The use of scanning laser ophthalmoscope microperimetry to detect visual impairment caused by macular photocoagulation

The use of Tamoxifen as treatment for breast cancer is well established and its use experimentally as prophylaxis is promising, but little has been written about mammographically detected changes in breast parenchyma as a result of Tamoxifen therapy. We report two cases of spontaneous regression of breast cysts after Tamoxifen therapy for premenopausal breast cancer in the contralateral breast.     

Tamoxifen restores the E-cadherin function in human breast cancer MCF-7/6 cells and suppresses their invasive phenotype

Tamoxifen is an antiestrogen used in adjuvant therapy of breast carcinoma and could potentially prevent the development of mammary cancer. While it is widely clinically used, its exact mechanisms of action are not yet fully elucidated. MCF-7/6 cells are estrogen receptor-positive invasive human breast cancer cells with a functionally inactive cell surface E-cadherin. In this study, we report that tamoxifen, and to a lesser extent its metabolites 4-OH-tamoxifen and N-desmethyltamoxifen, restore the function of E-cadherin in MCF-7/6 cells. In an aggregation assay, 10(-6) M tamoxifen significantly increases the aggregation of MCF-7/6 cells. This effect is abrogated by a monoclonal antibody against E-cadherin (HECD-1), is fast (within 30 min), and does not require de novo protein synthesis. Tamoxifen was also found to inhibit the invasion of MCF-7/6 cells in organ culture. Our data is the first demonstration that tamoxifen can activate the function of an invasion suppressor molecule and suggest that the restoration of E-cadherin function may contribute to the therapeutic benefit of tamoxifen in breast cancer patients.     

Hormonal treatment of advanced breast cancer

Endocrine therapy for breast cancer has been used for almost a century, but because of the enormous success of tamoxifen there has been a resurgence of interest by the pharmaceutical industry to develop new and innovative endocrine therapies. Overall, the strategy is quite simple. Estrogen stimulates growth; therefore, the goal is to deny the breast tumor estrogens. Tamoxifen accomplishes this by blocking the estrogen receptor. The new antiestrogens, toremifene and droloxifene, however, appear to have no greater activity than tamoxifen in the treatment of advanced disease and therefore may ultimately offer no advantages over current therapy. In contrast, the pure antiestrogens hold additional promise as they may produce a more profound inhibitory effect on the tumor, and the response may be maintained longer. An orally active, pure antiestrogen, however, would be an important advance. The strategy of using GnRH agonists for premenopausal patients clearly has merit to produce a chemical oophorectomy. The strategy could be integrated into the general treatment plan for the young premenopausal patient taking tamoxifen who may not have had her menstrual cycles stopped by combination chemotherapy. The GnRH agonists would block the reflex rise in estradiol caused by tamoxifen therapy and ultimately produce a more efficient antihormonal therapy. Indeed, the different specific aromatase inhibitors can also be integrated into the treatment plan to produce a complete estrogen blockade. Whether the use will be found to be superior to pure antiestrogens, however, must await the completion of comparative clinical studies. If all the results of endocrine therapy are therapeutically similar, the final strategy may depend on the acceptability by the patient of an individual delivery method for each pharmaceutical approach.     

Breast cancer: further metabolic-endocrine risk markers?

There is evidence that increased oestrogen receptor (ER) expression in normal mammary epithelium may be a risk marker for the development of breast cancer. Insulin-like growth factor 1 (IGF1) is a potent inducer of mitosis and has been shown to synergize with oestrogen in stimulating the growth of human breast cancer in vitro. In these cells oestradiol has been shown to upregulate IGF type 1 receptor (IGFR), and recently a similar effect has been reported in normal human breast tissue xenografts in vivo. It has been postulated that the combined effect of oestradiol and IGF1 may stimulate proliferation in normal mammary epithelium and increase breast cancer risk. The bioavailability of IGF1 to the tissues is modulated by IGF-binding proteins (IGFBPs), and higher circulating levels of IGF1 and lower levels of IGFBP3 have been reported in breast cancer patients. Breast cancer specimens show a positive correlation between ER status and IGF receptor status, and also a negative correlation between ER status and IGFBP3 expression. Finally, ectopic growth hormone expression has been shown in a majority of specimens of normal and malignant breast tissue, and this may contribute to breast cancer risk, possibly by increasing the local level of bioavailable IGF1. Expansion of such findings may provide clinically useful markers of increased risk to breast cancer in women.     

