Effects of the antiestrogens tamoxifen, toremifene, and ICI 182,780 on endometrial cancer growth.

BACKGROUND: Tamoxifen has been shown to promote the growth of human endometrial tumors implanted in athymic mice, and it has been associated with a twofold to threefold increase in endometrial cancer. Toremifene, a chlorinated derivative of tamoxifen, and ICI 182,780, a pure antiestrogen, are two new antiestrogens being developed for the treatment of breast cancer. The effects of these drugs on endometrial cancer are currently unknown. Our objective was to evaluate the effects of toremifene and ICI 182,780 on the growth of human endometrial cancer in athymic mice. METHODS: Athymic, ovariectomized mice were implanted with human endometrial tumors and treated with estrogen, tamoxifen, or the new antiestrogens. RESULTS: The effects of tamoxifen and toremifene on the growth of either tamoxifen-stimulated or tamoxifen-naive endometrial tumors in athymic mice were not substantially different. ICI 182,780 inhibited the growth of tamoxifen-stimulated endometrial cancer, in both the presence and the absence of estrogen. CONCLUSIONS: Toremifene and tamoxifen produce identical effects in our endometrial cancer models. Therefore, it is possible that toremifene, like tamoxifen, may be associated with an increased incidence of endometrial cancer. In contrast, ICI 182,780 inhibited tamoxifen-stimulated endometrial cancer, both in the presence and in the absence of estrogen, suggesting that this drug may be safe with regard to the endometrium, even if it is used following tamoxifen, and that it may not result in an increased incidence of endometrial cancer. Indeed, it is even possible that ICI 182,780 may prove useful as an adjuvant agent in early stage endometrial cancer.

Basic guide to the mechanisms of antiestrogen action.

Forty years ago, Lerner and coworkers (1958) discovered the first nonsteroidal antiestrogen and Jensen (Jensen and Jacobson, 1960) identified a target for drug action, the ER. This knowledge opened the door for the clinical development of tamoxifen which we now know provides a survival advantage in both node-positive and node-negative patients with ER-positive disease (Early Breast Cancer Trialists Collaborative Group, 1992, 1998). The drug has been studied extensively, and the results have provided an invaluable insight into possible ancillary advantages of "antiestrogens", i.e., maintenance of bone density and the prevention of coronary heart disease, and possible disadvantages, i.e., rat liver carcinogenesis and an increased risk of endometrial cancer. Most importantly, the identification of the target site-specific actions of tamoxifen caused a paradigm shift in the prospective uses of antiestrogens from a direct exploitation of the antitumor properties to the broader application as a preventative for osteoporosis, but with the beneficial side effects of preventing breast and endometrial cancer. Raloxifene, a second-generation SERM, has all the properties in the laboratory that would encourage development as a safe preventative for osteoporosis (Jordan et al., 1997). As a result, raloxifene has been evaluated in more than 11,000 postmenopausal women and found to maintain bone density with significant decreases in breast cancer incidence and no increase in endometrial thickness. Raloxifene is now available as a preventative for osteoporosis in postmenopausal women. There is every reason to believe that a multifaceted agent like raloxifene will find widespread use, and there will be continuing interest by the pharmaceutical industry in the development of new agents with even broader applications. The extensive clinical effort is augmented by past molecular innovations in the laboratory and the future promise of new discoveries. The cloning and sequencing of the ER (Green et al., 1986; Greene et al., 1986) has allowed the development of an ER knock-out mouse (Lubahn et al., 1993) that compliments Jensen's pioneering work (Jensen and Jacobson, 1962) and describes the consequences of the loss of ER alpha. However, ER beta (Kuiper et al., 1996), the second ER, has provided an additional dimension to the description of estrogen and antiestrogen action. For the future, the development of ER beta monoclonal antibodies, the classification of target sites for the protein around the body, and the creation of ER beta and ER alpha, beta knock-out mice will identify new therapeutic targets to modulate physiological functions. Clearly, the successful crystallization of ER alpha with raloxifene (Brzozowski et al., 1997) must act as a stimulus for the crystallization of ER beta. The central issue for research on antiestrogen pharmacology is the discovery of the mechanism (or mechanisms) of target site-specificity for the modulation of estrogenic and antiestrogenic response. The description of a stimulatory pathway for antiestrogens through an AP-1 ER beta signal transduction pathway (Paech et al., 1997), although interesting, may not entirely explain the estrogenicity of antiestrogens. The model must encompass the sum of pharmacological consequences of signal transduction through ER alpha and ER beta with the simultaneous competition from endogenous estrogens at both sites. This is complicated because estradiol is an antagonist at ER beta through AP-1 sites (Paech et al., 1997), so this is clearly not the pathway for estrogen-induced bone maintenance in women. Estrogen is stimulatory through ER alpha, but antiestrogens are usually partial agonists and may either block or stimulate genes. However, we suggest that the ER alpha stimulatory pathway could be amplified through selective increases in coactivators. The principle is illustrated with the MDA-MB-231 cells stably transfected with the cDNAs for the wild-type and the amino acid 351 mutan

Laboratory models of breast and endometrial cancer to develop strategies for antiestrogen therapy.

