Androgen ablation therapy induces apoptosis only in androgen-sensitive prostate cancer cells; therefore, other cytotoxic drugs are being used to induce apoptosis in androgen-refractory cells. Mifepristone, an antiprogestin used individually or together with the antiestrogen Tamoxifen, has been recommended for induction of cell death and treatment of several hormonal cancers. However, little is known about the mechanism of action of these drugs in prostate cancer. Therefore, we investigated the effect of Mifepristone on the tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) pathway, a newly identified and very effective member of tumor necrosis factor-α family. Mifepristone and Tamoxifen induced significant expression of death receptors in prostate cancer cells in vitro and in xenografts. However, Mifepristone in combination with Tamoxifen did not increase prostate cancer cell death compared with their individual values. The involvement of the TRAIL pathway was further confirmed by the activation of caspase-8 in Mifepristone-treated cells. This was followed by truncation of Bid, confirming that Mifepristone activates the TRAIL pathway. This knowledge is being used to design a combination treatment of TRAIL and Mifepristone to induce significant apoptosis in prostate cancer cells.
The prostate requires androgens for its growth, differentiation and maintenance (1). Surgical or chemical ablation of androgens is a commonly used therapy for androgen-sensitive prostate cancer. Because androgen-refractory prostate cancer cells continue to retain the capacity to undergo apoptotic cell death (2, 3, 4), treatment options for androgen-refractory prostate cancer involve induction of apoptosis. Several apoptotic agents are being investigated to induce apoptosis, but the majority of them have demonstrated several adverse effects (5, 6, 7, 8, 9, 10, 11).
Although antiprogestins such as Mifepristone were developed for the inhibition of progesterone-dependent reproductive processes (12), Mifepristone demonstrated antitumor activity in several hormone-dependent cancer models (13, 14, 15, 16, 17, 18, 19). In in vitro and xenograft models, Mifepristone was shown to induce apoptosis in breast cancer (18, 19), cervical carcinoma (20), endometrial cancer cells (21), lung cancer cells (22), meningioma, and leiomyoma (23). Mifepristone induced only 20–40% cell death, depending on the cancer and the model system used, which suggests the need for a better treatment regimen. It has been demonstrated that Mifepristone induces apoptosis through the Bcl2 pathway by increasing the expression of Bax and decreasing Bcl2 levels (18, 21, 24). Similar studies in prostate cancer cells are limited to those published by El Etreby et al. (25), who demonstrated that Mifepristone induces ∼20–40% cell death depending on the cell line used.
To enhance its apoptotic function, Mifepristone has been used in combination with other agents to induce tumor regression. In cervical carcinoma, Mifepristone treatment along with radiation therapy induced apoptosis in radioresistant cells (20). Similarly, Mifepristone increased sensitivity to Adriamycin in Adriamycin-resistant breast cancer cells (26). Another successful regimen for breast cancer included treatment with Mifepristone and an antiestrogen, Tamoxifen. However, little is known about the effects of these treatments on prostate cancer. Earlier studies demonstrated that Mifepristone treatment causes a reduction in Bcl2 levels, suggesting the involvement of this pathway (25). Because the effect of Mifepristone on other apoptotic pathways has not been determined, we investigated the activation of TRAIL pathway in prostate cancer cells treated with Mifepristone.
TRAIL3 is a new member of the tumor necrosis factor α family (27, 28, 29) with sequence similarities to tumor necrosis factor α and Fas ligand. TRAIL induces apoptosis through its interaction with the death domain receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2, TRICK2, or KILLER). The function of death receptors is blocked by the expression of decoy receptors DcR1 (TRID/TRAIL-R3) and DcR2 (TRUNDD/TRAIL-R4). DR4, DR5, DcR1, and DcR2 are structurally related except that death receptors have a cytoplasmic death domain responsible for initiating downstream events, whereas DcR1 and DcR2 lack this region. The extracellular domains of DcR1 and DcR2 compete with those of DR4 and DR5 for ligand binding, which prevents induction of apoptosis by TRAIL through death receptors. Activation of death receptors leads to the activation of caspase-8 (30, 31), which is responsible for the cleavage of BID (a Bcl2 family protein) into a truncated form. Truncated Bid is translocated into the mitochondria, which then release cytochrome c, leading to the formation of apoptosome.
TRAIL is potent inducer of apoptosis, which preferentially induces the death of cancer cells. However, TRAIL is most effective in combination with other apoptotic agents, such as etoposide, which significantly increased apoptosis in glioblastoma, breast cancer, and kidney cells (31, 32). Because both TRAIL and Mifepristone are more effective when treated in combination with other drugs, it was hypothesized that their combination may be very effective in inducing apoptosis in prostate cancer cells. However, it was first necessary to determine whether Mifepristone activated the same pathway affected by TRAIL. Therefore, in this study we used in vitro and xenograft models to demonstrate that Mifepristone is capable of activating key members of the TRAIL pathway.