Expression and immunolocalization of the oxytocin receptor in human lactating and non-lactating mammary glands

The milk ejection reflex is mediated by the release of pituitary oxytocin and its interaction with specific receptors within the mammary gland. Although up-regulation of the oxytocin receptor during lactation has been shown for the rat mammary gland by ligand binding assay, investigation of the receptor expression in human breast at the molecular level has not yet been carried out in detail. Here we report the expression and immunolocalization of the oxytocin receptor in the human breast. It appears that the expression level of the receptor-specific mRNA is not significantly elevated during lactation and the protein remains at a relatively low level. However, this lack of increase may be only a dilution effect because of the high level of milk protein expression. Immunohistochemistry and immunoelectron microscopy using three anti-oxytocin receptor antibodies raised against different epitopes of the receptor indicated the presence of receptor immunoreactivity only to a very limited extent in the myoepithelial cells; more specific expression appeared to occur in the ductal/glandular epithelium in both the non-lactating as well as lactating breast. This finding was also confirmed in a New World monkey, the common marmoset (Callithrix jacchus). These results suggest that, at least for human and marmoset, in addition to--or even instead of--myoid cells, the ductal/glandular epithelium is also a target for oxytocin action, not only during lactation but also in the non-lactating breast. Thus, there may be other physiological effects of oxytocin besides direct myoid cell contraction in the breast.     

Tamoxifen down-regulates CD36 messenger RNA levels in normal and neoplastic human breast tissues

Tamoxifen (TAM) exerts a long-term suppressive effect on human breast cancer cell proliferation. To determine whether the effects of TAM are mediated by specific gene activation or repression, normal and tumoral human breast tissues obtained before and during TAM treatment were analyzed by differential display technique. Total RNA for differential display analysis was obtained from breast tissues from two women with the diagnosis of estrogen receptor-positive stage II (T2N1M0) infiltrating ductal carcinoma, made by incisional biopsy, followed by modified radical mastectomy performed after a 30-day treatment with TAM (20 mg/day). One 202-bp cDNA band, AP5-1, was present in normal and tumoral biopsy samples, but was absent in breast tissue obtained during TAM treatment, and was confirmed by Northern hybridization, which showed a 2.7-kb band in both patients. The differentially expressed cDNA fragment showed 99% homology to Homo sapiens CD36 gene, a glycoprotein that acts as a receptor for the extracellular matrix proteins thrombospondin-1, collagen types I and IV, and oxidized low-density lipoprotein. These results indicate that the down-regulation of CD36 induced by TAM might represent alternative or additional mechanisms of action of this drug affecting the functions of thrombospondin-1, which is involved in hematogenous tumor spread, invasion and angiogenesis, and oxidized low-density lipoprotein, playing a role in inhibition of arteriosclerosis. The multiple functions affected by the down-regulation of CD36 by TAM warrant the need for additional studies.     

Binding characteristics of novel nonsteroidal antiestrogens to the rat uterine estrogen receptors

Tamoxifen (TAM), the only antiestrogen currently available for the endocrine therapy of breast cancer behaves as a mixed agonist/antagonist of estrogen action, thus limiting its therapeutic potential. We report the binding characteristics of a novel series of nonsteroidal antiestrogens to the rat uterine estrogen receptor. As measured by competition studies, the affinity of EM-652, the active metabolite of the prodrug EM-800, for the estrogen receptor is 7-11 times higher than that of 17beta-estradiol (E2), ICI 182780, and hydroxy-tamoxifen (OH-TAM), the active metabolite of Tamoxifen. EM-652 is 20x more potent than ICI 164384 and Droloxifene while it is 400 times more potent than Toremifene in displacing [3H]E2 from the rat uterine estrogen receptor. On the other hand, the prodrug EM-800 and Tamoxifen have respectively 150-fold and 410-fold less affinity for the estrogen receptor than the pure antiestrogen EM-652. No significant binding of EM-652, EM-800, TAM or OH-TAM was observed to the rat uterine progesterone receptor at concentrations up to 10,000 nM except for TAM that caused a 50% displacement of labeled R5020 at 4000 nM. No significant binding of EM-652 or EM-800 was observed on the rat ventral prostate androgen receptor or the rat uterine progesterone receptor. The present data demonstrate the high affinity and specificity of the new antiestrogen, EM-652, for the rat uterine estrogen receptor. The antiestrogen EM-652 thus becomes the compound having the highest known affinity for the estrogen receptor. Due to its unique potency and its pure antiestrogenic activity already demonstrated in many systems, this antiestrogen could well offer an important advance for the endocrine therapy of breast cancer, uterine cancer, and other estrogen-sensitive diseases in women.     

