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Molecular Endocrinology, doi:10.1210/me.2007-0426
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Molecular Endocrinology 22 (9): 2099-2115
Copyright © 2008 by The Endocrine Society

Androgen Receptor-Mediated Apoptosis Is Regulated by Photoactivatable Androgen Receptor Ligands

Boris Risek, Piotr Bilski, Annette B. Rice and William T. Schrader

Laboratory of Reproductive and Developmental Toxicology (B.R., A.B.R., W.T.S.) and Laboratory of Pharmacology and Chemistry (P.B.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: William T. Schrader, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, III T.W. Alexander Drive, Research Triangle Park, North Carolina 27709. E-mail: schrader{at}niehs.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have studied nonsteroidal ligands of the human androgen receptor (hAR) and have shown elsewhere that when photoactivated by visible light they collide with O2 to yield singlet oxygens (1O2) in vitro. Here we report cell killing after brief light activation (405 nm) of 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ) in human prostate tumor cells. TDPQ/AR complexes were required for the death response because AR-positive LNCaP cells were killed, whereas AR-negative PC-3 cells were resistant. Excess dihydrotestosterone (DHT) blocked the TDPQ effect when the two were added together; irradiation of cells containing DHT alone had no effect. When LNCaP AR expression was suppressed using small interfering oligonucleotides targeting AR, photocytotoxicity was diminished. Conversely, stable transfection of hAR into PC-3 cells made the cells photosensitive to TDPQ. Similar results were obtained using a structural isomer of TDPQ, and also the synthetic steroidal AR ligand R1881. Cell death occurred via apoptosis as demonstrated by annexin V immunostaining, nuclear condensation, and caspase inhibition. Death involved oxidative stress, because it was prevented by addition of the antioxidant ascorbic acid during photoactivation. Detection of elevated levels of 8-hydroxy-2'-deoxyguanosine in nuclei of irradiated cells indicated oxidative DNA damage. Apoptosis spread into adjacent nonirradiated cells by direct cell-cell contacts, indicative of a bystander effect. Other photoactivatable ligands are described, implying a general method for ablation of cells bearing specific nuclear hormone receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a ligand-activated transcription factor that belongs to a large family of nuclear receptor proteins (1). Testosterone (T) is the predominant androgen in mammals, and it is converted to a more potent form, dihydrotestosterone (DHT) by 5{alpha}-reductase in certain tissues. DHT or T binds with high affinity to AR and promotes its transcriptional activity that plays a critical role in development and maintenance of various target organs in healthy and pathological states, including muscle, bone, and prostate. Because the prostate undergoes apoptosis after androgen withdrawal (2, 3), the importance of AR in prostate cancer and its dependence on androgens is a focus of ongoing research and drug development (4). In preclinical models of prostate cancer, depletion of AR content results in antiproliferative and proapoptotic effects (5).

Valuable insights regarding the role of AR in prostate growth and cancer development have been provided using human prostate cancer cell lines. In the present study, we have used two prostate carcinoma cell lines, LNCaP and PC-3. LNCaP cells are AR positive, and their growth is stimulated by androgens (6, 7). In contrast, PC-3 cells are AR negative, and their growth is unaffected by androgens.

Recently, nonsteroidal selective AR modulators have been developed as candidate drugs (8). Two of these, 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ) and 4-ethyl-1,2,3,4-tetrahydro-6-(trifluoromethyl)-8-pyridino[5,6-g]quinoline (ETPQ) bind with high affinity (~30 nM) to human AR in vitro and show selective AR modulator activity in animal models (9, 10). In addition to their pharmacological properties, we have reported elsewhere (Bilski, P., B. Risek, C. F. Chignell, and W. T. Schrader, submitted) the physicochemical characteristics of TDPQ and ETPQ, which, upon excitation with visible light (405 nm), act as photosensitizers and generate singlet oxygen (1O2).

Photodynamic therapy (PDT) is a novel form of treatment of cancer and other diseases. This method involves a complex interaction between a photosensitizer, light, and molecular oxygen in a target tissue (10). In this process, 1O2 are generated by energy transfer from the photosensitizer upon excitation with light. Singlet oxygen can elicit other reactive oxygen species (ROS), thereby inducing diverse cellular responses, including oxidative DNA damage and apoptosis (11, 12, 13). Because apoptosis induced by PDT is considered to be one of the critical factors defining the treatment outcome, the data presented in this study provide a promising approach for developing effective photosensitizers for AR-dependent PDT. This mechanism also permits ablation of AR-positive cells for experimental purposes as well. In principle, other nonsteroidal ligands sharing the structural features of TDPQ as well as steroidal ligands photoproducing 1O2 can be used for similar ablations of cells bearing glucocorticoid, progesterone, mineralocorticoid, and other nuclear receptors.

In the present study, we provide evidence that visible light irradiation of AR-positive cells containing TDPQ caused apoptosis and required formation of TDPQ/AR complexes. The apoptotic cell death pathway involved nuclear ROS production, oxidative stress, and nuclear DNA damage.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We present an experimental system for cell irradiation and live cell imaging in combination with fluorescence microscopy using a motorized inverted microscope (Fig. 1Go). First, we established and optimized experimental conditions for irradiation studies by determining dosage parameters, including duration of irradiation, irradiation dose, concentration of the photosensitizer, and postirradiation incubation time. Then, experiments were carried out to test for a role of AR in mediating TDPQ/light-induced cell death.


Figure 1
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  		Fig. 1. Experimental Protocol Illustrating the Procedure for Cell Irradiation Studies

Routinely, cells were seeded and cultured on a cover glass in eight-well chambers at a density of 100,000 cells/cm2 1 d before treatment. Test compounds were added to cell cultures at different concentrations in fresh medium (0.5 ml/well), and cells were incubated for 30–60 min at 37 C before irradiation. A field of cells in each well was irradiated using an inverted microscope in combination with appropriate excitation filters and objectives. Nuclear dyes were added at defined time points after irradiation to stain nuclei of all (Hoechst 33342) or only of compromised cells (PI). Images were captured and analyzed using computer-assisted image analysis to quantify cell death.

 
Compounds Under Study
The structures and photochemical properties of the nonsteroidal and steroidal test compounds that were studied are listed in Table 1Go. All compounds were subjected to spectroscopic analysis and determination of 1O2 production as described in Materials and Methods, and results of these studies will be presented elsewhere (Bilski, P., B. Risek, C. F. Chignell, and W. T. Schrader, submitted). In view of the fact that naturally occurring steroids T and DHT do not absorb above 250 nm (14), they did not produce 1O2 when irradiated as described. Interestingly, synthetic steroids containing conjugated double-bond systems in the A and B rings (R1881, R5020, and RU486) produced 1O2. These compounds are known ligands for several steroid receptors, as indicated in Table 1Go.


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  		Table 1. Nuclear Receptor Ligands Exhibiting Photosensitizing Potential via Photogeneration of Singlet Oxygen

 
Nuclear Translocation of AR in LNCaP Cells by TDPQ
We used immunocytochemistry to assess whether ligands can translocate AR into nuclei of AR-positive cells. AR was detected in the cytoplasm and nuclei of LNCaP cells in the absence of ligands (Fig. 2AGo). However, AR was localized exclusively in nuclei after addition of 10 nM DHT or TDPQ, respectively (Fig. 2Go, B and C). These results confirm the distinctive feature of AR ligands to translocate AR into nuclei of AR-positive cells.