MATERIALS AND METHODS
Cell Culture and Treatments.
LNCaP cells were obtained from American Type Culture Collection (Rockville, MD). LNCaP C4-2 cells, derived from LNCaP, were purchased from Urocor Inc. (Oklahoma City, OK). Cells were grown in RPMI 1640, containing a 1% streptomycin-penicillin mixture and 10% fetal bovine serum (Hyclone, Logan, UT), in a 5% CO2 atmosphere at 37°C. Cells were treated with 10 μm Mifepristone (Sigma Chemical Co., St. Louis, MO) or 5 μm 4-hydroxy-Tamoxifen (Sigma) for various time intervals. When the experiment was completed, cells were harvested and total proteins or mitochondrial fractions were isolated as described below. Control cells were treated with similar volumes of vehicle (ethanol). At least three experiments were conducted with a minimum of four plates per treatment per experiment. Representative results are described below.
Morphological Assessment of Apoptosis.
The effect of treatment on apoptosis was assessed by trypan blue staining. Cells were harvested, washed with PBS, and stained with 0.2% trypan blue (Life Technologies, Inc., Grand Island, NY). Stained (apoptotic) and unstained (viable) cells were counted in a hemocytometer, and the results were expressed as percentage of apoptosis compared with vehicle-treated controls.
Generation of Xenografts in Nude Mice.
Viable LNCaP and LNCaP C4-2 cells (2 × 106) were suspended in 0.15 ml of Matrigel (Collaborative Research, Bedford, MA) and injected s.c. in the right flank of 6–8-week-old athymic male BALB/c-nu/nu nude mice (Harlan, Indianapolis, IN). Nude mice were housed under pathogen-free specific pathogen-free conditions in laminar free boxes in temperature-controlled rooms with a 12-h light/12-h dark schedule and were fed autoclave-sterilized chow and autoclaved water ad libitum. The nude mice were examined for tumors by palpation. Tumor size was measured in three dimensions in mm every 4 days, and tumor volumes were calculated according to the formula: volume = π/6 × length × width × height, expressed as mm3. Four weeks after inoculation with the cells, animals were treated with Mifepristone (50 mg/kg/day s.c.) suspended in 0.1 ml of vehicle (1 ml of vehicle consisted of 0.1 ml of ethanol, 0.0625 ml of Tween 80, and 0.8375 ml of 0.9% NaCl). Control animals were treated daily with 0.1 ml of the vehicle administered s.c. At the end of the experiments (28 days after the commencement of the treatment), all of the animals were sacrificed by spinal elongation, tumors were harvested, and wet weights were determined. The tumors were stored at −70°C until use.
Preparation of Cell Lysates for Western Blotting.
The cells were harvested by trypsinization and washed twice with PBS; the cell pellet was resuspended in lysis buffer [100 mm Tris-HCl (pH 8.0), 0.1% Triton X-100, and a cocktail of protease inhibitors (Boehringer Mannheim, Indianapolis, IN)]. The cells were incubated over ice for 30 min and centrifuged at 10,000 × g at 4°C for 10 min. The supernatant was collected, and the protein concentration was estimated using the Bio-Rad protein reagent (Bio-Rad Laboratories, Hercules, CA).
Lysates from Xenografts.
The tumor tissue was cut into 0.5-cm cubes and homogenized in 1 ml of lysis buffer in a Tekmar homogenizer (Tekmar Company, Cincinnati, OH) for 10 min on ice and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was used to estimate proteins and for Western analysis.
Separation of Mitochondrial Fractions.
Treated and untreated cells were collected by trypsinization and centrifuged at 600 × g for 10 min at 4°C. The cells were washed twice in ice-cold PBS and resuspended in buffer A [20 mm HEPES-KOH (pH 7.2), 10 mm KCl, 1.5 mm MgCl2, 1 mm sodium EDTA, 1 mm sodium EGTA, 250 mm sucrose, and a protease inhibitor cocktail]. Cells were homogenized on ice with a glass Dounce homogenizer with 30 strokes and were centrifuged at 750 × g for 5 min at 4°C. The supernatant was centrifuged at 10,000 × g for 15 min at 4°C. The mitochondrial pellet was washed in buffer and resuspended in lysis buffer, and the protein was separated by electrophoresis.
Assay for Caspase-8 Activity.
