathogenesis of this tumor. The aim of the present study is to investigate the presence of HPV in patients with breast carcinoma and the correlation of the viral infection with prognostic factors for the disease outcome. Between June 2001 and July 2002, 101 paraffin embedded breast carcinoma specimens were analyzed through polymerase chain reaction (PCR) and sequencing of HPV-E6 gene. Twenty specimens of reduction mammoplasty and 21 specimens of fibroadenomas were also studied as a non-malignant control group. Two different specific primer sets targeting E6 region of the HPVs 16 and 18 were used for the analysis. The HPV DNA was detected in 25 breast carcinomas (24.75%), but in none of the benign breast specimens ( p < 0.001). Out of the 25 positive cases, 14 were HPV-16 positive (56%) and 10 were HPV-18 positive (40%). An original finding was the detection of both HPV-16 and -18 in a single tumor (4%). The amplified viral sequences confirmed the presence of HPV-16 and -18. No correlation between the presence of HPV DNA and specific prognostic predictors for the disease outcome was observed. Our results suggest that the presence in the breast of either HPV-16 or -18 might be related to development of the malignant phenotype. Further studies are warranted.

Synergistic anti-cancer effects of grape seed extract and conventional cytotoxic agent doxorubicin against human breast carcinoma cells.

Abstract: With an approach to enhance the efficacy of chemotherapy agents against breast cancer treatment, here, we investigated the anti-cancer effects of grape seed extract (GSE) and doxorubicin (Dox), either alone or in combination, in estrogen receptor-positive MCF-7 and receptor-negative MDA-MB468 human breast carcinoma cells. GSE (25–200 µg/ml) treatment of cells resulted in 16–72% growth inhibition and 9–33% cell death, in a dose- and a time-dependent manner. In other studies, Dox (10–100 nM) treatment showed 23–96% growth inhibition and 10–55% cell death. Based on these results, several combinations of GSE (25–100 µg/ml) with Dox (10–75 nM) were next assessed for their synergistic, additive and/or antagonistic efficacy towards cell growth inhibition and death. In both MCF-7 and MDA-MB468 cells, a combination of 100 µg/ml GSE with 25–75 nM Dox treatment for 48 h showed a strong synergistic effect [combination index (CI) < 0.5] in cell growth inhibition, but mostly an additive effect (CI ∼ 1) in cell death. In cell-cycle progression studies, GSE plus Dox combination resulted in a moderate increase in G1 arrest in MCF-7 cells compared to each agent alone. GSE plus Dox combination showed a very strong and significant G1 arrest in MDA-MB468 cells when compared with Dox alone, however, it was less than that observed with GSE alone. In quantitative apoptosis studies, GSE and Dox alone and in combination showed comparable apoptotic death of MCF-7 cells, however, a combination of the two was inhibitory to Dox induced apoptosis in MDA-MB468 cells. This was further confirmed in another estrogen receptor-negative MDA-MB231 cell line, in which GSE and Dox combination strongly inhibited cell growth but did not show any increase in apoptotic cell death caused by Dox. Together, these results suggest a strong possibility of synergistic efficacy of GSE and Dox combination for breast cancer treatment, independent of estrogen receptor status of the cancer cell.

Energy balance in early breast cancer patients receiving adjuvant chemotherapy

Abstract: Weight gain is a common problem amongst women receiving adjuvant chemotherapy for early breast cancer. We undertook a study to determine the causes of this weight gain. Prospective measurements of body mass and composition (skinfolds, bioelectrical impedance, total body potassium), energy balance (resting energy expenditure dietary intake, and physical activity), were determined in 17 women during and in the 6 months after commencing adjuvant chemotherapy. Women gained significant amounts of weight (5.0 +/- 3.8; p < 0.01) and body fat (7.1 kg +/- 4.5; p < 0.01) over the year. Waist circumference (5.1 +/- 4.5 cm; p < 0.01) and abdominal skinfold (16.2 +/- 10 mm; p < 0.01) were also increased but there was a decline in fat free mass (FFM); 1.7 +/- 2.5 kg. Women due to receive adjuvant chemotherapy had a greater resting energy expenditure (REE) compared with healthy subjects (n = 21); 100.5 +/- 8.0% Harris Benedict compared to 94.5 +/- 8.4% Harris Benedict (p = 0.05). REE declined by 3% during adjuvant chemotherapy (p < 0.05), and remained depressed until at least 3 months posttreatment. There were no significant changes in dietary intake or physical activity over the year. Failure of women to reduce their energy intake to compensate for the decreased energy requirement may account for some of the weight gain. Treatment of adjuvant chemotherapy causes gain of body fat because of reduced energy expenditure, and the failure of women to reduce their energy intake to compensate for the decline in energy requirement during and in the 6 months posttreatment. Since weight gain impacts on survival, patients should be counselled to reduce energy intake and exercise during and after adjuvant treatment.