Laboratory models for breast and endometrial cancer have had an enormous impact on the clinical development of antiestrogens. Results from the DMBA-induced rat mammary cancer model has provided the scientific principles required to evaluate long-term adjuvant tamoxifen therapy. Similarly, the athymic mouse model allowed the identification of clinically relevant mechanisms of drug resistance to tamoxifen and a model system to test new agents for cross resistance. Additionally, the endometrial cancer model has allowed the identification of agents that cause a slight increase in the risk of endometrial cancer long before the data would have be available from clinical studies. However, it should be stressed that this model is really only relevant for agents to be tested as preventives in normal women. The risks of developing endometrial cancer during tamoxifen therapy are slight compared with the survival benefit in controlling breast cancer.Finally the discovery of the carcinogenic potential of tamoxifen in the rat liver, 20 years after it was first introduced into clinical practice, raises an interesting issue. If the studies of liver carcinogenicity had been completed and published in the early 1970's there would be no tamoxifen and tens of thousands of women with breast cancer would have died prematurely. In fact there would have been no incentive to develop new agents as alternatives to tamoxifen or following tamoxifen failure. Most importantly, we would not have any knowledge about the target-site or selective actions of antiestrogens. All the current interest in selective estrogen receptor modulators (SERMs) is based on the huge clinical data base obtained by studying tamoxifen. The success of tamoxifen as an agent that preserves bone density, lowers cholesterol and prevents contralateral breast cancer(43) has become a classic example of a multimechanistic drug. These concepts have acted as a catalyst to develop new agents for new applications. The laboratory studies of raloxifene(44-46)) provided the scientific rationale for the use of raloxifene as a preventive for osteoporosis(47)) but with the goal of preventing breast cancer in post-menopausal women(48,49)) (Fig 5). It is clear that the close collaboration between laboratory and clinical research has revolutionized the prospects for women's health care in the 21st century.

The PapG tip adhesin of P fimbriae protects Escherichia coli from neutrophil bactericidal activity.

Compared with Escherichia coli ORN103, a nonfimbriated K-12 strain, P-fimbriated E. coli ORN103/pPAP5 was found to interact poorly with human neutrophils and resist their bactericidal activity in vitro. PapG, the Gal alpha(1-->4)Gal binding moiety located at the distal end of the P fimbrial filament, appeared to be responsible for this effect because an isogenic PapG- mutant, E. coli ORN103/pPAP24, exhibited binding interactions with neutrophils that were similar to nonfimbriated E. coli ORN103. Although no direct evidence is available, the poor adherence mediated by PapG could be related to its electrostatic properties because the isolated PapG protein had a pI of 5.2, which indicated that in the physiological pH range it possessed a net negative charge. Antibodies against PapG overcame the protective effect of PapG and markedly enhanced the interactions of P-fimbriated E. coli with neutrophils resulting in bacterial killing. When a P-fimbriated clinical E. coli strain or its isogenic PapG- derivative was injected into the peritoneal cavities of mice, a similar number of neutrophils was recruited to the site of injection. After 2 h, the number of P-fimbriated E. coli organisms that survived the neutrophil influx in the mouse peritoneum was approximately four times more than the number of surviving PapG- bacteria. This result demonstrates that the PapG protein, which is strategically located at the distal region of the P-fibrillum structure, protects E. coli from the bactericidal action of neutrophils.

Mast cell phagocytosis of FimH-expressing enterobacteria.

Most studies of mast cells have been directed at their role in the pathophysiology of IgE-mediated allergic reactions with little recognition of their participation in bacterial infections. We report that mast cells can specifically bind FimH, a mannose-binding subunit on type 1 fimbriae expressed by Escherichia coli and other enterobacteria. This interaction triggers mast cell phagocytosis and killing of the bacteria within vacuoles and through the release of superoxide anions. Also, in view of the fact that mast cells have the capacity to release inflammatory mediators and are particularly abundant in the skin, mucosal surfaces, and around blood vessels, we suggest that these cells play an important role in host defense against microbial infection.

Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli.

Previous studies of type 1 fimbriae of Escherichia coli have implicated FimH, a minor subunit, as the determinant of its mannose binding property. Structure-function analysis of FimH has not been possible because of the difficulty in obtaining adequate amounts of the subunit from type 1 fimbriae. We have obtained nascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E. coli strain expressing the cloned fimH gene. Nascently translocated FimH was initially degraded in the periplasm; however, when co-expressed with FimC, a putative fimbrial chaperone, the FimH molecules were stabilized and readily isolated from the periplasmic extract by fractionation on a sodium dodecyl sulfate-polyacrylamide gel followed by electroelution of the FimH band from the gel. The eluted protein was purified to homogeneity by affinity chromatography on a mannose-Sepharose column. Purified FimH displayed the same mannose-inhibitable binding to human neutrophils as type 1 fimbriated bacteria, including triggering an oxidative burst with concomitant release of reactive oxygen metabolites. In addition, inert microspheres coated with FimH, but not those coated with bovine serum albumin, were phagocytosed by neutrophils. These data provide direct evidence that FimH is the determinant on type 1 fimbriae which is responsible for mediating mannose-specific adherence and that isolated FimH is a potent activator of human neutrophils.