Figure 2
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  		Fig. 2. Immunocytochemical Detection of AR in LNCaP Cells

Cellular distribution in the absence of exogenous androgens (A) and after addition of 10 nM DHT (B) or 10 nM TDPQ (C), respectively. Scale bar, 20 µm.

 
Determination of Photochemical Parameters
Effect of Light.
The effects of light and TDPQ were examined in detail for their ability to induce cell death in LNCaP cells. First, LNCaP cells were irradiated in the absence of TDPQ for periods from 0–5 min with light of 405 nm using HQ405 excitation filter and x40 long-distance (LD) objective. Irradiation for up to 3 min elicited no additional cell death response above the basal level (typically 3 ± 2%). After 5 min, limited cell death above the basal level was evoked (data not shown). To examine only compound-related cell effects, irradiation time was limited to periods of 0–3 min in all TDPQ experiments.

We tested for the effect of visible light irradiation (HQ405 excitation filter and x40 LD objective) on cells containing a high concentration of TDPQ (30 µM). Four hours after irradiation, cells were stained with nuclear dyes and subjected to image capture and analysis to calculate the cell death index. In the absence of light, no cell death above the basal level occurred despite the presence of 30 µM TDPQ (Fig. 3AGo). Thus, TDPQ itself is not toxic in the dark even at very high concentrations and does not induce cell death. The number of propidium iodide (PI)-positive cells progressively increased with duration of irradiation time. The highest photocytotoxicity, amounting to a cell death index of 57 ± 8% was observed after 3 min of irradiation. These conditions corresponded to an irradiation dose of 12 J/mm2 at 405 nm. Thus, a 3-min light irradiation regimen at 405 nm in combination with the x40 LD objective was chosen for further studies unless stated otherwise.


Figure 3
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  		Fig. 3. Effect of Photochemical Parameters on LNCaP Cell Death

A, Irradiation time. LNCaP cells were incubated with 30 µM TDPQ for 60 min and irradiated for different time intervals using the HQ405 excitation filter and x40 objective. Four hours later, cells were stained with nuclear dyes and subjected to image analysis for calculation of cell death index. The level of PI-positive cells was measured following irradiation times varying from 0 to 3 min. B, TDPQ concentration dependence. LNCaP cells were irradiated for 3 min with 12 J/mm2 at 405 nm using different concentrations of TDPQ. Twenty-four hours later, cells were stained with nuclear dyes and subjected to image analysis. Circles indicate the size of the irradiated area. Scale bars, 300 µm (A) and 1000 µm (B).

 
Effect of TDPQ.
The effect of TDPQ on LNCaP cell death is illustrated in Fig. 3BGo. Cells were irradiated with 12 J/mm2 at 405 nm and incubated for 24 h. The number of PI-positive cells increased with increasing concentrations of TDPQ. A concentration of TDPQ as low as 100 nM evoked a dramatic increase in cell death. At concentrations above 300 nM, dead cells were also sloughed off, resulting in regions devoid of cells. These regions ultimately expanded concentrically with time beyond the boundary of irradiated areas, resulting in propagation of cell death into nonirradiated bystander cells (see below).

Morphological Effect of TDPQ Photocytotoxicity
Using the irradiation conditions as defined in Fig. 3Go, we examined LNCaP cells that were incubated with 300 nM TDPQ and irradiated with 2.5 J/mm2 at 405 nm using a x20 microscope objective. Figure 4Go shows the results of irradiating the left half of the well while the right half was kept in the dark using an aluminum foil cover. Twenty-four hours after light irradiation, cells were stained with PI and analyzed by fluorescence microscopy. Light-irradiated cells were shrunken, contained condensed and lobular nuclei and were stained by PI. In contrast, aluminum foil-shielded cells retained their original morphology and were not stained by PI.


Figure 4
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  		Fig. 4. Differences in Cell and Nuclear Morphology and in Permeability to PI between Irradiated and Nonirradiated LNCaP Cells

Cells were incubated with TDPQ (300 nM) for 1 h and irradiated for 3 min with 2.5 J/mm2 at 405 nm. The right side of the chamber was covered with an aluminum foil, whereas the left side was exposed to the irradiation. The irradiated and the nonirradiated regions of the chamber are illustrated with a demarcation line. Twenty-four hours later, cells were stained with PI and assayed for nuclear staining using combined phase-contrast and fluorescence microscopy. Scale bar, 300 µm.

 
Irradiation of Either Cells or TDPQ Alone
We performed two control experiments to assess whether TDPQ and cells had to be irradiated together to induce cell death or whether either could be irradiated separately and recombined (data not shown). First we examined whether prior irradiation of LNCaP cells alone could sensitize them for subsequent killing when nonirradiated TDPQ was added. Cells were seeded in eight-well cover-glass chambers (100,000 cells per well) and irradiated with 12 J/mm2 at 405 nm. Immediately after irradiation, the culture medium was replaced with fresh medium containing TDPQ (0, 300, 1000, or 3000 nM). Twenty-four hours later, nuclear dyes were added and cell death was assessed by fluorescence microscopy. Cell death was not detected above the basal level (3 ± 2%) even at the highest concentration of TDPQ tested. This result demonstrated that the cells were not photosensitized by the light irradiation alone and that the preirradiation did not cause accumulation of an unknown photoproduct(s) able to initiate the apoptotic TDPQ effects at a later time point.

Next, a control experiment tested whether irradiated TDPQ could retain a persistent toxic effect that could be transferred subsequently to nonirradiated cells. We preirradiated 1000 nM TDPQ in 0.5 ml cell culture medium for 8 min through a x2.5 microscope objective at 405 nm to achieve the same energy input to the TDPQ solution as was used for the cell-irradiation experiments. This objective allowed irradiation of about 70% of the well area. (To verify that these conditions had photoactivated TDPQ in the cultures, LNCaP cells were included in one such irradiation. When 1000 nM TDPQ was used, nearly all of the cells were dead after 24 h.) The irradiated cell-free TDPQ was then added to recipient nonirradiated LNCaP cells grown in a separate eight-well cover-glass chamber either immediately or 30 min after irradiation. Twenty-four hours later, cell death was assessed by fluorescence microscopy. No cell death above the basal level was observed (data not shown).

These two experiments demonstrated that the death reaction required in situ irradiation of TDPQ directly within the cells and that the photoactivation was short-lived. These results were consistent with a photoactivation process involving transient species such as 1O2 as seen in cell-free experiments.

Blockade of TDPQ/Light-Induced Cell Death by DHT and Intracellular Distribution of TDPQ
Next, we tested whether nuclear AR/ligand complexes were required for mediating TDPQ/light-induced cell death. LNCaP cells were irradiated with 12 J/mm2 at 405 nm in the absence or presence of a large excess of DHT (3000 nM). Twenty-four hours after irradiation, cells were stained with nuclear dyes and the cell death index was determined after image capture and analysis. Irradiation of cells without TDPQ had no effect on cell death (Fig. 5AGo, top left panel). Similarly, irradiation of cells after addition of a high concentration of DHT alone had no effect (Fig. 5AGo, lower left panel). Hence, DHT, which does not absorb visible light and cannot produce 1O2 (see Table 1Go), could not trigger the cell death response. Cell death was observed in the irradiated region when TDPQ was used at either 100 or 300 nM in the absence of DHT. However, when excess DHT was added simultaneously as a competitor for AR, the cell death was completely blocked (Fig. 5AGo, middle and right panels). This finding is consistent with an essential role for AR/TDPQ complex formation in the cell death reaction induced by TDPQ and light.