Selective cytotoxicity of a red grape wine flavonoid fraction against MCF-7 cells.

Abstract: Red wine is a rich source of polyphenolic components such as anthocyanins and flavonoids. The inhibitory effects of red wine polyphenolics on human breast cancer cells have been demonstrated earlier, but their effects on normal cells have not been fully established. Red wine (Merlot) was fractionated by hydrophobic interaction chromatography and different flavonoid fractions with increasing hydrophobicity were obtained. These fractions were tested for their inhibitory effect on human breast cancer cells (MCF-7), normal human mammary epithelial cells (HMEC), and a non-tumorigenic MCF-10A cell line. By contrast to the authentic flavonoids such as quercetin, naringenin and catechin which inhibited the growth of HMEC much more than that of MCF-7 cancer cells, a red wine fraction, that was comprised mainly of the flavonoid aglycones, showed maximal inhibition of the growth of breast cancer cells, with relatively low cytotoxicity towards HMEC and MCF-10A cells. In the presence of this flavonoid fraction, the normal cells grew normally, whereas the breast cancer cells underwent a change in morphology into spherical forms. Cytotoxicity analyses suggested that these cells had become apoptotic. The efficiency of inhibition of cell proliferation by various flavonoid fractions appeared to be related to their inhibition of calcium and calmodulin-promoted phosphodiesterase activity, suggesting that flavonoids may interfere with calcium second messenger function. The results suggest that certain grape wine ingredients have anticancer properties and these ingredients may be helpful for developing designer functional foods with cancer-preventive properties.

Increased constitutive activity of PKB/Akt in tamoxifen resistant breast cancer MCF-7 cells.

Abstract: The tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line has been used as a model to identify the signalling pathways that enable resistant cancer cells to grow independently of steroid hormones. In TAM-R cells, peptide growth factor signalling pathways appear to be important in modified cell behaviour, growth and survival. The PI3 kinase signalling components Akt1 and Akt2 are expressed at similar levels by both parental wild-type MCF-7 and TAM-R cells, but Akt1 phosphorylation is significantly increased in TAM-R cells grown under basal conditions. High levels of basal Akt, GSK3α/β and p70S6 kinase phosphorylation are all inhibited by the PI3 kinase inhibitor, LY 294002. The ligands for the EGFR/erbB1 receptor, EGF (epidermal growth factor) and TGFα(transforming growth factor-α) demonstrate an increased ability to activate Akt in TAM-R compared with parental MCF-7 cells and it is proposed that the preferred autocrine or paracrine activation of Akt occurs via the erbB heterodimer EGFR/erbB2 in TAM-R cells. Akt phosphorylation is reduced by gefitinib (“Iressa”/ZD1839). The results suggest that the PI3 kinase pathway plays a role in proliferation of TAM-R cells and is important in the increased EGF induced membrane ruffling detected in the resistant cells. Increased Akt1 activation may contribute to the aggressive phenotype of tamoxifen resistant ER (oestrogen receptor) positive breast cancers.

The effect of environment on breast cancer risk.

