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。所采用的方法涉及利用独特的非甾体雄激素激动剂和拮抗剂，其可被可见光活化。小组成员使用基于细胞和动物的测定来研究这些化合物的药效学，特别是关于它们诱导雄激素受体（AR）依赖性细胞死亡的效用。 主要化合物是1，2，3，4-四氢-2，2-二甲基-6-（三氟甲基）-8-吡啶酮基-5，6-喹啉，或TDPQ。该化合物是雄激素受体的有效的组织选择性调节剂。TDPQ吸收400 nm的光，并可引发单态氧1 O2的形成。单线态氧又可以引发其他活性氧物质ROS的产生，这些活性氧物质可以通过多种机制在细胞中引起毒性作用。 该小组使用带有细胞生长室的荧光显微镜在活细胞测定中测试TDPQ的光细胞毒性。首先使用AR阳性人前列腺癌（LNCaP）细胞。免疫组化结果显示，TDPQ可引起AR向细胞核移位。在显微镜载物台上对培养物中的活细胞进行辐照产生细胞杀伤效应，该效应依赖于时间、光和化合物浓度。细胞通过碘化丙啶染色可见的可渗透阶段进展，随后是导致细胞死亡的细胞溶解阶段。小组成员正在研究这一途径的机制。 需要AR来引发TDPQ的光细胞毒性效应。在LNCaP细胞中使用siRNA抑制AR表达阻断了该效应，同时将细胞与大量过量的天然生理AR激素双氢睾酮一起孵育也是如此。PC 3，另一种缺乏AR的人前列腺癌细胞系。这些细胞对TDPQ光细胞毒性效应具有高度抗性。然而，当通过用AR表达质粒稳定转染将AR引入细胞中时，所得含AR的细胞对TDPQ杀伤的敏感性是原始PC 3细胞的10倍。这些发现表明，TDPQ作为光敏剂，以AR依赖性方式诱导细胞杀伤。 该小组还在研究细胞死亡途径。坏死性死亡的特征是细胞和细胞核膨胀，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促和渗透步骤发生。ROS在其他系统中通过该途径诱导细胞死亡的速度很慢。 该小组正在使用分子探针（碘化丙啶，Hoechst 33342和膜联蛋白V/FITC结合物）来定量细胞死亡参数，以测试TDPQ诱导细胞死亡的机制。用剂量为0至3 M的TDPQ处理细胞，然后使用0.1至1 kJ/cm 2的照射剂量在405 nm处照射3分钟。对照组由未暴露于光敏剂或UV照射或单独暴露于任一试剂的细胞组成。单独使用光敏剂或单独使用最大照射剂量1 kJ/cm ~ 2均无细胞杀伤作用。然而，在TDPQ存在下，光照射以时间和浓度依赖性方式诱导细胞死亡。 Annexin V染色、TUNEL染色和浓缩的核DNA的形态学外观证明大多数细胞死亡是由于凋亡。细胞凋亡的发生很快;早在照射后30分钟即可观察到。培养物的照射部分中的细胞凋亡的程度随着孵育时间而增加。到大约20小时，照射区域内的大部分细胞死亡。存在凋亡反应向孔的未照射部分的进行性同心侵入。这一观察结果表明存在“旁观者效应”，其中细胞凋亡通过细胞接触扩散。目前正在研究这一机制的后一个特点。 结合AR的TDPQ的光活化引起细胞凋亡的机制是未来工作的重点。活性氧物质可对DNA、AR本身或包括线粒体在内的其他细胞区室造成损伤。由于TDPQ-AR在辐射时位于细胞核中，该小组假设发生了AR介导的DNA损伤。目前正在努力绘制损害范围和地点的地图。 由于显示出仅杀死AR阳性细胞的选择性，这种光细胞毒性方法可以被认为是一种靶向方法，用于导致某些不需要的AR阳性细胞死亡，同时保留其AR阴性邻居。例如，TDPQ-AR凋亡效应对于皮肤的雄激素生成疾病（痤疮、男性型脱发和多毛症）的治疗具有潜在的治疗意义。该小组已经将其观察扩展到包括激活核激素受体家族其他成员的配体，从而也将靶向潜力扩展到其他疾病。

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.