Biology of Non-steroidal Androgen Receptor Modulators

该小组研究组织选择性雄激素受体调节剂的药理学。所采用的方法包括利用独特的非甾体雄激素激动剂和拮抗剂，其可被可见光光激活。小组成员使用基于细胞和动物的实验来研究这些化合物的药效学，特别是它们诱导雄激素受体（AR）依赖性细胞死亡的效用。

The group studies the pharmacology of tissue-selective androgen receptor modulators. The methods employed involve capitalizing upon unique non-steroidal androgen agonists and antagonists that are photo-activatable by visible light. Group members use both cell- and animal-based assays to investigate the pharmacodynamics of these compounds, with particular reference to their utility to induce androgen receptor (AR)-dependent cell death. The principal compound of interest is 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono5,6-gquinoline, or TDPQ. This compound is a potent, tissue-selective modulator of the androgen receptor. TDPQ absorbs light at 400 nm, and can elicit formation of singlet oxygens 1O2. Singlet oxygens can in turn elicit production of other reactive oxygen species ROS that can cause toxic effects in cells by a number of mechanisms. The group tested TDPQs photocytotoxicity in a live-cell assay using a fluorescence microscope with a cell-growth chamber. The AR-positive human prostate carcinoma (LNCaP) cells were used first. TDPQ caused translocation of AR to nuclei as shown by immunohistochemistry. Irradiation of the live cells in culture on the microscope stage generated a cell-killing effect that was dependent on time, light and compound concentration. Cells progressed through a permeable stage visualized via propidium iodide staining, followed subsequently by a cytolytic phase resulting in cell death. Group members are investigating the mechanism of this pathway. AR is required to elicit the photocytotoxic effect of TDPQ. Suppression of AR expression using siRNA in LNCaP cells blocks the effect, as does simultaneous incubation of the cells with a large excess of the natural physiologic AR hormone, dihydrotestosterone. PC3, another human prostate carcinoma cell line which lacks AR. These cells are highly resistant to the TDPQ photocytotoxic effect. However, when AR is introduced into the cells by stable transfection with an AR expression plasmid, the resulting AR-containing cells are ten times more sensitive to killing by TDPQ than the original PC3 cells. These findings demonstrate that TDPQ acts as a photosensitizer that acts in an AR-dependent manner to induce cell killing. The group is also studying the cell death pathway. Death by necrosis is characterized by bloating of the cells and nuclei, followed by disintegration of the cell membrane. Programmed cell death, termed apoptosis, takes place by a series of discrete enzymatic and osmotic steps. ROS have been slow in other systems to induce cell death via this pathway. The group is testing the mechanism of TDPQ-induced cell death using molecular probes (propidium iodide, Hoechst 33342 and annexin V/FITC conjugate) to quantify cell death parameters. Cells are treated with TDPQ in doses from 0 to 3 M and then irradiated for 3 min at =405 nm using irradiation doses from 0.1 to 1 kJ/cm2. Control groups consist of cells that were not exposed to either the photosensitizer or UV irradiation, or to either agent alone. Neither the photosensitizer alone nor the maximal irradiation dose of 1kJ/cm2 alone has a cell killing effect. However, cell death is induced by light irradiation in the presence of TDPQ in a time- and concentration-dependent manner. Annexin V staining, TUNEL staining and the morphologic appearance of condensed nuclear DNA proves that the majority of cell death occurs due to apoptosis. The onset of apoptosis is rapid; it is observed as early as 30 minutes after irradiation. The extent of apoptosis in the irradiated portion of the culture increases with incubation time. By about 20 hours, most of the cells within the irradiated area are dead. There is a progressive, concentric invasion of the apoptotic response into the non-irradiated portion of the well. This observation is indicative of a "bystander effect," in which cell apoptosis is spread via cell contacts. This latter feature of the mechanism is under study presently. The mechanism by which photoactivation of TDPQ bound to AR can cause apoptosis is the focus of the future work. Reactive oxygen species can cause damage to DNA, to the AR itself, or to other cell compartments including the mitochondria. Since the TDPQ-AR are located in the nucleus at the time of irradiation, the group hypothesizes that AR-directed DNA damage occurs. Efforts are under way to map the extent and site(s) of damage. Due to the selectivity shown to kill only AR-positive cells, this photocytotoxicity method can be thought of as a targeting method for causing death of certain unwanted AR-positive cells while sparing their AR-negative neighbors. For example the TDPQ-AR apoptotic effect has potential therapeutic implications for the treatment of androgenetic diseases of the skin (acne, male-pattern baldness and hirsutism). The group has extended its observations to include ligands that activate other members of the nuclear hormone receptor family, thereby also extending the targeting potential to other diseases as well.