LNCaP and LNCaP C4-2 cells were treated with vehicle or with 10 μm Mifepristone, 5 μm Tamoxifen, or a combination of Mifepristone and Tamoxifen for 3 days. Cells were harvested, and total proteins were extracted as described above. Caspase-8 activity was assayed using a colorimetric substrate, IETD-pNA, and a kit from Calbiochem-Novabiochem Corporation (San Diego, CA), as recommended by the manufacturer. Cleavage of the COOH-terminal peptide bond by the enzyme released p-nitroaniline, which was measured at 405 nm. Pure recombinant human caspase-8 supplied with the kit was used as a positive control, whereas Granzyme B Inhibitor II (Ac-IETD-CHO) was used as negative control.
Proteins (50 μg unless stated otherwise) were separated on NuPAGE 10% Bis-Tris gels (Novex precast mini gels; Invitrogen, Carlsbad, CA) at 100 V for 1.5 h in the presence of 1× MES-SDS running buffer (Invitrogen). Separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories) at 42 V for 2.5 h on a Novex XCell II blotting apparatus in NuPAGE transfer buffer in the presence of NuPAGE antioxidant. Transfer of the proteins to the polyvinylidene difluoride membrane was confirmed by staining with Ponceau S (Sigma). The blots were blocked in 5% nonfat dry milk in TBS, washed twice for 10 min each with TBS containing 0.01% Tween 20, and incubated for 2 h at room temperature with primary antibody diluted in TBS containing 0.5% milk. Antibodies against DR4 and DR5 were from Imgenex (San Diego, CA), and BID antibodies were from BioSource International (Camarillo, CA). The blots were washed and incubated with horseradish-conjugated antirabbit or antimouse secondary antibodies, and immunoreactive bands were visualized by the ECL detection system (Amersham, Pharmacia Biotech, Arlington Heights, IL). Signals were developed after exposure to X-ray film for varying periods of time (X-Omat films; Eastman Kodak Company, Rochester, NY).
RESULTS AND DISCUSSION
Recent therapeutic approaches for treatment of cancer have included induction of apoptotic pathways. Although androgen ablation therapy has been used to induce apoptosis in prostate cancer, this therapy is effective only in androgen-responsive cells. Therefore, the current research emphasis is to identify agents that induce apoptosis in both androgen-responsive and androgen-refractory cells. In our study, we investigated the effect of Mifepristone, an antiprogestin, on prostate cancer cells. Mifepristone was shown to induce apoptosis in both androgen-responsive and androgen-refractory prostate cancer cells and is undergoing clinical trials as a chemotherapeutic agent for induction of death of breast and ovarian cancer cells (34, 35, 36). Furthermore, because Mifepristone has been used in combination with Tamoxifen for the treatment of breast cancer, the effects of both Mifepristone and Tamoxifen on TRAIL pathway in prostate cancer cells were determined.
To determine whether a combination of Mifepristone and Tamoxifen induced cell death, LNCaP cells were treated with these drugs individually or together, and cell death was measured by trypan blue exclusion. Treatment of cells with Mifepristone resulted in 23% apoptosis, whereas Tamoxifen induced 26% cell death (Fig. 1). Combined treatment with Mifepristone and Tamoxifen yielded ∼31% cell death, which was very close to their individual effects (Fig. 1). It was surprising that, unlike in breast cancer cells (18, 19), the combination of Mifepristone and Tamoxifen was not more effective in inducing apoptosis, suggesting that it may be necessary to identify other treatment options in prostate cancer cells. Our trypan blue exclusion data were confirmed by combination staining with Hoechst 33258/propidium iodide and acridine orange/ethidium bromide (data not shown).
Mifepristone has been shown to induce apoptosis by increasing the expression of proapoptotic Bax and suppressing the expression of antiapoptotic Bcl2, thus affecting the ratio between the two proteins (18, 21, 24). To determine whether Mifepristone is capable of activating other apoptotic pathways, particularly the very effective TRAIL pathway, we examined the expression of TRAIL receptors. Our results demonstrated that Mifepristone and/or Tamoxifen induced expression of DR4 and DR5 in LNCaP cells (Fig. 2), although the induction of DR5 was more significant than that of DR4. Mifepristone treatment increased the expression of DR5 ∼10-fold compared with control but increased DR4 expression <2-fold. Tamoxifen did not induce significant changes in DR4 expression, although similar treatment increased DR5 expression ∼5-fold. Treatment of LNCaP cells with the combination of Mifepristone and Tamoxifen yielded little increase in DR4 expression compared with Mifepristone alone. DR5 levels increased ∼8-fold compared with control, although it was less abundant than in cells treated with Mifepristone alone (Fig. 2). These results indicate that in LNCaP cells, Mifepristone is more effective than Tamoxifen in inducing the expression of death receptors. The results shown in Figs. 1 and 2 suggest that the combination of Mifepristone with Tamoxifen did not improve their effectiveness and may not be a good treatment option for men, especially considering the estrogenic effects of Tamoxifen.