Figure 5
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  		Fig. 5. DHT Blockade and Intracellular Localization of TDPQ

A, DHT blockade. LNCaP cells were preincubated in the absence (top panels) or presence (bottom panels) of 3000 nM DHT for 1 h before addition of 0, 100, or 300 nM TDPQ (left to right panels, respectively). One hour later, cells were irradiated with 12 J/mm2 at 405 nm. Nuclear dyes were added 24 h after irradiation, and images were captured using Axiovision software. Circles denote the location of irradiated regions. B, Detection of intracellular TDPQ fluorescence. LNCaP cells were incubated with TDPQ and DHT at the indicated concentrations for 60 min. TDPQ fluorescence (top panels) was detected using the HQ405/20-nm excitation and HQ430-nm long-pass emission filters. Lower panels show corresponding phase-contrast images. Scale bars, 200 µm (A) and 50 µm (B).

 
Because TDPQ is strongly fluorescent, its distribution and localization within cells can be directly monitored by fluorescence microscopy using appropriate excitation and emission filters. LNCaP cells were incubated with 100 nM TDPQ with or without DHT (3000 nM) for 1 h, and the intracellular distribution of TDPQ was observed using the HQ405 nm excitation and HQ430 nm long pass emission fluorescence filters. TDPQ was detected throughout the cells, and its distribution was not affected by the presence of excess DHT (Fig. 5BGo). Hence, the blockade by DHT as shown in Fig. 5AGo occurred even though the cytoplasm of the cells contained TDPQ. Given that irradiation of cytoplasmic TDPQ did not elicit the cell-death response, these results demonstrate that the cell death pathway requires photoactivation of TDPQ that is bound to AR and localized within the nucleus.

Cell Sensitivity to TDPQ/Light Irradiation Is Dependent on Cellular Levels of AR
We investigated the effect of modulating AR levels on the potency and efficacy of TDPQ to elicit cell death using LNCaP, PC-3/AR, and PC-3/neo cell lines, each one containing different amounts of AR. Western blot analysis revealed that LNCaP cells expressed about 10 times more AR protein than PC-3/AR, whereas no AR was detected in PC-3/neo cells (Fig. 6AGo). The TDPQ/light concentration-dependent cell-killing curves are illustrated in Fig. 6BGo (the LNCaP cell-killing curve was obtained by quantitative analysis of the images shown in Fig. 3BGo). The potency (concentration for 50% efficacy) of TDPQ was approximately 300 nM for LNCaP. Efficacy reached 100% killing at 3000 nM TDPQ. In contrast, only a marginal cell killing with an efficacy of 20% was observed in PC-3/neo cells even at the highest concentration (3000 nM) of TDPQ. However, upon stable transfection of AR into PC-3 cells, potency was approximately 1000 nM, and the efficacy reached 90% at the highest concentration of TDPQ. These data demonstrate that both cell-killing potency and efficacy are related to the amount of AR in each cell line.


Figure 6
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  		Fig. 6. Alteration of AR Expression and Its Effect on TDPQ/Light-Induced Cell Death

Levels of AR were altered either by stable transfection of AR-deficient PC-3 cells or by gene silencing of AR-containing LNCaP cells. A, Detection of AR protein in three different human prostate tumor cell lines, LNCaP, PC-3/AR, and PC-3/neo. Twenty micrograms of WCL were analyzed by Western blotting using antibodies specific for AR or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as a loading control. B, Concentration response curves for TDPQ/light-induced cell death in LNCaP, PC-3/AR, and PC-3/neo cells. LNCaP (solid line), PC-3/AR (dashed line), and PC-3/neo cells (dotted line) were incubated with different concentrations of TDPQ for 1 h and irradiated for 3 min with 12 J/mm2 at 405 nm. Twenty-four hours later, nuclear dyes were added in fresh cell culture medium, and images were captured and analyzed for calculation of cell death index. Cell-killing concentration response curve for LNCaP was generated using images as shown in Fig. 3BGo. Each point on the graph represents the mean ± SD of three wells per concentration. C, Silencing of AR gene expression in LNCaP cells and detection of AR protein by Western blotting. LNCaP cells were transiently transfected using different concentrations of siRNAs, and 48 h later, 20 µg WCL was analyzed by Western blotting as described above. Bands were scanned densitometrically and expressed as relative amounts of vehicle-treated control, which was defined as 100%. β-Actin was used as loading control. Each point on the graph represents the mean ± SD of three wells per concentration. D, Silencing of AR gene expression in LNCaP cells and determination of cell death index after TDPQ/light irradiation. LNCaP cells were transiently transfected with 619, 620, or scrambled (scr) siRNA sequences, and 48 h later were subjected to TDPQ/light irradiation using 12 J/mm2 at 405 nm and 300 nM TDPQ. Cell death index was scored 24 h after irradiation. Basal level indicates the extent of cell death in the absence of any treatment. Each point on the graph represents the mean ± SD of three wells per concentration. Asterisks indicate statistical significance (P < 0.05) vs. control, as indicated.

 
In view of the fact that introduction of AR sensitized PC-3 cells to TDPQ, we asked whether suppression of AR would decrease TDPQ sensitivity in LNCaP cells. For this purpose, we used small interfering RNA (siRNA) gene silencing to down-regulate expression levels of AR in LNCaP cells. These cells were transiently transfected with two different siRNA sequences targeting two different regions within the ligand-binding domain (LBD) of AR. The target for the 619 siRNA sequence is within exon 4, and for 620 siRNA, it is within exon 6. Down-regulation of AR by both 619 and 620 siRNAs was detected at the protein level 48 h after transfection (Fig. 6CGo). Scrambled RNA sequences did not suppress AR protein expression. A concentration-dependent down-regulation of AR protein was observed for both 619 and 620 siRNA sequences. Expression levels amounted to 60 ± 15% relative to vehicle-treated control at the lowest concentration of siRNA 619 (1.5 nM) and declined further to 30 ± 10% (5 nM) and 15 ± 5% (15 nM), respectively. A more modest suppression was observed using the 620 siRNA sequence. A combination of both 619 and 620 siRNA sequences (5 nM each) significantly reduced AR protein expression down to 35 ± 10% of the vehicle-treated controls.

A TDPQ/light irradiation assay was performed in parallel experiments using LNCaP cells treated with these siRNAs. Both 619 and 620 siRNA decreased sensitivity to killing when the cells were irradiated with 12 J/mm2 at 405 nm in the presence of 300 nM TDPQ (Fig. 6DGo). Both 619 and 620 siRNA sequences reduced cell death by over 90%, suppressing the cell death indices to 10 ± 5% and 7.5 ± 3%, respectively. These low cell death indices were comparable with that observed in the absence of TDPQ (5 ± 2%) and approached the basal level of cell death. Similarly, the combination of siRNAs reduced the cell death index to 30 ± 3%. Conversely, there was no statistically significant effect of the scrambled RNA sequences on cell killing. These data confirm that TDPQ/light-induced cell death is an AR-mediated event in LNCaP cells and that the cell-killing efficacy correlates with the amount of AR.

