Estrogen and progestin bioactivity of foods, herbs, and spices.

In this study we report on the content and bioactivity of plant (phyto) estrogens and progestins in various foods, herbs, and spices, before and after human consumption. Over 150 herbs traditionally used by herbalists for treating a variety of health problems were extracted and tested for their relative capacity to compete with estradiol and progesterone binding to intracellular receptors for progesterone (PR) and estradiol (ER) in intact human breast cancer cells. The six highest ER-binding herbs that are commonly consumed were soy, licorice, red clover, thyme, tumeric, hops, and verbena. The six highest PR-binding herbs and spices commonly consumed were oregano, verbena, tumeric, thyme, red clover and damiana. Some of the herbs and spices found to contain high phytoestrogens and phytoprogestins were further tested for bioactivity based on their ability to regulate cell growth rate in ER (+) and ER (-) breast cancer cell lines and to induce or inhibit the synthesis of alkaline phosphatase, an end product of progesterone action, in PR (+) cells. In general, we found that ER-binding herbal extracts were agonists, much like estradiol, whereas PR-binding extracts, were neutral or antagonists. The bioavailability of phytoestrogens and phytoprogestins in vivo were studied by quantitating the ER-binding and PR-binding capacity of saliva following consumption of soy milk, exogenous progesterone, medroxyprogesterone acetate, or wild mexican yam products containing diosgenin. Soy milk caused a dramatic increase in saliva ER-binding components without a concomitant rise in estradiol. Consumption of PR-binding herbs increased the progestin activity of saliva, but there were marked differences in bioactivity. In summary, we have demonstrated that many of the commonly consumed foods, herbs, and spices contain phytoestrogens and phytoprogestins that act as agonists and antagonists in vivo.

Prognostic value of Cathepsin D expression in breast cancer: immunohistochemical assessment and correlation with radiometric assay.

BACKGROUND: The prognostic significance of Cathepsin D and optimal methodologies to measure Cathepsin D in breast cancers are controversial. PATIENTS AND METHODS: Quantitative (immunoradiometric) and semiquantitative (immunohistochemical) assays for Cathepsin D expression were compared using 25 breast carcinomas. Immunohistochemical Cathepsin D results were derived using 3 different anti-Cathepsin D antibodies and significant associations between immunohistochemical and radiometric Cathepsin D data were observed for each reagent. Immunohistochemical analysis of Cathepsin D expression was performed on nearly 500 fixed-embedded archival breast cancers with long-term patient follow-up using 2 anti-Cathepsin D antibodies (CDR2-11/23, IC11). RESULTS: The immunohistochemical reagents recognized generally overlapping subsets of Cathepsin D positive tumors (correlation co-efficient 0.54; p = 0.00016). Correlations between Cathepsin D data and clinical, histologic or biologic features differed for each antibody. For the node-negative patient subset, Cathepsin D immunopositivity correlated with erbB-2 and stress-response protein 27 overexpression but not survival. Cathepsin D positivity was associated with subsequent distant metastasis and estrogen receptor positivity in node positive patients. Univariate analysis of all patients suggested that Cathepsin D immunopositivity may be predictive of a reduced metastasis-free but not overall survival. Multivariate analysis, however, failed to confirm an independent prognostic value for Cathepsin D in breast cancer patients. CONCLUSIONS: These data do not confirm an independent prognostic significance for Cathepsin D using immunohistochemical methods on breast cancers.

Preliminary correlations of clinical outcome with in vitro chemosensitivity of second passage human breast cancer cells.

These studies describe the clinical correlations of 63 in vitro chemosensitivity assays on breast cancer cells after short-term monolayer culture. Forty-five of the assays were single agent correlations. Based on cut-off values determined empirically, the test accurately predicted resistance for 36 of 41 patients (88%) who did not respond to the drug. It also predicted sensitivity with a high degree of accuracy: 21 of 22 patients (95%) who responded to the drug tested had a sensitive assay. In five cases, two biopsies were evaluated from the same patient. Whenever assays were performed before and after treatment with a given drug, tumor cells from the second biopsy were more resistant in vitro if the patient failed on therapy. If the patient did not fail, but stopped therapy for other reasons, or if there was no intervening therapy with the tested drug, the two biopsies remained similar in drug sensitivity. These results suggest that in vitro chemosensitivity assays which accurately predict both sensitivity and resistance can be obtained with breast cancer cells after short-term culture and that further prospective trials are warranted.

Loss of heterozygosity on chromosome 1q in human breast cancer.

Cytogenetic markers involving the long arm of chromosome 1 are the most frequently observed karyotypic changes seen in breast cancer. Based on cytogenetic data, we have used polymorphic DNA markers to search for allelic losses at this chromosome region among 48 breast carcinomas. For SPTA1, allelic losses were seen in 6 of 26 (23%) informative carcinomas, while 3 of 13 (23%) and 5 of 19 (26%) informative patients showed losses at AT3 and D1S53, respectively. The background frequency of allelic loss was obtained from data using 3 other loci on the 1q arm and 2 on the p arm of chromosome 1. With these markers, only 6 of 62 informative patients (8%) showed an allelic loss, with the range being 0-13%. The allelic losses seen on 1q, which were found in 9 carcinomas, comprised an overlapping set; the common region deleted was approximately 26 centimorgans on the q arm of chromosome 1 (bands q23-32 between AT3 and D1S53). These results suggest that inactivation of a gene(s) located on 1q23-32 might contribute to the genesis of breast cancer.

