Antagonism of oestrogen action in human breast and endometrial cells in vitro: potential novel antitumour agents.

PURPOSE: There is a need to find novel oestrogen receptor (ER) ligands that antagonize oestrogen action in the reproductive tissues and would therefore have therapeutic potential in oestrogen-dependent tumours. We tested novel ER ligands in both breast and endometrial cells to profile agonism/antagonism in these oestrogen target reproductive tissues. METHODS: Novel analogues of the ER antagonist ICI 182,780 were synthesized and tested for their ability to inhibit gene expression dependent on oestrogen response elements (ERE) in human breast (MCF-7) and endometrial (Ishikawa) cell lines. This activity was correlated with inhibition of oestrogen-induced cell proliferation and ER binding. RESULTS: The sulphide analogue (compound 1) and sulphone analogue (compound 2) had no intrinsic ERE-dependent agonism in either breast cancer or endometrial cells in culture. All three compounds dose-dependently inhibited ERE-mediated oestrogen agonism. Moreover, these ER ligands inhibited oestrogen-stimulated proliferation of breast cancer and endometrial cells. ICI 182,780, compound 1 and compound 2 were all able to bind both isoforms of the ER (ER alpha and ER beta). In endometrial cells, the relative binding to ER beta correlated with the ERE-dependent antioestrogenic effect of these ligands, suggesting that in this tissue this receptor is the predominant isoform that determines antioestrogenic activity. CONCLUSIONS: The ability of these analogues of ICI 182,780 to inhibit oestrogen-stimulated transcriptional activity and cell proliferation suggests that these agents, in particular the sulphone analogue, have therapeutic potential in the treatment of breast cancer without exhibiting the unwanted oestrogenic effects in the endometrium.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Role of caspases in human renal proximal tubular epithelial cell apoptosis.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells.

Raloxifene and idoxifene are selective estrogen receptor modulators (SERMs) that exhibit tissue-specific agonist or antagonist properties via interactions with the estrogen receptor (ER). Both compounds are similarly osteoprotective in the ovariectomized rat in vivo as assessed by measurement of bone mineral density, urinary pyridinium cross-links, and serum osteocalcin, suggesting a similar mechanism of action. However, we have identified a fundamental difference in this mechanism via the estrogen response element (ERE) in osteoblast-like cells. With the use of ERE-luciferase reporter constructs, raloxifene, like the complete ER-antagonist ICI-182780, acts as an antagonist via the ERE in osteoblastic cells. In contrast, idoxifene, like 17beta-estrogen itself and 4-OH-tamoxifen, acts as an agonist in osteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780 and raloxifene inhibited the ERE-dependent agonist activity of 17beta-estradiol and idoxifene in osteoblastic cells. In contrast, in breast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 had no agonist activity and, indeed, raloxifene and idoxifene were potent antagonists of ERE-mediated 17beta-estradiol action, indicating an ERE-dependent mode of action in these cells. Although these SERMs exhibit a similar antagonist activity profile in breast cells, they can be distinguished mechanistically in osteoblastic cells.

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Identification and cloning of a human urea transporter HUT11, which is downregulated during adipogenesis of explant cultures of human bone.

Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.

Idoxifene: a novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats.

Idoxifene, a novel selective estrogen receptor modulator, was tested for its effects on bone loss, serum cholesterol, and uterine wet weight and histology in the ovariectomized (Ovx) rat. Idoxifene (0.5 mg/kg x day) completely prevented loss of both lumbar and proximal tibial bone mineral density (BMD). In an intervention study, idoxifene (0.5 and 2.5 mg/kg x day) completely prevented further loss of both lumbar and proximal tibial BMD during a 2-month treatment period commencing 1 month after surgery, when significant loss of BMD had occurred in the Ovx control group. Idoxifene reduced total serum cholesterol, which was maximal at 0.5 mg/kg x day. Idoxifene alone displayed minimal uterotrophic activity in Ovx rats and inhibited the agonist activity of estrogen in intact rats. Histologically, myometrial and endometrial atrophy were observed in both idoxifene and vehicle-treated Ovx rats. In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.

Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders.

The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by increased marrow adipose tissue formation. Reversal of this process may provide a novel therapeutic approach for osteopenic disorders. We have shown that cells cultured from human trabecular bone are not only osteogenic, but are able also to undergo adipocyte differentiation under defined culture conditions. Osteoblast differentiation was induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and adipocyte differentiation by dexamethasone (dex) plus 3-isobutyl-1-methylxanthine (IBMX) treatment. Adipogenesis was characterized by lineage-specific enzyme and gene activities, alpha-glycerophosphate-3-dehydrogenase activity, fatty acid binding protein, aP2 and lipoprotein lipase expression. Osteoblastogenesis was assessed by osteoblast characteristic 1,25(OH)2D3 induction of alkaline phosphatase activity and osteoblast-specific 1,25(OH)2D3-induced osteocalcin synthesis and release. We provide evidence for a common pluripotent mesenchymal stem cell that is able either to undergo adipogenesis or osteoblastogenesis, using clonal cell lines derived from human trabecular bone cell cultures. Adipogenesis can be induced also by long chain fatty acids and the thiazolidinedione troglitazone. Dex plus IBMX-induced adipogenesis can be inhibited by interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. Interestingly, and in contrast to extramedullary adipocyte differentiation as shown by mouse 3T3L-1 and a human liposarcoma SW872 cell line, trabecular bone adipogenesis was unaffected by insulin. Also, the formation of fully differentiated adipocytes from trabecular bone cells after troglitazone treatment and long chain fatty acids was dependent on increased expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma2 caused by dex plus IBMX. Specific inhibition of marrow adipogenesis and promotion of osteoblastogenesis of a common precursor cell may provide a novel therapeutic approach to the treatment of osteopenic disorders.