Other Ligands Also Induce Cell Death upon Photosensitization
Steroidal AR ligand R1881.
A steroidal compound was also tested in the LNCaP cell death paradigm. R1881 is a commonly used steroidal research ligand that binds to human AR (hAR) (15, 16). Unlike DHT, R1881 contains a conjugated double-bond system in the A and B rings that causes it to absorb in the UVA region (Amax= 335 nm); moreover, R1881 generates 1O2 with a high quantum yield ({varphi}) of 80% (Table 1Go). Due to these molecular and physicochemical properties, we tested it for the photo-killing effect in LNCaP cells. A new irradiation time course was carried out in the absence of R1881 to determine the optimal exposure time using a UVA excitation filter (365/12 nm) and a x40 LD microscope objective. Cells were more sensitive to irradiation at this shorter wavelength of light (data not shown). Because no UVA light-induced cell death was observed for up to 30 sec of irradiation, 30 sec was chosen for the R1881 studies. These parameters corresponded to an irradiation dose of 2 J/mm2 at 365 nm. The R1881 concentration-dependent cell-killing curve is illustrated in Fig. 7AGo. The cell death index increased with R1881 concentration, reaching 37 ± 2% at 300 nM 24 h after irradiation. No cell death was observed in the presence of 300 nM R1881 in the nonirradiated regions of the cell culture well (data not shown).


Figure 7
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  		Fig. 7. Effect of R1881 and ETPQ on LNCaP Cell Death

A, LNCaP cells were incubated with a steroidal AR ligand, R1881, at different concentrations for 1 h and irradiated for 30 sec using an UV excitation filter (365/12 nm). These settings correspond to an irradiation dose of 2 J/mm2. Nuclear dyes were added 24 h after irradiation, and images were captured and analyzed to calculate the cell death index. Cell death increased proportionally with R1881 concentration. Each point on the graph represents the mean ± SD of three wells per concentration. B, LNCaP cells were incubated with ETPQ at two different concentrations for 1 h and irradiated for 3 min with 12 J/mm2 at 405 nm. Twenty-four hours later, cells were stained with nuclear dyes and subjected to image capture and analysis. Circles indicate the size of irradiated area. Scale bar, 1000 µm.

 
Nonsteroidal ETPQ.
We also tested cell-killing properties of ETPQ, which is a structural isomer of TDPQ. The ETPQ preparation is a racemic mixture with identical photochemical properties as described for TDPQ (see Table 1Go). The cell-killing activity of ETPQ is illustrated in Fig. 7BGo. At a concentration of 1000 nM, about 90% of the cells were killed within the irradiated area, whereas at 3000 nM, the cell death amounted to 100%. At this concentration, the cell death was also observed in the nonirradiated bystander cells. This structural isomer thereby also retains the property of a photosensitizer. We have not compared full dose-response curves to determine the relative potencies. Future experiments using the two resolved ETPQ enantiomers are planned to address this question.

Pathway of Cell Death in LNCaP Cells
We tested cell cultures exposed to TDPQ and light to determine the mechanism of cell death. The assays included cell morphology analysis, permeability of cells to PI, externalization of phosphatidylserine (PS), and inhibition of caspases. LNCaP cells were treated with 300 nM TDPQ, irradiated with 12 J/mm2 at 405 nm and incubated for 24 h before analysis. Phase-contrast and fluorescence microscopy revealed shrunken cells with lobular nuclei, and these cells were also permeable to PI (Fig. 8Go, A and B). These morphological features are characteristic of apoptosis. To confirm the apoptotic cell death pathway, a parallel set of cells was stained for externalized PS using annexin-V fluorescein isothiocyanate (FITC) conjugate. All annexin-V-positive cells were also permeable to PI (Fig. 8CGo).


Figure 8
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  		Fig. 8. Tests for Apoptosis in LNCaP Cells

LNCaP cells were incubated with 300 nM TDPQ for 1 h and irradiated with 12 J/mm2 at 405 nm. Twenty-four hours later, cells were stained with annexin V-FITC conjugate and counterstained using nuclear dyes Hoechst 33342 and PI. Apoptosis was assayed by phase-contrast microscopy (A), PI nuclear staining (B), annexin V reactivity (C), and caspase inhibition (D). For caspase inhibition, LNCaP cells were incubated with 300 nM TDPQ in absence or presence of 20 µM general caspase inhibitor z-VAD-fmk for 1 h and irradiated as described above. One hour later, cells were stained with nuclear dyes and cells death index calculated. Asterisk indicates statistical significance (P < 0.05), as indicated. Scale bar, 50 µm.

 
Apoptosis was also evaluated by measuring inhibition of caspase activity using the general inhibitor z-VAD-fmk. LNCaP cells were incubated with 300 nM TDPQ in the presence or absence of 20 µM z-VAD-fmk for 1 h and irradiated with 12 J/mm2 at 405 nm. One hour later, cells were stained with nuclear dyes and subjected to image capture and analysis. In the absence of caspase inhibitor, the cell death index was 7.5 ± 0.7%. This index was reduced to 1.8 ± 0.4% when the cells were irradiated in the presence of the inhibitor (Fig. 8DGo). The results of these four assays demonstrate that TDPQ/light irradiation of LNCaP cells induces cell death via apoptosis.

Test for Photoinduced Oxidative Stress
Table 1Go shows ligands that photogenerated 1O2 when irradiated in the cell-free system. To test for the intracellular production of ROS, we used the major physiological antioxidant ascorbic acid (AA), a hydrophilic low-molecular weight molecule that belongs to a network of cellular antioxidant defense mechanisms (17, 18). LNCaP cells were grown as described above and incubated with or without AA (100–1000 µM) in the presence or absence of 300 nM TDPQ for 1 h in the dark. After 12 J/mm2 irradiation at 405 nm, cells were incubated overnight at 37 C, stained with nuclear dyes, and subjected to fluorescence microscopy. The protective effect of AA is illustrated in Fig. 9AGo. The cell death index was calculated from several wells and is illustrated in Fig. 9BGo. In the absence of AA, TDPQ/light caused a cell death index of 24 ± 6%; this effect was significantly reduced to 5 ± 2% in the presence of 300 µM AA. Control cultures containing AA alone with or without irradiation showed no increase in cell death above the basal level (data not shown). This protective antioxidant effect confirmed a role for ROS and oxidative stress in the photosensitization effects of TDPQ.


Figure 9
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  		Fig. 9. Preventive Effect of AA on TDPQ/Light-Induced LNCaP Cell Death

LNCaP cells were incubated with 300 nM TDPQ in absence or presence (300 µM) of AA for 1 h and irradiated with 12 J/mm2 at 405 nm. After a 16-h incubation, cells were stained with Hoechst 33341 and PI nuclear dyes and subjected to image analysis for calculation of cell death index. The circle denotes the area of irradiation. Each point on the graph represents the mean ± SD of three wells per concentration. Asterisk indicates statistical significance (P < 0.05%) vs. control. Scale bar, 400 µm.

 
Oxidative Stress-Induced DNA Damage
Oxidative stress caused by ROS can produce mutations and lesions in genomic and mitochondrial DNA, including formation of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG; (19). This substituted nucleotide is a predominant and stable product of earlier short-lived DNA oxidized intermediates. For this reason 8-oxo-dG is widely used as a marker of oxidative stress and oxidative DNA damage (20). We tested whether TDPQ/light treatment of LNCaP cells resulted in accumulation of this oxidative DNA adduct using either of two commercially available fluorescence methods for detection of 8-oxo-dG. The simpler method, applying a single reagent (OxyDNA assay kit) was used in combination with fluorescence microscopy.