Estrogen-replacement therapy in younger women with breast cancer

Approximately 25% of breast cancers occur in premenopausal women. In addition to local therapy, surgery or surgery plus irradiation, systemic chemotherapy administration has become the standard of care for all node-positive and many node-negative patients. Systemic adjuvant chemotherapy can result in ovarian dysfunction or failure. This renders many women prematurely estrogen deficient. The consequences of menopause, genitourinary atrophy, bone loss, and increased risk of cardiovascular disease, have not been routinely assessed in clinical trials. The risks of estrogen deficiency have not been assessed in comparison to improved disease-free and overall survival benefits of adjuvantly treated premenopausal breast cancer patients. Estrogen-replacement therapy in postmenopausal women has been shown to prevent osteoporosis and reduce fracture risk. The majority of studies also show a marked reduction in cardiovascular disease and mortality. Estrogen-replacement therapy has been considered a disease-prevention strategy rather than a therapeutic intervention. The risks and benefits of estrogen-replacement therapy in women with primary breast cancer are unknown. It is unknown how the well-known benefits accrued from reduction in skeletal and cardiovascular morbidity/mortality compare with the potential risks of increased breast cancer morbidity/mortality. Carefully designed prospective clinical trials with well-defined objectives and endpoints are required to learn if more harm than good is done by the withholding of estrogen therapy in breast cancer patients.     

New muscle relaxants

Recurrent bleeding occurred in a premenopausal breast cancer woman during tamoxifen therapy. This bleeding required three curettages. Atypical hyperplasia and carcinoma were excluded, and the patient was prepared for endometrial ablation with two GnRH agonist injections. An endometrial ablation using the roller ball technique was carried out without complications. Tamoxifen therapy was continued postoperatively. During 24 months' follow-up the patient experienced no bleeding and no endometrium was seen by sonography.     

Target volume and indications of radiotherapy in breast carcinoma

Breast-conserving surgery is now commonly used to treat breast cancer. While mastectomy has been the traditional treatment for ductal carcinoma in situ, it is felt to be excessive in most cases. A great effort has been made to identify pathobiological characteristics of DCIS that can be used to identify patients best suited for wide local excision vs. wide local excision and breast irradiation. Mastectomy and conservative surgery plus irradiation offer a similar outcome in patients with early stage invasive breast cancer. Radiotherapy has not been shown to improve survival but is able to reduce significantly the rate of local recurrence, which is regarded as a very undesirable outcome. There is still controversy concerning the necessity of irradiation of the breast in all patients. Analysis of predictors of outcome cannot identify a subgroup of patients with a very low risk for local breast recurrence who might not require radiation therapy. In premenopausal, node-positive breast cancer patients XRT has a beneficial effect not only on locoregional but also on systemic recurrences. Radiotherapy has to be integrated for a pre- or postoperative consolidation in new treatment concepts for locally advanced breast cancer which use primary chemotherapy and immediate or subsequent breast radiation. Radiation of internal mammary chain and supraclavicular fossa after conservative surgery does not lead to an increase in clinically important skin or pulmonary complications. Its role is being evaluated in current multicentre studies.     

