Raloxifene.

Efforts to interfere with the initiation and promotion of breast and other cancers by endocrine manipulation are not new. It is of obvious benefit to cancer patients to administer substances that combine minimal general toxicity with maximal oestrogen inhibition. Raloxifene is a relatively recent addition to a group of compounds loosely designated as antioestrogens, which implies their ability to antagonize oestrogen effects via competitive binding to the various receptors. This is a reductionist simplification, since their effect varies and ranges from interaction with lipid transduction cascades, covalent binding to proteins and DNA, regulation of growth factors, erbB2, mdr1 and probably p53 expression, complexing with E-cadherin/catenin to active induction of apoptosis and many other effects on the genome. Also, the action of most antioestrogens is not solely antagonistic and different compounds do exert some agonistic effects in various tissues. Apart from some "pure" antioestrogens, the benzothiophene derivative Raloxifene has been found to combine a high degree of selective oestrogen suppression with several other desirable characteristics, such as reduction of bone demineralisation and antiatherogenic effects without endometrial stimulation. It is well tolerated, has been successfully tested as a chemopreventive agent for breast cancer in certain groups of the population and does not prevent ovulation in women with normal menstrual cycles. Certainly, Raloxifene is only another forerunner of upcoming "designer" oestrogen modulators, but it represents a welcome addition to the therapeutic choices available for the control of some menopausal problems as well as for the prevention and treatment of breast cancer, as outlined in the following brief review.

Raloxifene: another selective estrogen modulator.

The role of estrogens as one of the prime stimulators of tumor cell proliferation is well recognized; efforts to interfere with the initiation and promotion of breast and other cancers by endocrine manipulation have a long and successful past. The benzothiophene derivate Raloxifene is a relatively recent newcomer into a heterogeneous family of compounds loosely called antiestrogens, which implies their ability to act as antagonists to estrogen effects via competitive binding to various steroid receptors. This is a reductionist explanation, since their action is colorful and varied; they interact with lipid transduction cascades, covalently bind to DNA and to different proteins and regulate growth factors and the expression of various genes, such as erB2, mdr1 and p53; they complex with E-cadherin/catenin and are thus able to induce apoptosis, actively or indirectly. Raloxifene not only modulates estrogen effects, but has been found to reduce bone demineralization and atherogenesis, without carcinogenic stimulation of the endometrium.

Chemoprevention of cancer: an ongoing saga.

The trivial adage "an ounce of prevention..." is certainly appropriate in oncology; cancer has and continues to have an enormous impact on morbidity, suffering, socioeconomics and mortality. Curative therapy is elusive--cancer remains a mainly lethal disease, which makes the objective of prevention even more important and attractive. Sober estimates put the potentially avoidable or preventable cancers in the Western World at 80% (1): the effects of smoking and alcohol, being overweight, diet promiscuity and other lifestyle choices are well known, yet at the individual level, corrective measures are disappointingly ignored. Lately, this issue is being further weakened by our acceptance of inherited susceptibility--why change our habits and indulgences, if we can not escape our genetic destiny? However, there is a massive and growing amount of information on chemoprevention which needs to be carefully evaluated, in the hope that someday, we should be able to avoid or at least delay cancer by the use of natural or synthetic compounds which intervene in the early precancerous process.

Genomic effects of tamoxifen.

Tamoxifen exerts a variety of genomic effects which explains, in part, its efficacy in both hormone-responsive and independent tumours. The above quotation expresses this in a timeless and elegant way: our understanding of antiestrogen action has been narrowly fettered by the simplistic interpretation of this drug as an antihormone. The regulatory and controlling influence of Tamoxifen on numerous genes involved in apoptosis (p53, Bcl 2, c-myc, erb-B2 and others) will be discussed in this review.

The effect of toremifene on the expression of genes in a rat mammary adenocarcinoma.

Toremifene, apart from its partial estrogen antagonism, exerts multiple and varied effects on a variety of genes involved in the control of signalling and apoptosis. After three weeks of exposure of R3230AC hosting rats to therapeutical oral doses of toremifene distinct changes in steroid receptors, P-glycoprotein, p53 and Bc1-2 expression and protein S100 levels occurred, which may contribute to our understanding of the mechanisms of action of this antiestrogen.

The effect of Toremifene on the expression of some genes in human mononuclear cells.