Abstract: Environmental factors are believed to explain a large proportion of breast cancer incidence. Known risk factors for breast cancer, which are related to the reproductive life of women, and other factors, such as inheritance and socioeconomic status, explain only about half of the breast cancer cases in the US. Ionizing radiation is a well established environmental risk factor for breast cancer. Chemicals that induce mammary cancer in rodents have served as leads for studies in humans, but occupational and environmental exposure to these chemicals have for the most part lacked association with breast cancer risk. However, there is recent evidence in rats that cadmium at very low doses acts as an estrogen mimic, indicating a need to investigate the effects of metals on breast cancer risk. Studies suggest that circadian rhythm disruption is linked with breast cancer, but too few studies have been done to be conclusive. Over the years, cigarette smoking as a risk factor for breast cancer has remained controversial. However, recent research has found passive smoke exposure to be associated with increased breast cancer risk, which is hypothesized to be accounted for on the basis of an antiestrogenic effect of smoking. Solar radiation has been noted to be associated with reduced breast cancer, supporting the hypothesis that vitamin D plays a protective role in reducing this risk. Although, most of the environmental factors discussed in this review have not been convincingly found to influence breast cancer risk, research suggests that environmental exposure in combination with genetic pre-disposition, age at exposure, and hormonal milieu have a cumulative effect on breast cancer risk.

Naringenin inhibits glucose uptake in MCF-7 breast cancer cells: a mechanism for impaired cellular proliferation.

Abstract: Certain flavonoids inhibit glucose uptake in cultured cells. In this report, we show that the grapefruit flavanone naringenin inhibited insulin-stimulated glucose uptake in proliferating and growth-arrested MCF-7 breast cancer cells. Our findings indicate that naringenin inhibits the activity of phosphoinositide 3-kinase (PI3K), a key regulator of insulin-induced GLUT4 translocation, as shown by impaired phosphorylation of the downstream signaling molecule Akt. Naringenin also inhibited the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK). Inhibition of the MAPK pathway with PD98059, a MAPK kinase inhibitor, reduced insulin-stimulated glucose uptake by approximately 60%. The MAPK pathway therefore appears to contribute significantly to insulin-stimulated glucose uptake in breast cancer cells. Importantly, decreasing the availability of glucose by lowering the glucose concentration of the culture medium inhibited proliferation, as did treatment with naringenin. Collectively, our findings suggest that naringenin inhibits the proliferation of MCF-7 cells via impaired glucose uptake. Because a physiologically attainable dose of 10 µM naringenin reduced insulin-stimulated glucose uptake by nearly 25% and also reduced cell proliferation, naringenin may possess therapeutic potential as an anti-proliferative agent.

Osteoprotegerin (OPG) produced by bone marrow stromal cells protects breast cancer cells from TRAIL-induced apoptosis.

Abstract: Advanced breast cancer is often associated with metastatic bone disease, causing a number of serious complications for the patients such as hypercalceamia, pain, nerve compression and fractures. The formation of bone metastases depends on complex interactions between tumour cells and the cells of the bone microenvironment, but the precise molecular mechanisms involved in the development of tumour-induced bone disease have not been identified. We have investigated the ability of bone marrow stromal cells (BMSC) isolated from breast cancer patients to generate osteoprotegerin (OPG), a molecule involved both in bone turnover and cell survival. The potential survival effects of OPG are mediated through binding to a member of the TNF super family, TNF-related Apoptosis Inducing Ligand (TRAIL), preventing association between TRAIL and its death-inducing receptors present on a number of tumour cell types. In the present report we show that bone marrow stromal cells isolated from breast cancer patients produce OPG when grown in culture. The levels of OPG present in BMSC conditioned medium is sufficient to protect breast cancer cells from undergoing TRAIL induced apoptosis. Our data suggest that bone-derived OPG may increase survival of breast cancer cells that reach the bone microenvironment as part of the metastatic process.

HER-2/neu as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel

Abstract: Purpose. To evaluate the predictive value of HER-2 in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin (A) or with single-agent docetaxel (T).