Inhibitory effect of combined treatment with the aromatase inhibitor exemestane and tamoxifen on DMBA-induced mammary tumors in rats

The antitumor effect of exemestane (FCE 24304), an irreversible aromatase inhibitor, given alone or in combination with tamoxifen, was investigated in rats with 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors. The compounds were given once daily, 6 days a week for 4 weeks. Exemestane, given at the dose of 20 mg/kg/day s.c., induced 26% complete (CR) and 18% partial (PR) tumor regressions, compared to 0% CR and 6% PR observed in controls. Tamoxifen, given at 1 mg/kg/day p.o., induced 16% CR and 13% PR. The combined treatment caused 41% CR and 16% PR, thus resulting in a higher antitumor effect than either single treatment. The appearance of new tumors was reduced by each single treatment and almost totally prevented by the combined treatment. Serum prolactin (PRL) levels, assayed 4 h after the last dose, were unchanged in the group treated with the combination, whereas tamoxifen alone caused a slight increase of serum PRL. These results indicate that estrogen deprivation through aromatase inhibition and estrogen receptor antagonism causes a better inhibition of DMBA-induced mammary tumors than either treatment modality alone.     

Polysaccharide vaccines. II. Meningococcal vaccines

A variety of indirect evidence suggests that mammary tumors can synthesize free estrogens in situ via the sulfatase enzyme. The present study utilized an isotopic kinetic technique to provide direct confirmation of local tumor synthesis. Animals bearing nitrosomethylurea (NMU)-induced rat mammary tumors were infused with 14C-estrone as well as 3H-estrone sulfate and plasma:tissue gradients for each steroid measured. Liver, serving as a control tissue, uniformly synthesized free estrone from estrone sulfate with local synthesis in this organ providing an average of 78 +/- 1.0% of the estrone in this tissue. In rat mammary tumors, five out of seven synthesized estrone locally with individual values ranging from 19 to 50% synthesized in tissue. These data indicate that liver uniformly converts estrone sulfate to free estrone, whereas the majority, but not all, breast tumors synthesize estrogen locally via this pathway.     

Tamoxifen attenuates glucocorticoid actions on bone formation in vitro

Tamoxifen is a synthetic estrogen analog which may regulate osteogenesis in vivo by virtue of its antiglucocorticoid properties. We have examined tamoxifen regulation of glucocorticoid-induced osteogenesis in two different in vitro bone systems: the chicken periosteal osteogenesis model (CPO) and rat bone marrow stromal cells (RBMC). Hormone uptake studies were conducted with the osteosarcoma cell line, ROS 17/2.8. In the CPO model, alkaline phosphatase (AP) activity and collagen synthesis were stimulated by the glucocorticoid dexamethasone (Dex; 0.1 microM). These Dex-mediated effects were inhibited by increasing concentrations of tamoxifen (10-100 microM). Similarly, in the RBMC model, Dex-dependent (0.01 microM Dex) mineralized tissue formation and AP activity were blocked by tamoxifen (0.1 microM). Although tamoxifen inhibited Dex-mediated increases of AP activity in ROS 17/2.8 cells, it did not inhibit uptake of 3H-Dex or of 3H-estrogen. Northern analyses showed that tamoxifen did not affect messenger RNAs (mRNAs) for AP. Tamoxifen did seem to reduce mRNA for collagen type I, but not bone sialoprotein, osteopontin, and osteocalcin. Dex-induced increases for all proteins mRNAs in the RBMC model were not reduced by tamoxifen. Similarly, tamoxifen had no effects on cellular proliferation. We conclude that tamoxifen has no direct effect on gene expression of bone-related proteins of osteoblastic cells. Further, in the ROS 17/2.8 cell line, the antiglucocorticoid properties of tamoxifen do not appear to be mediated through either Dex or estrogen receptors.     

Is there a basis for prevention of breast cancer with drugs?

The anti-oestrogen drug tamoxifen has led to one of the most important improvements in therapy for breast cancer patients achieved during the last decades. Tamoxifen reduces risk of relapse and improves survival in women with breast cancer. In addition, tamoxifen has favourable effects on the lipoprotein metabolism, reduces morbidity and death from myocardial infarction, and stabilizes bone density in women after menopause. Owing to the good therapeutic results in breast cancer patients and the additional favourable effects of tamoxifen, studies were started among healthy women with elevated risk of breast cancer. The intention was to examine whether tamoxifen could reduce the risk of breast cancer development in healthy women. Recent studies, however, have demonstrated higher risk of endometrium cancer in breast cancer patients treated with tamoxifen, and higher risk of histological abnormalities in the endometrium in healthy women. Prevention trials, when tamoxifen is given to healthy women, are disputed, owing to the apparent carcinogenic effect of tamoxifen.     