Exposure of LNCaP cells to TDPQ/light irradiation (300 nM TDPQ, 12 J/mm2 at 405 nm) resulted in increased levels of 8-oxo-dG DNA lesions in nuclei of irradiated cells (Fig. 10Go). The intensities were considerably higher than those seen in the nuclei of adjacent nonirradiated cells or in cells that were irradiated in the absence of TDPQ. The increase in 8-oxo-dG staining was prominent as early as 2 h after irradiation. These data, together with AA protection show that treatment of cells with TDPQ/light led to the formation of ROS and accumulation of oxidative DNA damage in nuclei.


Figure 10
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  		Fig. 10. Detection of 8-oxo-dG in LNCaP cells by fluorescence microscopy

LNCaP cells were incubated with vehicle (0.1% DMSO) or TDPQ (300 nM) for 1 h and irradiated with 12 J/mm2 at 405 nm. The 8-oxo-dG was detected 2 h after irradiation using the commercially available OxyDNA assay kit. The circle denotes the area of irradiation. Scale bar, 300 µm.

 
Concentric Propagation of Apoptotic Cell Death
During the course of irradiation studies, we noticed that cell death often spread beyond the initial irradiated area. This effect was particularly pronounced at higher concentrations of TDPQ and ETPQ (see Figs. 3BGo and 7BGo). To test the spreading of cell death more closely, LNCaP cells were incubated with 3000 nM TDPQ for 1 h and irradiated with 12 J/ mm2 at 405 nm. Twenty-four hours later, cells were stained with nuclear dyes and subjected to fluorescence microscopy. Images were captured either immediately or after 2 or 4 h of additional incubation. The results are illustrated in Fig. 11AGo. The irradiated area of the well is outlined by the inner circle (solid line). A concentric progression of cell death was observed into the adjacent nonirradiated area as shown by the outer circle (dotted line). The progression spread slowly with time, as shown by the two insets on Fig. 11AGo taken at 26 and 28 h, respectively. The detection of annexin-V-positive cells in these areas indicated that cell death was due to apoptosis (data not shown). The propagation of cell death into the nonirradiated region is indicative of a bystander effect.


Figure 11
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  		Fig. 11. Concentric and Time-Dependent Propagation of Cell Death into Nonirradiated Regions

A, LNCaP cells were incubated with 3000 nM TDPQ for 1 h and irradiated with 12 J/mm2 at 405 nm. Twenty-four hours after irradiation, cells were stained with nuclear dyes, and images were taken every 2 h over a period of 4 h. The smaller circle (solid line) denotes the size and location of the irradiated area, whereas the larger circle (dotted line) denotes the adjacent area of the same size. The square on the bottom right of the image composite illustrates the region that was monitored over a period of 4 h after addition of nuclear dyes. B, Effect of cell density on cell death in irradiated and adjacent nonirradiated regions. LNCaP cells were incubated at low (25,000 cells per well, left panel) or high (120,000 cells per well, right panel) density with different concentrations of TDPQ for 1 h and irradiated with 12 J/mm2 at 405 nm. Twenty-four hours later, cells were stained with nuclear dyes and subjected to image capture and analysis. The smaller circle (solid line) denotes the size and location of the irradiated area, whereas the larger circle (dotted line) denotes the adjacent area of the same size. Images shown in B contain cells that were incubated with 3000 nM TDPQ. C, Determination of the cell death index in the irradiated (solid line) and in the adjacent nonirradiated (dotted line) regions of low-density (open circles) and high-density (full circles) plated cells. Each point on the graph represents the mean ± SD of three wells per concentration. Asterisks indicate statistical significance (P < 0.05) vs. control, as indicated. Scale bars, 250 µm (A) and 500 µm (B).

 
Because the bystander effect could have been mediated either through the cell culture medium or by direct transfer between contacting cells, experiments were carried out to test whether direct cell-cell contact is required. LNCaP cells were seeded at low (25,000 cells per well) and high (120,000 cells per well) densities, incubated with TDPQ (0, 300, 1000, and 3000 nM) for 1 h and irradiated with 12 J/mm2 at 405 nm. Twenty-four hours later, cells were stained with nuclear dyes and subjected to fluorescence microscopy. Images were captured and analyzed, and cell death index was calculated as described above. Very few cell contacts were established at low seeding density (~10% confluency; Fig. 11BGo, left panel), whereas almost all cells were in contact at high density (90–100% confluency; Fig. 11BGo, right panel). In low-density cultures, cell death was confined within the irradiated area (inner circle, solid line) and was not observed in the adjacent area (outer circle, dotted line). In contrast, death spreading was observed into the adjacent nonirradiated area of high-density seeded cells. The cell death index was calculated for the irradiated areas as well as for the adjacent nonirradiated regions of the same surface area (Fig. 11CGo). Although the cell-killing curves were essentially the same for low and high cell densities within the irradiated regions, killing was observed in the nonirradiated region only when cell-cell contact was prevalent. A statistically significant increase in cell killing above the basal level was observed at 1000 nM TDPQ, and it reached 45 ± 5% at 3000 nM TDPQ. Death in this nonirradiated area was a consequence of the bystander effect. These findings demonstrate that direct cell-cell contacts were required for the propagation of cell death signals into the nonirradiated bystander cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The observation that nonsteroidal AR ligands TDPQ and ETPQ can act as photosensitizers upon irradiation with visible light (405 nm) in cell-free systems prompted us to hypothesize that these compounds could act as photosensitizers in living cells as well. If so, this will lead to elaboration of ROS and potentially trigger cell death reactions. A second finding, that AR-positive LNCaP cells were sensitive to TDPQ-induced killing, whereas AR-negative PC-3 cells were not, led us to hypothesize that formation of AR/TDPQ complexes in nuclei was required to cause the cell death reaction. We developed the methodology described in the current studies and documented that AR and its complexing with TDPQ in nuclei are required to evoke apoptosis. In addition, we employed several assays to demonstrate that apoptosis predominates in our experimental paradigm. This is because there are conflicting literature reports on AR perturbation concerning cell death. Some reports describe proapoptotic effects of androgens and AR (21, 22, 23), whereas others present data for antiapoptotic effects (24, 25, 26).

To investigate the role of AR in inducing apoptosis in our test system, we used multiple approaches to perturb the function and concentration of AR. Our findings demonstrate that blockade of TDPQ/AR by DHT as well as down-regulation of AR gene expression by siRNA resulted in attenuation of the apoptotic cell death response in LNCaP cells. We also demonstrated that both potency and efficacy of cell killing depended upon content of AR in the different cell lines.

Owing to the fluorescent nature of TDPQ, we could directly monitor its cellular presence during the DHT blockade studies. Although DHT blocked TDPQ association with AR, it could not change the overall TDPQ concentration in the cells. Thus, we assume that cytoplasmic TDPQ was also photoactivated but could not trigger the apoptotic response. Moreover, even if TDPQ was generating toxic photoinduced cytoplasmic events, they were not sufficiently deleterious to affect cell viability, at least not during the extent of our observations up to 24 h. Given that TDPQ was able to translocate AR to nuclei, we concluded that it was the photoactivation of nuclear TDPQ/AR complexes that triggered apoptosis. The detection of oxidative DNA damage in the form of 8-oxo-dG is consistent with the photochemically induced reactions in nuclei. The accumulation of oxidative DNA damage in nuclei within the TDPQ/light-irradiated regions was manifested in noticeably higher intensities of 8-oxo-dG staining as compared with adjacent nonirradiated cells. Low-level detection of 8-oxo-dG in nuclei of nonirradiated cells is consistent with reports that 8-oxo-dG is present in all growing cells (27, 28). The observed protective effect of AA, which is ascribed to its antioxidant and free radical scavenging capacities (29, 18) has also been reported by other investigators (30, 31).

