Ester and carbamate ester derivatives of Biochanin A: synthesis and in vitro evaluation of estrogenic and antiproliferative activities.

Biochanin A (BCA), a major isoflavone in red clover and many other legumes, has been reported to display estrogenic as well as cancer chemopreventive properties. Ingested BCA is known to display low bioavailability due to poor solubility, extensive metabolism and rapid clearance. Esters of bioactive isoflavones are known to increase metabolic stability and bioavailability following local rather than systemic administration. We synthesized BCA from phloroglucinol and p-methoxy-phenylacetic acid by a Friedel-Crafts reaction and cyclization. We also synthesized esters (1, 3) and carbamate esters (2, 4, 5) at position 7 of BCA using short aliphatic chains bearing a chlorine (1, 2) or a bromine atom (3, 4) or long aliphatic chains without such atoms (5). We tested the estrogenic and antiproliferative activities of 1-5 and BCA using human breast and endometrial adenocarcinoma cells. We found that 5 affects MCF-7 and Ishikawa cells in a manner providing for induction of gene expression to a level similar to 17ß-estradiol and BCA but, unlike both of the latter, for suppression of cell proliferation as well. In addition, 5 appeared to display higher stability compared to 1-4 and BCA in both MCF-7 and Ishikawa cells. The inference is that 5 may represent a safer than BCA alternative to hormone replacement therapy.

Heat-induced degradation of overexpressed glucocorticoid receptor Separate protective roles of hsp90 and hsp70.

The glucocorticoid receptor (GR) occurs in cells in the form of a hormone-responsive complex (HRC) with hsp90. The HRC is dynamic, with hsp90 constantly directing disassembly, and hsp70, assisted by hsp90, driving reassembly. WCL2 cells stably overexpress GR to an extent that reduces the excess of hsp90 and hsp70 over GR by about 10-fold, compared to the ratio in HeLa cells. Yet the half-lives of the HRC in WCL2 and HeLa cells are comparable. As a result, the rate of assembly in WCL2 is overwhelmed by accumulation of the non-hormone-binding form of GR in its complex with hsp70 and hsp90. This form comprised some 50% of total GR in WCL2 cells. When the cells were heated to 44 degrees C, the hormone-binding activity and solubility of GR fell in parallel, and the receptor formed heavy aggregates by sequestering large amounts of hsp70. About 40% of this aggregated receptor was degraded in cells recovering at 37 degrees C in the presence of cycloheximide. Concentration of GR protein increased with increasing induction of hsp70 following exposure to 41-44 degrees C. However, balance between hormone-binding and inert forms of GR could shift in either direction in response to the increase or decrease of hsp90 induction, depending on the temperature. Suppression of degradation following re-exposure of the cells to 44 degrees C correlated better with induction of hsp90 than hsp70. We infer that sequestration of hsp70 by heat-unfolded receptor is the primary factor opposing degradation, while induction of hsp90 acts to further suppress degradation by accelerating HRC assembly.

Maintenance of glucocorticoid receptor function following severe heat-shock of heat-conditioned cells.

The competence of the glucocorticoid receptor to regulate gene expression is thought to depend on Hsp70-driven continuous reactivation following spontaneous inactivation of its hormone-binding state. We show here that the glucocorticoid-binding capacity of HeLa cells fell with increasing temperature in the range 43-45 degrees C in a manner that closely paralleled the loss of soluble receptor protein. Receptor activity was maintained during moderate (43 degrees C) but not severe (45 degrees C) heat shock. Hsp70 was rapidly rendered insoluble and was replenished by soluble chaperone at 43 but not 45 degrees C. In heat-conditioned cells expressing different levels of Hsp70, we observed a positive correlation between the concentration of active receptor and the amount of Hsp70 rendered insoluble by heat shock. Much higher amounts of Hsp70 were rendered insoluble and receptor competence to regulate gene expression was preserved after severe heat shock of appropriately heat-conditioned cells. An excess of Hsp90 was found associated with resolubilized heat-inactivated receptor from severely heat-shocked cells. The data indicate that GR activity is maintained, provided that denaturation and/or aggregation of the receptor is prevented by Hsp70; and that the concentration of the chaperone is the limiting determinant of receptor activity in heat-shocked HeLa cells.

Overexpressed glucocorticoid receptor negatively regulates gene expression under conditions that favour accumulation of non-hormone-binding forms of the receptor.

Previous reports have suggested that the native hormone-responsive glucocorticoid receptor is a heterocomplex with hsp90 and that the receptor constantly cycles between the hormone-responsive and an inactive state, with complex assembly and turnover being driven by hsp70 and hsp90, respectively. Since hsp70 appears to be titrated in cells that transiently overexpress the receptor, assembly intermediates may accumulate when more receptor is produced than can be assembled to hormone-responsive complex. Comparison of receptor protein and hormone-binding levels in extracts from transiently transfected COS-7 cells revealed the presence of non-hormone-binding receptor forms in addition to the native heterocomplex. The receptor was predominantly nuclear in the majority of the transfected cells even in the absence of hormone, with the DNA-binding domain (DBD) being necessary for nuclear localisation. Moreover, the unliganded receptor exhibited constitutive DNA-binding activity and reactivity towards antibodies against the hinge region where NLS1 is known to reside. By comparing fluorography to immunoblotting of two-dimensional SDS-PAGE of cross-linked [3H]dexamethasone-mesylate-labelled receptor, we detected non-hormone-binding receptor species capable of binding DNA in vitro. In addition, using a constitutively active receptor mutant, we found that the overexpressed wild-type receptor was capable of repressing mutant-activated transcription of transiently and stably transfected reporter genes alike in a DBD-dependent manner.