The Role of MRI in Invasive Lobular Carcinoma

Abstract: Purpose. To determine the value of MR imaging in the detection and measurement of tumor size in patients with invasive lobular carcinoma (ILC) compared to mammography and ultrasound. Materials and methods. From 36 cases of ILC in 34 patients who were surgically treated, the pre-operative imaging measurements, being mammography, ultrasound and contrast enhanced MR, were retrospectively re-evaluated for tumor detection and size. Findings were compared with pathology. Two radiologists were used for evaluation of the mammograms, the other imaging modalities were only evaluated by one radiologist. The Pearsons correlation test was used to determine the correlation between histopathological and imaging measurements for each imaging modality. Results. For mammography, ultrasound and MRI the false negative scores were respectively 14%, 3% and 0%. The percentage for underestimated, correctly estimated and overestimated measurements on imaging were 56%, 33% and 11% for radiologist 1 and 50%, 33% and 17% for radiologist 2 on mammography. For ultrasound and MRI these percentages were respectively 53%, 47%, 0% and 14%, 75%, 11%. The correlation coefficients for mammography were respectively r= 0.34 (p 0.05) for both radiologists, for Ultrasound r= 0.24 (p > 0.05) and for MRI r= 0.81 (p < 0.01). Conclusion. Of the three imaging modalities contrast enhanced MR has the lowest false negative rate in detecting ILC and has the highest accuracy in measuring the size of the ILC. MR could play a key role in the pre-operative work-up for accurate tumor size determination.

The role of the AP-1 transcription factors c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of mammary carcinomas.

Abstract: Members of the Fos family of AP-1 transcription factors (c-Fos, FosB, FosB2, Fra-1 and Fra-2) are able to form dimers with Jun proteins which bind to the regulatory sequences of target genes. As many proteases involved in tumor invasion are AP-1-regulated, we assumed that Fos family members might be important for invasion of mammary carcinomas. Therefore, we performed transient transfections with expression vectors for c-Fos, FosB, FosB2, Fra-1 and Fra-2, followed by matrigel invasion assays. Fra-1 transfection resulted in a 2-4-fold increase of invasive cells in both cell lines. In a less degree, the invasive potential of MDA-MB231 cells was stimulated by Fra-2, whereas MCF7 invasion was enhanced by c-Fos and FosB. By double-labelling immunocytochemistry, PAI-1 up-regulation was observed in cells transfected with c-Fos, Fra-1 and Fra-2 expression vectors, whereas MMP1 and MMP9 expression was not affected. Results of cotransfection with a MMP9 promoter construct and AP-1 expression vectors do not indicate a direct up-regulation of MMP9 expression by Fos proteins except a positive effect of c-Fos in MCF7 cells. In parallel, expression of Fos family members as determined by Western Blot analysis in 75 mammary carcinomas was correlated with MMP1, MMP9, PAI-1 and uPAR protein levels in the tumors. Interestingly, high FosB levels were significantly associated with MMP1 overexpression, whereas expression of c-Fos and phosphorylated Fra-1 correlated with MMP9 protein levels. Strong Fra-2 expression correlated with high levels of MMP9, PAI-1, the uPA/PAI-1 complex and early recurrence. These data indicate that Fos proteins, especially Fra-1, c-Fos and Fra-2, might be involved in invasion of breast cancer cells.

Enumeration of circulating tumor cells in the blood of breast cancer patients after filtration enrichment: correlation with disease stage.

Abstract: The biological and clinical significance of circulating tumor cells (CTC) in the peripheral blood of breast cancer patients is not known To study this question, we used a direct visualization assay to correlate the number of CTC with disease stage and progression The CTC were enriched from the nucleated cell fraction by filtration and enumerated visually following immunostaining with anti-cytokeratin 8 (CK8) antibody CAM 52 In mixing experiments, we achieved a limit of detection of 5 MCF7 cells per 5 ml of blood or 5 × 107 peripheral blood leukocytes (PBL) We did not detect CTC in any control subjects (0/20) In 131 breast cancer patients, we found a higher incidence of CTC in patients with distant metastatic 36/51 (71%) than those with node-positive 17/36 (47%)(p= 0026), or node-negative 17/44 (39%)(p= 0001) disease The distribution of the highest numbers of CTC observed in individual patients by repeated sampling over time ranged from 1 to 700 per 5 ml of blood with a trend toward higher numbers in those with distant metastases In comparison with previous studies of equal specificity, based on a similar absence of CTC in controls, we report a higher incidence of CTC in node-negative and node-positive patients, suggesting a more frequent detection of CTC by our approach This higher incidence was achieved, in part, by repeated sampling of our study population over time Our results support the concept that CTC can be detected and enumerated in peripheral blood and that this minimally invasive assay merits further evaluation as a potential prognostic indicator and marker of disease progression

Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue

Abstract: cDNA arrays provide a powerful tool to identify gene expression pattern that are potentially associated with tumor invasion and metastasis However, genes work at the protein level and, since the transcriptional activity of a gene does not necessarily reflect cellular protein expression, the identification and quantification of proteins is essential for the understanding of molecular events leading to malignant transformation We have therefore employed a high-throughput protein microarray system which contains 378 well-characterized monoclonal antibodies in order to compare the gene expression pattern of malignant and adjacent normal breast tissue in a patient with primary breast cancer Using this technique, we have identified a number of proteins that show increased expression levels in malignant breast tissues such as casein kinase Ie, p53, annexin XI, CDC25C, eIF-4E and MAP kinase 7 The expression of other proteins, such as the multifunctional regulator 14-3-3e was found to be decreased in malignant breast tissue, whereas the majority of proteins remained unchanged when compared to the corresponding non-malignant samples The protein expression pattern was confirmed by immunohistochemistry, in which antibodies against 8 representative proteins known to be involved in carcinogenesis were employed in paraffin-embedded normal and malignant tissue sections deriving from the same patient In each case, the results obtained by IHC matched the data obtained by antibody microarray system Taken together, we have described for the first time a tumor cell specificity protein expression pattern by use of a novel commercially available antibody microarray system We have thus demonstrated the feasibility of high-throughput protein arrays in the proteomic analysis of human breast tissue We hypothesize that the use of protein arrays will not only increase our understanding of the molecular events, but could prove useful in evaluating prognosis and in determining optimal antineoplastic therapy

Her-2/neu gene amplification and protein expression in primary male breast cancer.

Abstract: The role of HER-2/neu in male breast cancer is not well defined. The purpose of the current study was to measure the frequency of HER-2/neu expression in primary male breast cancer, to demonstrate HER-2/neu gene amplification in cases found to be positive for protein overexpression, and to correlate HER-2/neu positivity with clinicopathological variables. Formalin-fixed, paraffin-embedded archival material from 99 primary male breast carcinomas was evaluated by immunohistochemistry (IHC) using the HercepTest™ (DAKO Corp., Hamburg, Germany). Scoring was performed according to established guidelines. All cases demonstrating HER-2/neu staining by IHC (1+/2+ and 3+) were analyzed for HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) utilizing the PathVysion™ assay (Vysis Corp., Downers Grove, Illinois) to assess HER-2/neu amplification status. The immunohistochemical staining of the HER-2/neu protein revealed HER-2/neu positivity in 15/99 (15.1%) cases, eight tumors showed 2+ and 7 tumors 3+ staining. HER-2/neu gene amplification was observed in 11/99 cases (11,1%), and all of the 3+ and 4/8 from the 2+ cases were amplified. HER-2/neu gene amplification/protein overexpression did not correlate with tumor state, histological grade or estrogen/progesterone receptor status nor the axillary lymph node status. This is the first comprehensive study of HER-2/neu gene amplification by FISH analysis in primary male breast cancer. Compared to female primary breast cancer the percentage of HER-2/neu positivity in our study was lower. Our data provide first evidence for HER-2/neu gene amplification in male breast cancer. Further studies should be addressed on the potential application of the monoclonal rhuMAB HER-2/neu antibody for treatment of HER-2/neu positive male breast cancer.

Molecular basis for Rho GTPase signaling specificity.

Abstract: There is now considerable evidence for the involvement of aberrant Rho GTPase activation in breast cancer development. Like Ras, Rho GTPases function as signaling nodes regulated by diverse extracellular stimuli. Rho GTPase activation is facilitated by multiple regulatory proteins, in particular guanine nucleotide exchange factors (GEFs) such as Dbl family proteins. Activated Rho GTPases in turn interact with and regulate a spectrum of functionally diverse downstream effectors, initiating a network of cytoplasmic and nuclear signaling cascades. Thus, Rho GTPases represent points of signaling convergence as well as relay switches that disseminate signaling divergence. In this review, we highlight issues relating to the structural basis by which Dbl family GEFs facilitate signaling convergence and Rho GTPase activation, and how Rho GTPases promote signal dissemination through downstream effectors.

Growth inhibitory activity of extracts and purified components of black cohosh on human breast cancer cells.