The in vitro combination-effect of toremifene with CAF (cyclophosphamide, adriamycin, 5-fluorouracil) on growth of various human mammary carcinomas

Toremifene (TOR) is a new antiestrogenic agent, a triphenylethylene derivative that was developed as an alternative to tamoxifene (TAM). TOR has been observed to be more effective than TAM with milder toxicity at high doses. We examined the in vitro combination-effect of TOR with cyclophosphamide, adriamycin, 5-fluorouracil and three drug mixture (CAF) on the growth of various human mammary carcinomas. The combination shows a semi-additive or additive growth inhibitory effect on all estrogen positive cells used here except one cell line. In particular, the additive or synergic combination-effect was observed on TAM resistant cells. Furthermore, TOR exhibits a chemosensitizing activity in ADR-resistant cells by expressing P-glycoprotein coded by MDR-1 (multidrug resistance gene). The chemosensitizing activity is dose-dependent of TOR. As described above, the combination of TOR with CAF shows more than a semi-additive effect in this experiment. In conclusion, the addition of high-dose TOR to CAF therapy might be useful for advanced/recurrent breast cancer.     

Expression of cytosolic sulfotransferases in normal mammary epithelial cells and breast cancer cell lines

Breast cancers require the presence of estrogens for the maintenance of growth at some time in the course of their development, as does normal breast tissue. Sulfation is an important process in the metabolism and inactivation of steroids, including estrogens, because the addition of the charged sulfonate group prevents the binding of the steroid to its receptor. Also, many of the therapeutic and chemopreventive agents used in the treatment of breast cancer are substrates for the sulfotransferases (STs). The activity and expression of four cytosolic STs, which are the human phenol-sulfating and monoamine-sulfating forms of phenol ST (PST), dehydroepiandrosterone ST, and estrogen ST (hEST), were assayed in normal breast cells and in breast cancer cell lines. ST activities and immunoreactivities were assayed in the estrogen receptor-positive human breast cancer cell lines ZR-75-1, T-47D and MCF-7; in the estrogen receptor-negative breast cancer cell lines BT-20, MDA-MB-468, and MDA-MB-231; and in normal human mammary epithelial cells. The PSTs were the most highly expressed ST activities in the breast cancer cell lines, although the levels of activity varied significantly. ZR-75-1 and BT-20 cells possessed the highest levels of activity of the human phenol-sulfating form of PST. The breast cancer cell lines showed only trace levels of dehydroepiandrosterone ST and hEST activities. In contrast, hEST was the only ST detectable in human mammary epithelial cells. Understanding the regulation of ST activity in these breast cancer and normal breast cell lines will improve our knowledge of the role of sulfation in breast cancer and provide a model with which to study the mechanism of action of estrogens in mammary cells.     

Epithelial proliferation and hormone receptor status in the normal post-menopausal breast and the effects of hormone replacement therapy

The proliferation rate (as assessed by Ki67 expression) and expression of oestrogen-regulated progesterone receptor (PR) was studied in normal post-menopausal breast epithelium. Normal breast epithelium from patients receiving hormone replacement therapy (HRT) at the time of surgery containing either oestrogen alone (E2) or oestrogen and progesterone combined activities (E2 + P) was also studied, as HRT has been linked to an increased breast cancer risk. Samples of breast tissue, containing normal epithelium, from 185 patients undergoing surgery for benign or malignant disease were immunocytochemically stained for PR and Ki67. The percentage of labelled cells was expressed as the labelling index (LI). The median Ki67 LI in normal post-menopausal breast epithelium was 0.19 and median PR LI was 4.75, and both were unaffected by patient age, duration of menopause or if the tissue sample originated from a breast with benign or malignant disease. Proliferation did not alter significantly in patients taking HRT (P = 0.61); however, PR expression was up-regulated in both E2 and E2 + P users (P = 0.01). The dose and duration of HRT had no effect on either parameter. A possible attenuation of sensitivity to oestradiol-induced proliferation but not to PR expression occurs in the post-menopausal breast.