Our data suggest that oxidative DNA damage via ROS plays an important role in TDPQ/light-evoked apoptosis. Consequently, the underlying mechanisms will be subjected to more detailed studies by investigating whether or not DNA interactions by AR are required, and whether the ligand’s pharmacology as a transcriptional AR agonist or antagonist plays a role in these interactions. Currently, we favor the idea that the effects reported here are independent of transcriptional activity per se.

It has been reported that LNCaP cells bear a specific mutation (T877A) that causes drugs such as flutamide to act as agonists (32, 33). Similarly, unpublished cotransfection studies have shown that TDPQ behaves like flutamide and is an AR agonist in LNCaP cells. However, this compound is an antagonist in the stably transfected PC-3/AR cell line whose hAR lacks this mutation (34, 35). Our data show that both cell lines, one for which TDPQ is an AR agonist (LNCaP) and one for which it is an AR antagonist (PC-3/AR) are killed by the photosensitization reaction. Hence, the transcriptional pharmacology of TDPQ is probably not a factor in the photosensitization reported here. This distinction may also help to explain why the effective concentration of these compounds is about 10-fold higher than their published potencies in cell-based cotransfection assays (12, 13). The potency of TDPQ in both AR-containing cell lines was higher than the published ligand-binding constant (26 ± 5 nM) (13) and its potency as a transcriptional modulator (27 ± 5 nM) (13). Other unpublished work by us and others has demonstrated a separation between potencies when diverse cellular endpoints are measured. In addition, the cell culture medium used for irradiation experiments contained fetal calf serum, which was used without charcoal treatment to remove endogenous steroids. Hence, the potencies observed for the added ligands may also be correspondingly elevated due to the role of endogenous compounds competing for AR. In any event, we can only speculate as to the reason for this separation. Because cells were incubated for up to 24 h before death evaluation, the cumulative effects of multiple pathways may cause the separation to be widened. Alternatively, as DNA oxidation studies presented here show, it is conceivable that AR is acting as a carrier to deliver TDPQ to sensitive DNA sites, rather than as a conventional transcriptional regulator.

Although most studies of apoptosis have focused predominantly on intracellular signaling pathways, evidence is emerging that intercellular communication via gap junctions might influence the development and the extent of apoptosis as well (36, 37, 38). Our findings, showing a slow, concentric spreading of the apoptotic cell death response into the nonirradiated region of the high but not low cell densities, are consistent with a bystander effect mediated via gap junctions. It is of interest to note that gap junction-mediated cell communication can also be modulated by irradiation (39). An alternative mechanism, spreading of the apoptotic cell death signal by paracrine processes, appears to be less likely because the bystander effect was not observed after 24 h in sparsely populated cultures, i.e. when cells were not in direct cell-cell contact. However, additional cell-cell mechanisms, including oxidative metabolism, plasma membrane-bound lipid rafts and calcium fluxes could also contribute to the observed bystander effect. The participation of these AR-independent intercellular mechanisms might explain why a bystander effect was observed only at high concentrations (1000–3000 nM) of photosensitizers. In contrast, lower concentrations (100–300 nM) were sufficient to induce cell killing in individual cells within the irradiated area using AR-dependent intracellular pathways. Although it is currently unclear what the molecular mediators of bystander effects are, there are studies pointing toward ROS, including long-lived radicals (40, 41, 42, 43).

Our current understanding of the AR-mediated photoactivation cell death pathway is presented in Fig. 12Go. Upon addition of a photosensitizing ligand, AR-ligand complexes will accumulate in the nucleus, bound to androgen response elements on chromosomal DNA. The next step is the photoactivation of ligand in these DNA-bound AR complexes, thereby producing ligand in an excited triplet state. Excited ligand may collide with O2 generating 1O2. Although the highly reactive 1O2 has a very short lifetime (<40 nsec) and a short radius of action (<20 nm) in biological systems (44), it is sufficient to attack substrates such as DNA directly. More likely, it is able to initiate the generation of other ROS, which in turn can interact with other biological molecules, including DNA and AR. The oxidative pathway involving 1O2 is termed type II oxidation (45). Metastable oxidative adducts to DNA can form that have very short lifetimes and ultimately decay to stable oxidative products like 8-oxo-dG. This latter entity represents DNA damage that, when sensed by DNA repair and replicative enzymes, could trigger apoptosis.


Figure 12
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  		Fig. 12. A Model for Triggering Apoptosis via AR-Mediated Photosensitization

Certain AR ligands can be photoactivated resulting in generation of 1O2 in presence of O2. The type II oxidation pathway (shown in bold) is the one for which direct experimental evidence has been presented. The oxidations proceed through production of ROS that can oxidize DNA and lead to apoptosis. The alternative type I oxidative pathway involves either formation of stable adducts of the activated ligand interacting directly with DNA or other substrates or elaboration of H2O2 followed by subsequent oxidations.

 
An alternative type I oxidative reaction that may act through the production of H2O2 has not been excluded, however (45). Downstream molecular events of either type I or type II can lead not only to DNA damage as we have demonstrated but also perhaps to attacks and modifications of the AR protein itself. Earlier studies (46) had shown that photoactivation of certain ligands can lead to cross-linking of an activated ligand directly to a nuclear receptor protein’s LBD. This alternate pathway could also occur, at least for steroidal photoactivatable compounds. At this time, we cannot assess the extent to which these two independent fates of excited state TDPQ, ETPQ, and R1881 would occur.

In conclusion, our data demonstrate for the first time that certain AR ligands can be used in combination with light to act as photosensitizers for targeting and killing AR-positive cells. This mechanism opens a promising approach for targeted ablation of receptor-positive cells for experimental purposes. It also could be extended for use in the treatment of AR-associated disorders by PDT. Our identification of additional 1O2-photosensitizing nuclear receptor ligands suggests that this concept can be applied to other members of the nuclear receptor superfamily as well.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Steroid Receptor Ligands
The chemical structures and photosensitizing properties of TDPQ and ETPQ are presented in Table 1Go. The compounds were obtained from Dr. Tim Willson at GlaxoSmithKline (Research Triangle Park, NC). Stock solutions (1 mM) were prepared in dimethylsulfoxide (DMSO) and kept in the dark at –30 C. Commercially available steroid receptor ligands were purchased from Steraloids Inc. (Newport, RI; DHT and testosterone), PerkinElmer Life And Analytical Sciences, Inc. (Waltham, MA; R1881 and R5020) and Sequoia Research Products (Pangbourne, UK; RU486).

