Potential role for retinoic acid receptor-gamma in the inhibition of breast cancer cells by selective retinoids and interferons.

Retinoids are known to inhibit the growth of a wide variety of cancer cells, including breast cancer cells. Advances made in recent years in the understanding of the molecular mechanisms of retinoid action have allowed the design of retinoids with selective activities. Such selective retinoids are of particular interest, because they may reduce the number of undesirable side effects observed with natural compounds. Here, we have compared the growth-inhibitory activities of natural retinoids with various selective retinoids, including anti-activator protein (AP)-1 selective compounds on estrogen receptor-positive and -negative breast cancer cell lines. In addition, we have investigated cooperativity between selective retinoids and IFNs and have begun to analyze the pathways that these two different growth inhibitors use for antagonizing breast cancer cell proliferation. We observe that several selective retinoids can inhibit breast cancer cells as efficiently as the natural compounds. Anti-AP-1-selective retinoids are as effective as retinoic acid receptor (RAR)-beta/gamma-selective compounds. This lets us conclude that retinoid-induced inhibition of breast cancer cell growth does not require retinoid receptor transactivation. Several synthetic retinoids including anti-AP-1-selective compounds show synergism with IFNs. However, true synergism between the two different types of growth regulators was seen only when both classes of molecules were used at low concentrations. RAR-beta/gamma and anti-AP-1-selective retinoids, but not RAR-alpha-selective compounds, induced increased RAR-gamma mRNA levels. Interestingly, IFNs at elevated concentrations (100 units/ml and higher) also induced increased RAR-gamma expression. Thus, when used at high concentrations, IFNs may activate growth-inhibitory pathways overlapping with those activated by retinoids. Because increased RAR-gamma expression is induced by the two different classes of breast cancer cell inhibitors, it is likely to have an important role in controlling the growth fo these cancer cells.

Apoptosis induction and potent antiestrogen receptor-negative breast cancer activity in vivo by a retinoid antagonist.

Close to 180,000 women will be diagnosed with breast cancer this year in the United States and more than 43,000 will die from this disease. Antiestrogens have shown promise, but they can only be effective against estrogen-dependent stages of the disease. We identify here a retinoid antagonist, MX781, that is effective against estrogen receptor-positive and -negative breast cancer cells. Although classical retinoids show limited efficacy and significant side effects, this novel compound kills breast cancer cells by inducing apoptosis and is effective against estrogen receptor-negative human breast cancer tumors in vivo. Remarkably, MX781 is well tolerated and does not seem to have significant toxicity. This novel retinoid antagonist, therefore, represents a promising new candidate for the treatment of breast cancer.

A novel selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis.

Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11beta-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11beta-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1.0 microM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11beta-HSD1 inhibitor PF-877423. 11beta-HSD1 mRNA expression increased across adipocyte differentiation (P<0.001, n=4), which was paralleled by an increase in 11beta-HSD1 oxo-reductase activity (from nil on day 0 to 5.9+/-1.9 pmol/mg per h on day 16, P<0.01, n=7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312-fold (P<0.001) and glycerol-3-phosphate dehydrogenase 47-fold (P<0.001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11beta-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11beta-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.

Novel cold-sensitive cytosolic 3,5,3'-triiodo-L-thyronine-binding proteins in human red blood cell. Isolation and characterization.

Four cytosolic 3,5,3'-triiodo-L-thyronine-binding proteins (CTBP) were isolated from hemoglobin-free human erythrocyte on DEAE-cellulose column by linear gradient of NaCl (0-0.4 M). CTBP I, II, and IV underwent rapid loss of their activities at low temperatures, whereas CTBP III was cold-insensitive. Reactivation of cold-inactivated CTBPs by warming was obtained at 20 and 37 degrees C. CTBP I, II, and IV were not inhibited by thiol-blocking agents, whereas CTBP III was blocked. Scatchard analysis of L-3,5,3'-triodo-thyronine binding showed a high affinity site with Kd on the order of 10(-10) M for CTBP II and Kd values of about 10(-9) M for CTBP I and IV and of about 10(-8) M for CTBP III. The order of affinity of iodothyronine analogues to CTBPs was similar in CTBP I, II, and IV but different in CTBP III. Chromatography on Sephacryl S-200 HR showed the elution of a single peak for each CTBP. The apparent molecular weights were about 200,000, 200,000, 25,000, and 60,000 for CTBP I, II, III, and IV, respectively. The physiological relevance of these CTBPs is discussed.