Hyaluronic acid-stimulating activity in sera from the bovine fetus and from breast cancer patients.

The sine qua non of malignancy is the ability of tumor cells to migrate and invade surrounding tissue. There are many substances that have been described that enhance cell motility and hyaluronic acid is prominent among these. Hyaluronic acid is a high molecular weight alternating disaccharide polymer found in abundance in extracellular matrices whenever rapid cell proliferation or tissue regeneration and repair occur. It creates a permissive environment for cell motility during embryogenesis, and high levels of hyaluronic acid also correlate with increased tumor cell invasion and aggressiveness. Little is known about the regulation of hyaluronic acid production, either in normal tissue or in malignancy. In this study, we characterize a hyaluronic acid-stimulating activity in fetal calf serum and describe a similar activity in the sera of breast cancer patients. The stimulating activity was measured by placing aliquots of test substance on fibrosarcoma cells. These indicator cells, which synthesize copious quantities of hyaluronic acid, respond to stimulation in a time- and dose-dependent fashion. The fetal calf serum hyaluronic acid-stimulating activity is maximum early in gestation and then falls rapidly to essentially no activity at term. This activity was partially purified from 120-day fetal calf serum by concanavalin A-Sepharose affinity and ion exchange chromatography and is accounted for by a glycoprotein with a molecular weight of 150,000 on gel filtration under native conditions. The sera of breast cancer patients with measurable burden of disease also contained hyaluronic acid-stimulating activity, which was not present in normal serum donors or in breast cancer patients without evidence of disease. The production of this stimulating activity may contribute to the development of the malignant phenotype by inducing hyaluronic acid-rich microenvironments that are permissive to tumor cell invasion and metastases.

Incidence of activating ras oncogene mutations associated with primary and metastatic human breast cancer.

To test the hypothesis that ras activation is involved in the final stages of breast cancer progression, we analyzed tumor DNA derived from 60 different patients and extracted from 40 invasive primary breast tumors, seven lymph node and skin metastases, nine metastatic effusions, and five established breast cancer cell lines. The polymerase chain reaction technique was used to amplify DNA fragments containing Kirsten-(Ki-), Harvey-(Ha-), and N-ras codons 12, 13, and 61 which were then probed on slot-blots with labeled synthetic oligomers to detect nonconservative single base mutations. Activating mutations were found in one of 40 primary tumors (Ki-ras codon 13), zero of seven lymph node and skin metastases, one of nine metastatic effusions (Ki-ras codon 12), and two of five cell lines (Ki-ras codons 12 and 13). These results indicate that activating ras mutations are rarely involved in either the initiation or metastatic progression of human breast cancer.

Molecular lesions involved in the progression of a human breast cancer.

Specific genetic alterations are observed in human breast cancer, but little is known about when these occur in the evolution of the disease. Three breast cancer effusions that occurred sequentially in a single patient were examined. Common cytogenic abnormalities were found in all effusion samples suggesting a single progenitor metastatic cell. Only the last effusion, however, exhibited a mutation of the c-K-ras gene and a loss of heterozygosity at the c-H-ras locus. These specific genetic abnormalities detected in the last effusion was correlated with improved in vitro growth of the primary cells, and with the ability to establish breast cancer cell lines. Thus a mutant ras gene and the loss of heterozygosity at the c-H-ras locus were associated with a more aggressive tumor phenotype occurring late in the course of this patient's disease and not with the initiation of the primary breast cancer, or in the establishment of metastases.

Specificity of tumor necrosis factor toxicity for human mammary carcinomas relative to normal mammary epithelium and correlation with response to doxorubicin.

By using a unique short-term culture system capable of growing both normal and malignant breast epithelial tissue, human recombinant tumor necrosis factor (TNF) showed preferential cytotoxicity to malignant cells as compared to the corresponding nonmalignant cells. Most of the malignant specimens were sensitive to TNF with 13 of 18 specimens showing 90% inhibition of clonal growth (ID90) by less than 500 units of TNF per ml of culture fluid. In contrast, all 13 nonmalignant specimens tested clustered at the resistant end of the TNF response spectrum, with ID90 values being greater than 5000 units of TNF per ml of culture fluid. This differential sensitivity to TNF was seen in three cases in which malignant and nonmalignant breast epithelial tissues from the same patient were studied. To investigate the mechanism of resistance to TNF by normal cells, the presence of receptors for TNF was determined. Five of six cultures showed specific binding of 125I-labeled TNF and there was no relationship between the degree of resistance and the degree of specific binding. Simultaneous comparison of tumor responsiveness to doxorubicin and TNF revealed a positive correlation in ID90 values; these results may have important implications for the clinical use of TNF in cancer patients heavily pretreated with doxorubicin.

An in vitro model of neoplastic progression in murine epithelial cells.

We have developed an epithelial cell line derived from mouse liver which represents a spectrum of malignant progression. When inoculated into mice at low passage, the cells induced benign cysts; after extensive subcultures, the same line induced low grade adenocarcinomas. A variant of these cells with increased invasive and metastatic potential was selected. In culture, growth in methocel correlated with acquisition of malignant potential while increased homotypic aggregation correlated with metastatic ability. These cultures should be extremely valuable for studies on the nature of epithelial cell malignancy, the most common form of human cancer.