Toremifene exerts multiple and varied effects on the gene expression of human peripheral mononuclear cells. After short-term, in vitro exposure to therapeutical levels, distinct changes in P-glycoprotein, steroid receptors, p53 and Bcl-2 expression take place. In view of the increasing use of antiestrogens in cancer therapy and prevention, there is obvious merit in long-term in vivo studies to be conducted.

Prevalence of serum antibodies against the p53 tumor suppressor gene protein in various cancers.

We have developed 2 new quantitative methods for measuring anti-p53 antibodies in human serum. Using these methods we analyzed 1,392 sera from patients with various malignancies and 230 sera from individuals without malignancy. Highest prevalence of anti-p53 antibodies was associated with ovarian and colon cancers (15%), followed by lung (8%) and breast (5%) cancers. Prevalence in other malignancies was lower (< 4%). In hospitalized patients and apparently healthy individuals, prevalence was very low (< 2 and 1% respectively). Extremely high antibody concentrations (> 10(5) U/L) were found in 5 ovarian, 2 breast, 1 lung and 1 colon cancers. Sequential analysis of 6 positive samples has shown that the p53 antibody test may have potential for patient monitoring. The p53 antibody-positive sera from breast cancer patients were associated with tumors that were steroid hormone receptor-negative (p < 0.002). We propose that the measurement of p53 antibodies is a relatively specific serological test for cancer, which can be performed with easily automatable and quantitative methodologies and may be further exploited for patient monitoring, prognosis, diagnosis and probably screening for selected cancers.

Molecular interrelationships in multidrug resistance (review).

A brief review of the "state of the art" (1993) knowledge concerning the interrelationships of oncogenes and tumour suppressor gene p53 in multidrug resistance is presented. Gene products which induce malignant transformation and uncontrolled proliferation may play a role in activating gene(s) involved in multidrug resistance. In view of the fact that drug resistance remains the major reason for chemotherapy failure in cancer therapy, a better understanding of the genetic connections which influence the final outcome of cancer could help rationalizing treatment.

The reversal of multidrug resistance in cancer (review).

A brief review of the "state of the art" (1992) knowledge concerning various strategies and tactics to overcome multidrug resistance is presented. Multidrug resistance remains the main obstacle to long-term successful cancer chemotherapy; its prevention or elimination is probably one of the most promising avenues in cancer research.

Modulation of biological responses of normal human mononuclear cells by antiestrogens.

The effect of three non-steroidal antiestrogens, Tamoxifen, Toremifene and ICI 164, 384, on various aspects of the immune response was studied in cultures of normal peripheral mononuclear blood cells. The drugs differ somewhat in their effect on the functions tested (pokeweed mitogen-induced immunoglobulin synthesis and cell proliferation, mixed lymphocyte reaction, Interleukin 2 (IL 2) synthesis, IL 2 receptor expression and Tumor Necrosis Factor (TNF alpha) synthesis). However, all three are immunosuppressive. On the other hand, ICI 164, 384 and Tamoxifen were stimulatory for TNF alpha production by adherent cells which may prove as an additional feature in antiestrogen treatment.

Toremifene resistance in a rat mammary tumour model.

The effect of Toremifene on cell growth in vitro was tested on the R3230AC rat mammary adenocarcinoma as model. Toremifene differed from Tamoxifen in that it did not induce resistance; some cross-resistance was observed in Tamoxifen tolerant tumour cell lines. Toremifene does not influence P-glycoprotein expression in this model and at the levels studied.

Immunohistochemical determination of P-glycoprotein in a rat mammary tumour treated with tamoxifen.

The effect of treatment with the antiestrogen Tamoxifen on the distribution and localization of P-glycoprotein was determined by immunohistochemistry in a rat mammary tumour model, the R3230AC. Both in the untreated and the treated tumours, P-glycoprotein is unevenly expressed with numerous negative tumour cells and located predominantly in the cytoplasmic membranes. Administration of Tamoxifen significantly lowers P-glycoprotein content of the tumour studied.

Plasminogen activator in human breast cancer cytosols: revisited after five years.

The outcome after five years in 63 women with breast cancer was correlated with biochemical parameters (estrogen and progesterone receptors, plasminogen activator activity) determined in the cytosol of tumour specimens obtained at surgery. The information from tumour cell products and their regulatory proteins may provide additional prognostic indicators, independent of subsequent treatment.

Decreased P-glycoprotein in a tamoxifen-tolerant breast carcinoma model.