Cold-sensitive cytosolic 3,5,3'-triiodo-L-thyronine-binding protein and pyruvate kinase from human erythrocytes share similar regulatory properties of hormone binding by glycolytic intermediates.

Similar cold-sensitive properties, values of dissociation constants (Kd = 1 x 10(-10) M), and regulatory effectors were found for the cold-sensitive cytosolic 3,5,3'-triiodo-L-thyronine (L-T3)-binding protein (CTBP) and pyruvate kinase from human erythrocyte. Various metabolites of the blood cell were assayed for their effects on CTBP activity after heat and cold preincubation treatments. Among these compounds, five- and six-carbon phosphorylated sugars were effective in protecting the CTBP activity against cold inactivation, whereas only ATP and dATP blocked activation by heat treatments. The effects of fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, and ATP were obtained at physiological concentrations. Three-carbon phosphorylated intermediates of glycolysis, ADP, AMP, cAMP, and GTP had no effect on cold and heat treatments. The monomer-tetramer interconversion of the enzyme was also regulated by fructose 1,6-bisphosphate and ATP. The association is under the control of fructose 1,6-bisphosphate, whereas the dissociation is under ATP control. This regulation may have physiological relevance since the hormone binds to the tetrameric form of the enzyme at a site other than the active site.

Molecular interconversion of cold-sensitive cytosolic 3,3',5-tri-iodo-L-thyronine-binding proteins from human erythrocytes: effect of cold, heat and pH treatments.

Cytosolic 3,3',5-tri-iodo-L-thyronine-binding proteins (CTBP I, II and IV species) from human red blood cells undergo rapid loss of activity at low temperatures. Cold treatment of CTBPs was accompanied by dissociation of the polymeric protein to the 60 kDa inactive monomer. Re-activation of the cold-inactivated CTBP IV by warming resulted in association of the monomer to the active polymeric form. A similar association-dissociation phenomenon was also obtained isothermically, though pH changes. We conclude that CTBP I and CTBP II are polymeric forms of CTBP IV.

4-Hydroxyphenyl retinamide is a highly selective activator of retinoid receptors.

Retinoids have shown promise as anti-cancer and cancer preventative agents. All-trans-N-(4-hydroxyphenyl)retinamide (4HPR) belongs to a new group of retinoids that not only inhibit the proliferation of cancer cells but also can induce apoptosis in certain cancer cells. Because of its increased efficacy against cancer cells and its low toxicity it has been entered into a number of clinical trials. However, its mechanism of action is not known, and it had been assumed that it is not a true retinoid. Here we analyze its ability to function as an activator of nuclear retinoid receptors (RARs and RXRs). We observe that, in transactivation assays, 4HPR is a potent transactivator with RARgamma and a moderate activator with RARbeta but is not an activator with RARalpha and RXRalpha. Furthermore, RARgamma-selective transactivation by 4HPR is enhanced on some response elements and reduced on others when compared to natural retinoids. In contrast to transactivation, 4HPR in transrepression assays functions mostly with RARalpha, RARbeta, and RXRalpha. Optimal receptor activation is seen at 4HPR concentrations at which it is a potent growth inhibitor and inducer of apoptosis. We conclude that 4HPR is a highly selective activator of retinoid receptors. We propose that this selective activation of the nuclear receptors is likely to be the basis for its specific biological activities and its favorable pharmaceutical properties.