Singlet Oxygen Measurements
The best characterization of 1O2 production is to determine the quantum yield ({varphi}) of its photogeneration, where {varphi} is defined as a ratio of the number of the photo-produced 1O2 molecules to the number of absorbed photons. The {varphi} of 1O2 production was measured from the 1O2 phosphorescence spectra as described previously (47) using a steady-state 1O2 laser spectrometer featuring an optimized optical system as in our pulse 1O2 spectrophotometer (48). Briefly, the apparatus used a germanium diode (model 403 HS; Applied Detector Corp., Fresno, CA), a light chopper with a monochromatore in conjunction with an efficient optical system, and a digital lock-in amplifier (Stanford Research, Sunnyvale, CA) for signal detection. Samples were excited from a 500-W mercury lamp through an appropriate interference filter combination. The 1O2 phosphorescence spectra were recorded during one approximately 30-sec scan over the range of 1200–1350 nm and were normalized to the same number of absorbed photons at the excitation wavelengths selected by the filters (Table 1Go). The relative number of absorbed photons at the excitation wavelengths was calculated using the Lambert-Beer law. The absorption spectra of each compound dissolved in DMSO or cell culture medium were acquired using an HP Diode Array Spectrophotometer model 8452A (Hewlett Packard Co., Palo Alto, CA). The air-equilibrated samples were prepared in a Suprasil fluorescence cuvette (0.5 cm path length), and the {varphi} of 1O2 production were calculated using perinaphthenone (phenalenone) as a reference (49).

Cell Cultures
Cell culture medium RPMI 1640 was obtained from GIBCO-BRL (Gaithersburg, MD) and was supplemented, unless otherwise noted, with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine to obtain complete growth medium. Cell cultures (see below) were maintained at 37 C in a humidified atmosphere containing 95% air and 5% CO2 and reseeded into fresh medium every 7 d. One day before treatments, cells were seeded at a density of 100,000 cells/cm2 and cultured on a cover glass in eight-well chambers (Lab-Tek II Chamber Coverglass; Nalge Nunc International, Rochester, NY). Unless stated otherwise, all experiments were carried out on subconfluent cell densities. The LNCaP human prostate carcinoma cell line was obtained from the American Type Culture Collection (Rockville, MD). LNCaP is androgen sensitive and expresses AR containing a mutation (T877A) in the LBD (50, 51). PC-3 cells, which are also derived from human prostate carcinoma, do not express detectable levels of AR, and growth is not regulated by androgen in vitro (21, 52, 53). Two different PC-3 sublines, PC-3/AR and PC-3/neo, were established by stably transfecting AR-deficient PC-3 cells with a vector bearing the human AR gene or the empty vector (35). Both PC-3 sublines were provided by Dr. Kerry Burnstein (University of Miami, Miami, FL). The stable clones were selected in G418 (GIBCO-BRL, Gaithersburg, MD) and maintained in complete RPMI 1640 cell growth medium.

Experimental Conditions for Cell Irradiation Studies
A generalized experimental protocol illustrating the procedure for cell irradiation studies is summarized in Fig. 1Go. Live cell imaging and cell irradiation studies were performed using an inverted microscope (Zeiss Axiovert 200M; Zeiss, Oberkochen, Germany) equipped with an incubation chamber, epifluorescence, and a phase-contrast system. During irradiation, cells were kept on a heated stage at 37 C in a humidified atmosphere and were irradiated with a mercury light source (Osram GmbH, Munich, Germany; HBO100) in combination with appropriate excitation filters. Based on absorption and emission spectra of TDPQ and ETPQ, a set of custom-made microscope fluorescence filters (Chroma Technology Corp., Rockingham, VT) was used for excitation (HQ405/20 nm) and emission (HQ430LP nm) of these compounds in cell cultures. Irradiation doses were defined by the properties of microscope objectives and exposure time of cells to light irradiation at the focal plane of the specimen. A laser power meter, Field-Max II-Top (Coherent, Inc., Portland, OR) was used to measure the power output of mercury light at the focal plane of the specimen in combination with appropriate microscope objectives and fluorescence excitation filters. The power output varied between 15 and 25 mW, depending on the properties of microscope objectives. The light dose (joules per square millimeter) was calculated as the product of fluence rate (milliwatts per square millimeter) and irradiation time (seconds). Relative irradiation doses of 1, 2.5, and 12 J/mm2 were achieved by irradiating samples for 3 min using x10, x20, and x40 achroplan objectives, respectively. Unless stated otherwise, cells were routinely irradiated for 3 min using a LD x40 objective, corresponding to an irradiation dose of 12 J/mm2. Test compounds were added to cell cultures at different concentrations in fresh medium (0.5 ml/well in eight-well chambers), and cells were incubated for 30–60 min at 37 C before irradiation. Different irradiation times (from 0–5 min) and concentrations up to 3000 nM were used to determine photocytotoxic effects of the test compounds. Control wells lacked photosensitizer, irradiation, or both stimuli.

Calculation of Cell Death Index
Image analysis was applied for quantitative determination of cell death index. Two nuclear dyes, PI and Hoechst 33342, were purchased from Molecular Probes (Invitrogen, Carlsbad, CA) and were used at final concentrations of 10 µM (Hoechst 33342) or 30 µM (PI), respectively. Nuclei were stained with Hoechst 33342 and PI for 30 min and examined by fluorescence microscopy using appropriate dual-band filter sets (Chroma Technology) suitable for excitation and emission of PI (BP 546/12; LP 590) and Hoechst 33342 (BP 365/12; LP 397). Carl Zeiss’ proprietary software Axiovision 4.4 was used to control the motorized microscope as well as for image capture and analysis. Images were captured using a monochrome digital camera (AxioCam MRm; Zeiss). The percentage of cell death was calculated by counting the number of PI-positive nuclei and normalizing it with respect to the number of Hoechst 33342-positive nuclei. Cell death index was used for determination of cell-killing efficacies and potencies. Every experiment was performed in triplicate wells, and all experiments were carried out at least three times.

Apoptosis Assays
Several assays were employed to investigate cell death pathways, including changes in cellular morphology, permeability to PI, annexin V staining, and caspase inhibition.

Cell Morphology.
Cell shrinkage, nuclear condensation, and fragmentation of nuclei were used as morphological criteria of apoptosis (54). These changes were observed using phase-contrast light microscopy.

PI Staining.
PI was used to detect cells that have lost membrane integrity and as a consequence accumulated PI red staining throughout the nucleus. Routinely, PI staining was used in combination with a second nuclear dye, Hoechst 33342, to differentiate between compromised and healthy cells.

Annexin V Staining.
The appearance of PS residues on the surface of the cell is an early indication of apoptosis. PS was analyzed using an annexin V-FITC apoptosis detection kit (BioVision Research Labs, Mountain View, CA) with a fluorescent conjugated annexin V anticoagulant that has a high affinity to PS. Annexin V-FITC was visualized using fluorescence microscopy equipped with a green fluorescence filter set (BP450–490 nm excitation, BP515–565 nm emission).

Caspase Inhibition.
A general caspase inhibitor, z-VAD-fmk, was purchased from R&D Systems Inc. (Minneapolis, MN) and was used to assay for inhibition of apoptosis. The assay was performed according to the manufacturer’s protocol using a final concentration of 20 µM caspase inhibitor in 0.1% DMSO in the culture medium. LNCaP cells were grown in complete RPMI 1640 medium on a cover glass in eight-well chambers as described above. Twenty-four hours later, cells were incubated in fresh RPMI 1640 medium for 1 h in the presence of 300 nM TDPQ and 20 µM z-VAD-fmk. One hour later, cells were irradiated with 12 J/mm2 at 405 nm and incubated for 1 h. PI and Hoechst 33342 were added in fresh RPMI 1640 medium and incubated for 30 min. Detection of nuclear staining, image capture, and analysis were the same as described above.