The initial benefits of antiestrogen treatment in mammary cancer tend to decrease with time as antiestrogen resistant subpopulations of cancer cells predominate. The role of P-glycoprotein in this phenomenon is not known. In the tumour model investigated, estradiol increased this efflux pump, while prolonged Tamoxifen exposure resulted in cell populations tolerant to the drug and with decreased P-glycoprotein expression. While Tamoxifen resistance is clinically undesirable, selective pressure by this drug may result in cancer cell lines with increased vulnerability to other chemotherapeutic modalities.

R-verapamil decreases anti-estrogen resistance in a breast cancer model.

Drug resistance eventually limits the effectiveness of antiestrogens in breast cancer treatment. Pharmacological reversal of this refractoriness has been attempted with R-Verapamil, a well tolerated calcium channel blocker. This drug significantly decreased the incidence of lung foci after intravenous seeding of the R3230AC rat adenocarcinoma; this effect was correlated with reduction in the expression of P-glycoprotein. The simultaneous administration of antiestrogens with a non-toxic enantiomer of Verapamil was beneficial in the tumour model investigated.

Drug resistance, the last frontier?

A brief review of the (1990) "state of the art" knowledge concerning multidrug resistance and P-glycoprotein function is presented. Modification and manipulation of the efflux pump by calcium channel blockers and antiestrogens are discussed.

Arterial resistance to neoplastic invasion.

The relevance of proteinase inhibitors to the resistance of arterial walls against neoplastic invasion was investigated in human and porcine vessels using plasminogen activators as model. No significant differences in the inhibitory potential of extracts from the vascular walls were found between pulmonary and systemic arteries. It is concluded that the proteinase - inhibitor system studied is not responsible for the immunity of arterial walls against neoplastic invasion. The lack of antineoplastic resistance of pulmonary arteries may be explained by lesser intraarterial blood pressure and hence a lower pressure gradient across the vessel wall in pulmonary as opposed to systemic arteries.

Antimetastatic effect of amiloride in an animal tumour model.

The diuretic Amiloride competitively inhibits the catalytic activity of the urokinase-type plasminogen activator on plasminogen in vitro. This effect was tested on a rat adenocarcinoma model and its invasive potential in the host lung. While the inhibitory effect on intact cell cultures of the tumour was slight, continuous exposure of tumour-injected animals to the drug completely prevented the formation of lung metastasis.

Reversible myocardial damage in gerbil brain ischaemia and its prevention by beta-adrenergic blockade.

Acute cerebral infarction in gerbils, produced by unilateral carotid ligation, was used as a model to investigate secondary myocardial changes. The extent of the myocardial damage revealed by succinic dehydrogenase (SDH) histochemistry and by release of myocardial creatine phosphokinase (MB-CK) was measured in gerbils sacrificed from 3 to 48 h after either carotid ligation, carotid isolation only or skin incision only. For technical reasons dead animals were excluded from analysis. Of surviving ligated animals 74% developed neurological deficits related to brain ischaemia. A significant weight increase in the ipsilateral hemisphere was found at 6-10 h, and maximal histological damage at 16 h, both partially reversible thereafter. Non-ligated animals did not develop neurological changes, and showed neither brain swelling nor cerebral histopathology. Extensive cardiac damage was shown by the SDH method from 3 h postoperatively, and confirmed by the elevated serum levels of MB-CK in the carotid-ligated group. The SDH changes were identical with those described in the hearts of patients with acute intracranial lesions, and appeared to be reversible. The effect of beta-adrenergic blockade was assessed in this model. Metoprolol tartrate injected intraperitoneally 3 h before and 1 h after carotid ligation (10 mg/kg each dose) significantly decreased the extent of myocardial damage as estimated both with SDH histochemistry and MB-CK serum levels. It had no effect on the ischaemic brain changes. These results strongly support the concept of catecholamine mediation of myocardial injury resulting from acute brain lesions.

Tamoxifen tolerance and steroid receptors in a tumor model.

In a rat mammary adenocarcinoma model, prolonged exposure to the antiestrogen Tamoxifen results in a Tamoxifen-tolerant tumor cell line which is readily transplantable and grows under continuous oral intake of the drug. The solid tumor contains steroid receptors; however, the level of Tamoxifen needed for effective displacement of diethylstilbestrol cannot be achieved with therapeutical doses; resistance to Tamoxifen is not the result of diminished or absent estrogen receptors.