Abstract: The purpose of this study was to determine whether black cohosh contains constituents that inhibit the growth of human breast cancer cells, and therefore might eventually be useful in the prevention or treatment of breast cancer. Black cohosh rhizomes were extracted with methanol/water and fractionated by solvent-solvent partitioning to yield three fractions: hexane, ethyl acetate and water. The ethyl acetate fraction displayed the highest potency in two cell-based assays, growth inhibition and cell cycle analysis. This fraction inhibited growth of both the ER+ MCF7 and ER-MDA-MB-453 human breast cancer cell lines with IC50 values of about 20 and 10 micro g/ml, respectively. It also induced cell cycle arrest at G1 when tested at 30 micro g/ml and at G2/M at 60 micro g/ml in MCF7 cells. This suggests that the extract contains a mixture of components with the more active (or more abundant) causing G1 arrest and the less active causing G2/M arrest. We then examined specific components in this extract. The triterpene glycoside fraction obtained by polyamide column chromatography, and the specific triterpene glycosides actein, 23-epi-26-deoxyactein and cimiracemoside A, inhibited growth of the MCF7 human breast cancer cells and induced cell cycle arrest at G1. The most potent compound, actein, decreased the level of cyclin D1, cdk4 and the hyperphosphorylated form of the pRb protein and increased the level of p21cip1 in MCF7 cells, changes that may contribute to the arrest in G1. Further studies are in progress to identify the mechanisms by which actein and related compounds present in black cohosh inhibit growth of human breast cancer cells.

Quality of life of breast cancer survivors after a recurrence: a follow-up study.

Abstract: Previous studies on breast cancer recurrence provide a mixed picture of the quality of life of women following a recurrence. To clarify the picture, the present study addresses some previous methodological concerns by offering a multidimensional assessment with follow-up, a matched comparison group of disease-free survivors, and a closer look at the nature of recurrence (local versus metastatic). Fifty-Four of 817 women who participated in an earlier study experienced a recurrence at follow-up, and are compared to a matched sample of 54 women who remained disease-free. Analyses indicate that women who had a recurrence report significantly poorer functioning on various health-related quality of life (HRQOL) domains compared to women who remained disease-free. However, the differences appear to be largely due to the poorer HRQOL of women with metastatic disease. Although women who had a recurrence report good mood, low stress, and good quality of interpersonal relationships, they report significantly higher cancer-specific stress compared to disease-free women at follow-up. Women who had a recurrence also report experiencing both more meaning and vulnerability as a result of breast cancer than disease-free women, but report similar levels of spirituality and benefit-finding at follow-up as disease-free women. These results suggest that although women report relatively good psychosocial adjustment following a recurrence, cancer-specific domains of quality of life are most likely to be negatively affected. These results may be useful in identifying individuals with breast cancer recurrences who are most in need of psychosocial services.

Cimicifuga racemosa extract inhibits proliferation of estrogen receptor-positive and negative human breast carcinoma cell lines by induction of apoptosis.

Abstract: Hormone replacement therapy is contraindicated in women with breast cancer. Extracts from the rhizomes of Cimicifuga racemosa, have gained acceptance as a natural alternative for the treatment of menopausal symptoms. In the present study we investigated the antiproliferative activity of C. racemosa extracts (isopropanolic and ethanolic) on the estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB231 breast cancer cells by WST-1 assay. Down regulation of the proliferative activity and cell killing by isopropanolic and ethanolic extracts occurred in a clear dose-dependent response with a 50% growth inhibitory concentration of 54.1 ± 11.4 and 80.6 ± 17.7 µg/ml in MCF-7 cells and of 29.5 ± 3.0 and 58.6 ± 12.6 µg/ml in MDA-MB231 cells, respectively. Further, the mode of cell death was identified as apoptosis by microscopic inspection and confirmed by light scatter characteristics and by detection of Annexin V adherence to phosphatidylserine by flow cytometry. In addition, the involvement of activated caspases was supported by the cleavage of cytokeratin 18 detected with M30 antibody. Increases in the level of M30-antigen of about 4-fold and 2-fold over untreated controls were observed in C. racemosa-treated MCF-7 and MDA-MB231 cells. These results indicate that C. racemosa extract exerts no proliferative activity, but kills the estrogen receptor positive MCF-7 as well as estrogen receptor negative MDA-MB231 cells by activation of caspases and induction of apoptosis.