Western Blot Analysis
Whole-cell lysates (WCL) were prepared from LNCaP and PC-3 cells by scraping and homogenization in modified RIPA buffer [150 mM sodium chloride, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecyl sulfate, 5 µg/ml aprotinin, 5 µg/ml leupeptin]. Cell debris was removed by centrifugation. Protein concentration was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). The WCL was boiled for 5 min in 1x SDS sample buffer [50 mM Tris-HCl (pH 6.8), 12.5% glycerol, 1% sodium dodecyl sulfate, 0.01% bromophenol blue] containing 5% β-mercaptoethanol. The WCL protein extracts (10–20 µg) were separated on a 4–12% gradient NuPage Bis Tris gel and transferred to polyvinylidene fluoride membrane according to the NuPage protocol (Invitrogen). Membranes were processed for Western blotting using standard procedures. Briefly, membranes were incubated in 5% nonfat milk, 0.25% Tween 20 in PBS (PBST) blocking solution for 1 h at room temperature, followed by incubation with primary antibody for 1–2 h at room temperature in PBST. Primary antibodies raised against hAR (N-20), glyceraldehyde-3-phosphate dehydrogenase, and β-actin were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA) and were used at 3 µg/ml final concentration. After washing, blots were incubated for 1–2 h with the secondary horseradish peroxidase-conjugated goat antirabbit IgG (Santa Cruz) using a 1:5000 dilution in PBST. Immunolabeled proteins were visualized using the enhanced chemiluminescence system (Amersham, Inc., Piscataway, NJ) following the supplier’s instructions. Blots were scanned and signal intensities quantified using IS-1000 digital imaging system (Alpha Innotech Corp., San Leandro, CA).

Immunocytochemistry
Detection of AR.
Immunocytochemical staining was performed to detect and localize AR in LNCaP cells in absence or presence of AR ligands. LNCaP cells were grown in complete RPMI 1640 medium in an eight-well Lab-Tek chamber slide (Nalge Nunc) as described above. Twenty-four hours later, medium was replaced with fresh medium containing 10% charcoal/dextran-treated FBS (HyClone, Logan, UT), and cells were kept in androgen-depleted medium for the next 24 h. Then, cells were incubated in fresh RPMI 1640 medium containing 10% charcoal/dextran-treated FBS at 37 C for 1 h in the presence of DHT (10 nM), TDPQ (10 nM), or vehicle alone (0.1% DMSO). The cell culture medium was aspirated, and cells were washed twice with ice-cold PBS and fixed for 10 min with 4% paraformaldehyde on ice. Next, cells were permeabilized with 1% Triton X-100 in PBS for 6 min, washed twice with PBS, and blocked with 3% normal goat serum in PBS for 1 h at room temperature. Blocking solution (3% normal goat serum in PBS) was aspirated, and cells were incubated with a primary antibody (PG-21; 3 µg/ml in 3% blocking solution) for 1 h at room temperature. The PG-21 primary antibody is raised against AR N-terminal peptide sequence and was purchased from Abcam, Inc. (Cambridge, MA). After three washes in PBS, cells were incubated with a secondary FITC-conjugated goat anti rabbit (Abcam, Inc., Cambridge, MA) at a final concentration of 10 µg/ml in blocking solution for 1 h at room temperature. After three washes in PBS, chamber and gaskets were removed, and slides were air dried and mounted with antifade medium and a coverslip. Specimens were examined using a fluorescence microscope equipped with FITC filter sets.

Detection of 8-Oxo-dG.
Two commercially available fluorescence-based reagents were used according to their manufacturer’s protocols. The OxyDNA assay kit (Calbiochem Inc., San Diego, CA) is based upon the direct binding of a fluorescent probe to 8-oxo-dG in the DNA of fixed cells, whereas another immunoassay kit (Trevigen Inc., Gaithersburg, MD) employs primary and secondary antibodies. Although both assay kits resulted in similar observations, Calbiochem’s OxyDNA assay kit was routinely used for the detection of 8-oxo-dG due to technical simplicity and fewer washing steps required. LNCaP cells were grown in eight-well Labtek cover glass chambers as described above and were incubated with or without TDPQ (300 nM and 0.1% DMSO, respectively) for 1 h. After the irradiation step using 12 J/mm2 at 405 nm, cells were incubated for different times (2, 4, 8, and 16 h) at 37 C and subjected to detection of 8-oxo-dG using the OxyDNA assay kit.

AR Knockdown in LNCaP Cells Using siRNAs
The siRNA sequences targeting hAR gene as well as scrambled siRNA sequence negative controls were purchased from Invitrogen. The AR-HSS100619 siRNA sequence (5'-GACTCCTTTGCAGCCTTGCTCTCTA-3') targets the exon 4 of the LBD, and the siRNA sequence AR-HSS100620 (5'-TCTCAAGAGTTTGGATGGCTCCAAA-3') targets exon 6 of the LBD. LNCaP cells were seeded in eight-well cover glass chambers at a density of 100,000 cells per well in 0.5 ml complete RPMI 1640 growth medium. Twenty-four hours later, when cells reached 80–90% confluence, growth medium was replaced with 0.4 ml/well Opti-MEM transfection medium (Invitrogen) and 0.1 ml siRNA transfection mix. Transfections were carried out according to Invitrogen’s oligofectamine protocol using 1.5, 5, or 15 nM siRNA 619 or 620 and 3 µl oligofectamine for every 0.1 ml transfection mix. After transfection, cells were incubated for 48 h at 37 C in a 5% CO2 humidified atmosphere and used either for Western blotting or for photocytotoxic studies using TDPQ and light irradiation protocol.

Statistical Analysis
All experiments were repeated two or three times using separate preparations of cells, sometimes months apart. Each experiment consisted of multiple wells, independently and identically plated in triplicate. Each well was irradiated and scored separately. Representative results of each experimental design are shown. Triplicate values, each from an individual well within a single day’s experiment, are shown. Numerical values are presented as mean ± SD. Statistical significance was calculated using Student’s t test to compare individual means. A P value of <0.05 was considered significant.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Tim Willson (GlaxoSmithKline) for providing TDPQ and ETPQ compounds and Dr. Kerry Burnstein (University of Miami) for providing PC-3/AR and PC-3/neo cell lines. Dr. Colin Chignell (National Institute of Environmental Health Sciences) was helpful in discussion of the photochemistry of TDPQ and related compounds. We also appreciate reviewers’ helpful comments.


   FOOTNOTES
 
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences.

Disclosure Summary: The authors have nothing to disclose.

First Published Online June 18, 2008

Abbreviations: AA, Ascorbic acid; AR, androgen receptor; DHT, dihydrotestosterone; DMSO, dimethylsulfoxide; ETPQ, 4-ethyl-1,2,3,4-tetrahydro-6-(trifluoromethyl)-8-pyridino[5,6-g]quinoline; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; hAR, human AR; LBD, ligand-binding domain; LD, long distance; 8-oxo-dG, 8-hydroxy-2'-deoxyguanosine; PBST, 5% nonfat milk, 0.25% Tween 20 in PBS; PDT, photodynamic therapy; PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species; siRNA, small interfering RNA; TDPQ, 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono [5,6-g]quinoline; WCL, whole-cell lysate.

Received for publication September 14, 2007. Accepted for publication June 10, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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Nuclear Receptors:   AR
Ligands:   Dihydrotestosterone  |  R1881




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