The results in LNCaP C4-2 cells demonstrated little difference in the expression of DR4 and DR5 in cells treated with Mifepristone. Treatment of these cells with Tamoxifen or Tamoxifen with Mifepristone increased the expression of both DR4 and DR5. Although it is interesting to note differences in the response of the two cell lines to Tamoxifen, it is difficult to explain these changes because the expression of estrogen and progesterone receptors in LNCaP C4-2 cells has not been determined. The present experimental design does not delineate receptor-mediated or receptor-independent effects of these antihormones in either cell line.
To corroborate the results obtained in cell cultures, we treated mice carrying xenografts of prostate cancer cells with Mifepristone. LNCaP and LNCaP C4-2 cells were injected into nude mice, and xenografts were allowed to grow before the animals were treated with vehicle or Mifepristone. Our earlier experiments (36) indicated that treatment of mice with Mifepristone for 28 days produced 55% and 52% reductions in tumor weights in LNCaP and LNCaP C4-2 xenografts, respectively. Western-blot analysis of xenografts generated with LNCaP cells demonstrated significant increases in the expression of DR4 and DR5 in Mifepristone-treated mice (Fig. 3), confirming the in vitro data. Similar experiments using LNCaP C4-2 xenografts revealed no significant differences in DR4 and DR5 in treated versus control mice, which is similar to the results shown in Fig. 2. Thus, both in vitro and xenograft models demonstrate that Mifepristone induced significant apoptosis in prostate cancer cells, suggesting its usefulness as a drug for prostate cancer treatment.
To further determine the mechanism of action of Mifepristone, especially the involvement of TRAIL pathway, we determined the expression of key proteins that respond to the increased expression of death receptors. Because activation of procaspase-8 is a critical event in the involvement of TRAIL-induced cell death, prostate cancer cells were treated, and the protein was analyzed for caspase-8 activity. To obtain quantitative data for the caspase-8 activity, we used a colorimetric assay system. The results demonstrated that Mifepristone induced caspase-8 activity, compared with controls, in both LNCaP and LNCaP C4-2 cells (Fig. 4). A comparison of Fig. 4,A with Fig. 4 B indicates higher caspase-8 activity in LNCaP C4-2 cells compared with LNCaP in both control and treated cells. Treatment of LNCaP cells with Tamoxifen reduced caspase-8 activity, whereas similar treatment of LNCaP C4-2 cells did not significantly affect caspase-8 activity. However, the combination of Tamoxifen and Mifepristone significantly reduced caspase-8 activity in both cell lines. The reasons for the differential response of caspase-8 to Tamoxifen are unknown, and further experimentation is needed to explain these results. However, the observations described here were confirmed by assays of multiple samples obtained from different experiments.
Activated caspase-8 is responsible for the cleavage of Bid into a smaller NH2-terminal and a larger COOH-terminal fragment (tBid), which is translocated into mitochondria, where it binds to either Bax or Bak, a necessary step for the release of cytochrome c into the cytoplasm (37). Cytochrome c binds to Apaf1 in the presence of ATP to form the apoptosome with procaspase-9, which is responsible for the cascade of events resulting in cell death. To further determine the involvement of the TRAIL pathway and to confirm the activation of caspase-8, we determined the presence of truncated Bid in LNCaP and LNCaP C4-2 cells, using mitochondrial fractions. Analysis of LNCaP cells demonstrated the expression of tBid only in cells treated with Mifepristone for 3 days (Fig. 5), whereas similar experiments with LNCaP C4-2 did not show tBid in mitochondrial fractions. These results support the observations that LNCaP cells are more sensitive to Mifepristone treatment than are LNCaP C4-2 cells (see above). However, even in LNCaP cells, tBid was noted only in cells treated for 3 days, suggesting that Mifepristone is not a powerful inducer of apoptosis in prostate cancer cells, an observation supported by the data presented in Fig. 1. These results further suggest that to induce effective cell death in prostate cancer cells, it may be necessary to treat cells with Mifepristone and another drug that activates the TRAIL pathway. The recruitment of the TRAIL pathway by Mifepristone is of great importance in view of our plans to use TRAIL with or without Mifepristone for the treatment of prostate cancer cells.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: TRAIL, tumor necrosis factor α-related apoptosis-inducing ligand; DR, death receptor; TBS, Tris-buffered saline.