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类固醇雄激素激动剂和拮抗剂，这些激动剂和拮抗剂可以被可见光激活。小组成员使用基于细胞和动物的分析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)依赖的细胞死亡的效用。 主要的感兴趣化合物是1，2，3，4-tetrahydro-2，2-dimethyl-6-(trifluoromethyl)-8-pyridono5，6-gquinoline，或TDPQ。这种化合物是雄激素受体的一种有效的组织选择性调节剂。TDPQ在400 nm处吸收光，并能诱导单线态氧的形成。单线态氧可以通过多种机制诱导其他活性氧物种ROS的产生，从而对细胞产生毒性影响。 该小组使用带有细胞生长室的荧光显微镜，在活细胞试验中测试了TDPQS的光细胞毒性。首先使用AR阳性的人前列腺癌(LNCaP)细胞。免疫组织化学显示，TDPQ可使AR移位至核内。在显微镜下照射培养中的活细胞产生一种依赖于时间、光照和化合物浓度的细胞杀伤效应。通过碘化丙啶染色可见细胞进入渗透期，随后进入溶细胞期，导致细胞死亡。研究小组成员正在研究这一途径的机制。 需要AR才能诱导TDPQ的光细胞毒效应。使用siRNA抑制LNCaP细胞中AR的表达可以阻断这种作用，同时将细胞与大量过剩的天然生理AR激素双氢睾酮孵育也是如此。PC3，另一种缺乏AR的人前列腺癌细胞系。这些细胞对TDPQ的光细胞毒作用具有高度的抵抗力。然而，当通过稳定的AR表达载体将AR导入细胞中时，所得到的含有AR的细胞对TDPQ杀伤的敏感性是原始PC3细胞的10倍。这些发现表明，TDPQ作为光敏剂，以AR依赖的方式诱导细胞杀伤。 该小组还在研究细胞死亡途径。坏死死亡的特征是细胞和细胞核肿胀，随后细胞膜解体。程序性细胞死亡，称为细胞凋亡，通过一系列离散的酶和渗透步骤发生。在其他系统中，ROS通过这一途径诱导细胞死亡的速度一直很慢。 该小组正在使用分子探针(碘化丙啶、Hoechst 33342和Annexin V/FITC结合物)来量化细胞死亡参数，以测试TDPQ诱导细胞死亡的机制。用TDPQ处理细胞，剂量从0到3M，然后以0.1到1kJ/cm2的照射剂量，在=405 nm处照射3min。对照组由不暴露于光敏剂或紫外线照射，或仅暴露于任何一种试剂的细胞组成。光敏剂单独作用和1kJ/cm2的最大照射剂量单独作用都不具有杀伤细胞的作用。然而，在TDPQ存在的情况下，光照射以时间和浓度依赖的方式诱导细胞死亡。 Annexin V染色、TUNEL染色和核DNA凝集的形态观察表明，大多数细胞死亡是由细胞凋亡引起的。细胞凋亡发生迅速，最早可在照射后30分钟观察到。随着培养时间的延长，受照部位的细胞凋亡程度增加。大约20小时后，受辐射区域内的大部分细胞死亡。有一个渐进的，同心侵袭的凋亡反应进入井的非辐射部分。这一观察表明了一种“旁观者效应”，即细胞凋亡通过细胞接触传播。这一机制的后一特征目前正在研究中。 与AR结合的TDPQ的光激活导致细胞凋亡的机制是未来工作的重点。活性氧物种会对DNA、AR本身或包括线粒体在内的其他细胞室造成损害。由于TDPQ-AR在辐射时位于细胞核内，该小组假设发生了AR导向的DNA损伤。目前正在努力绘制损坏范围和地点(S)的地图。 由于显示的选择性只杀死AR阳性细胞，这种光细胞毒性方法可以被认为是一种靶向方法，导致某些不想要的AR阳性细胞死亡，同时保留它们的AR阴性邻居。例如，TDPQ-AR的凋亡效应对治疗皮肤雄激素性疾病(粉刺、男性型秃顶和多毛症)具有潜在的治疗意义。该小组将观察范围扩大到包括激活核激素受体家族其他成员的配体，从而也将靶向扩展到其他疾病。

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.