Chemical genetic identification of the IGF-linked pathway that is mediated by STAT6 and MFP2.

Insulin-like growth factor 2 (IGF2) is a potent mitogen whose deregulation plays a role in developing liver, breast, and prostate cancers. Here, we take a small-molecule approach to investigate molecular pathways that modulate IGF2 signaling, by using chromeceptin, a synthetic molecule that selectively impairs the viability and growth of IGF2-overexpressing hepatocellular carcinoma cells. Affinity purification revealed that chromeceptin binds to multifunctional protein 2 (MFP-2), a seemingly multifunctional enzyme implicated in peroxisomal beta-oxidation. The small molecule-protein interaction stimulates the expression of IGF binding protein 1 (IGFBP-1) and suppressor of cytokine signaling-3 (SOCS-3), two cellular attenuators of the IGF signals, through activation of signal transducers and activators of transcription 6 (STAT6). The results underline the importance of STATs in IGF/insulin regulation, and they implicate a new pathway for STAT6 activation that is amenable to small-molecule intervention.

In vitro studies of tibolone in breast cells.

OBJECTIVE: To investigate the effects of tibolone and its main metabolites on breast homeostasis. DESIGN: In vitro studies in primary cultures of normal breast cells and in breast cancer cell lines. SETTING: Hospital-based academic research center. PATIENT(S): Human breast cells were obtained from women undergoing surgery for hypermastia. Breast cancer cell lines (MCF-7, T47-D, and ZR75-1) were routinely obtained from subcultures. INTERVENTION(S): Cells were incubated with tibolone, its various metabolites, the pure pregnane Org 2058, and the androgen dihydrotestosterone. MAIN OUTCOME MEASURE(S): Proliferation was determined by using a morphometric growth index, apoptosis by using morphologic analysis and flow cytometry, and antiapoptotic proteins bcl-2 and bclx(L) by using Western blot assay. Activity of 17beta-hydroxysteroid dehydrogenase was measured as an epithelial differentiation marker. RESULT(S): Tibolone and its delta(4) isomer were antiproliferative in normal breast cells. Tibolone and its delta(4) isomer increased apoptosis in breast cells. These proapoptotic effects were at least partially mediated through decreased expression of the antiapoptotic proteins bcl-2 and bclx(L). An increase in HSD activity was observed after tibolone administration. CONCLUSION(S): Tibolone is antiproliferative and proapoptotic and induces differentiation in normal breast cells. It is also proapoptotic in breast cancer cell lines.

Functional site of human placental estradiol 17 beta-dehydrogenase involves tyrosine residues, as evidenced by reaction to p-nitrobenzenesulfonyl fluoride.

Native estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from human placenta was inactivated in time dependent manner by p-nitrobenzenesulfonyl fluoride (NBSF), which is a reagent for chemical modification of tyrosine. The sulfhydryl-blocked enzyme by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) was also reacted with NBSF more slowly in pseudo-first-order kinetics. After the sequential treatments with DTNB, NBSF and dithiothreitol (DTT), the enzyme in which tyrosine residues alone were modified was isolated, and its activity was decreased. These results suggest that tyrosyl residues of the estradiol 17 beta-dehydrogenase from human placenta are located at or near its catalytic site, and play a functional role in the enzyme reaction.

Steroidal control of rat uterine 17 beta-hydroxysteroid dehydrogenase activity.

The interconversion of estradiol-17 beta and estrone in the rat uterus is due to the action of 17 beta-hydroxysteroid dehydrogenase. Whole uteri or 800 x g supernatant fractions of the uteri were incubated in the presence of [3H] estradiol-17 beta and NAD at 37 degrees C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17 beta and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17 beta-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17 beta, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17 beta-hydroxysteroid dehydrogenase activity of estradiol-17 beta treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17 beta administration. These results suggest that estradiol-17 beta caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17 beta-hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17 beta.