Our appreciation of the physiological functions of estrogens and the mechanisms through which estrogens bring about these functions has changed during the past decade. Just as transgenic mice were produced in which estrogen receptors had been inactivated and we thought that we were about to understand the role of estrogen receptors in physiology and pathology, it was found that there was not one but two distinct and functional estrogen receptors, now called ER alpha and ER beta. Transgenic mice in which each of the receptors or both the receptors are inactive have revealed a much broader role for estrogens in the body than was previously thought. This decade also saw the description of a male patient who had no functional ER alpha and whose continued bone growth clearly revealed an important function of estrogen in men. The importance of estrogen in both males and females was also demonstrated in the laboratory in transgenic mice in which the aromatase gene was inactivated. Finally, crystal structures of the estrogen receptors with agonists and antagonists have revealed much about how ligand binding influences receptor conformation and how this conformation influences interaction of the receptor with coactivators or corepressors and hence determines cellular response to ligands.

In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor ␤ (ER␤). In rat ventral prostate, 530-and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ER␤ does not contain the same protein chaperones that are associated with ER␣. Estradiol (E2) binding and ER␤ immunoreactivity coincide on the gradient, with no indication of ER␣. In prostates from mice in which the ER␤ gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigen Ki-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells express Ki-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5␣-androstane-3␤,17␤-diol (3␤Adiol). This compound, which competes with E 2 for binding to ER␤ and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ER␤ knockout (BERKO) mice. Thus ER␤, probably as a complex with 3␤Adiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ER␤ may be useful in the prevention and͞or clinical management of prostatic hyperplasia and neoplasia. E pidemiological and experimental studies indicate that estrogenic hormones are involved in both the induction and prevention of prostatic cancer (1-7), but their precise role is not well understood. Excessive exposure to estrogens during critical stages of development or long-term treatment of adult animals with estrogens or androgens leads to prostatic neoplasia (8, 9). In apparent contrast, diets rich in phytoestrogens, particularly soy products, are associated with a low risk of prostate cancer (10-12) and have chemopreventive properties in experimental tumor models (12, 13). Some of these conflicting observations may be explained by the fact that there are two distinct estrogen receptors, ER␣ and ER␤, which have unique and sometimes opposing roles (14). For example, recent studies have demonstrated that, in the rodent uterus, ER␤ acts to restrain the stimulatory action of ER␣ (15).Early studies, using both ligand-binding and immunochemical techniques, detected two types of estrogen-binding substances in human prostate (16), one of which is the classical estrogen receptor now known as ER␣. Low levels of this receptor are present in the stroma of rodent prostates, but none is detectable in the epithelium (17, 18). Because of this difference in the levels of this receptor, it was proposed that the effects of estrogen on the epithelium are indirect via an initial interaction with the stroma (18). But after the discovery of ER␤ in rat prostatic epithelium ...

It is widely accepted that drug discovery often requires a systems-level polypharmacology approach to tackle problems such as lack of efficacy and emerging resistance of single-targeted compounds. Network pharmacology approaches are increasingly being developed and applied to find new therapeutic opportunities and to re-purpose approved drugs. However, these recent advances have been relatively slow to be translated into the field of natural products. Here, we argue that a network pharmacology approach would enable an effective mapping of the yet unexplored target space of natural products, hence providing a systematic means to extend the druggable space of proteins implicated in various complex diseases. We give an overview of the key network pharmacology concepts and recent experimental-computational approaches that have been successfully applied to natural product research, including unbiased elucidation of mechanisms of action as well as systematic prediction of effective therapeutic combinations. We focus specifically on anticancer applications that use in vivo and in vitro functional phenotypic measurements, such as genome-wide transcriptomic response profiles, which enable a global modelling of the multi-target activity at the level of the biological pathways and interaction networks. We also provide representative examples of other disease applications, databases and tools as well as existing and emerging resources, which may prove useful for future natural product research. Finally, we offer our personal view of the current limitations, prospective developments and open questions in this exciting field.