Pro-survival Function of Akt/Protein Kinase B in Prostate Cancer Cells

Tumor necrosis factor superfamily member TRAIL/Apo-2L has recently been shown to induce apoptosis in transformed and cancer cells. Some prostate cancer cells express constitutively active Akt/protein kinase B due to a complete loss of lipid phosphatase PTEN gene, a negative regulator of phosphatidylinositol 3-kinase pathway. Constitutively active Akt promotes cellular survival and resistance to chemotherapy and radiation. We have recently noticed that some human prostate cancer cells are resistant to TRAIL. We therefore examined the intracellular mechanisms of cellular resistance to TRAIL. The cell lines expressing the highest level of constitutively active Akt were more resistant to undergo apoptosis by TRAIL than those expressing the lowest level. Down-regulation of constitutively active Akt by phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. Treatment of resistant cells with cycloheximide (a protein synthesis inhibitor) rendered cells sensitive to TRAIL. Transfecting dominant negative Akt decreased Akt activity and increased TRAIL-induced apoptosis in cells with high Akt activity. Conversely, transfecting constitutively active Akt into cells with low Akt activity increased Akt activity and attenuated TRAIL-induced apoptosis. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage, as caspase-8 activity was not affected. Enforced expression of anti-apoptotic protein Bcl-2 or Bcl-XLinhibited TRAIL-induced mitochondrial dysfunction and apoptosis. We therefore identify Akt as a constitutively active kinase that promotes survival of prostate cancer cells and demonstrate that modulation of Akt activity, by pharmacological or genetic approaches, alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to treat prostate cancer.

Tumor necrosis factor superfamily member TRAIL/ Apo-2L has recently been shown to induce apoptosis in transformed and cancer cells. Some prostate cancer cells express constitutively active Akt/protein kinase B due to a complete loss of lipid phosphatase PTEN gene, a negative regulator of phosphatidylinositol 3-kinase pathway. Constitutively active Akt promotes cellular survival and resistance to chemotherapy and radiation. We have recently noticed that some human prostate cancer cells are resistant to TRAIL. We therefore examined the intracellular mechanisms of cellular resistance to TRAIL. The cell lines expressing the highest level of constitutively active Akt were more resistant to undergo apoptosis by TRAIL than those expressing the lowest level. Down-regulation of constitutively active Akt by phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. Treatment of resistant cells with cycloheximide (a protein synthesis inhibitor) rendered cells sensitive to TRAIL. Transfecting dominant negative Akt decreased Akt activity and increased TRAIL-induced apoptosis in cells with high Akt activity. Conversely, transfecting constitutively active Akt into cells with low Akt activity increased Akt activity and attenuated TRAILinduced apoptosis. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage, as caspase-8 activity was not affected. Enforced expression of anti-apoptotic protein Bcl-2 or Bcl-X L inhibited TRAIL-induced mitochondrial dysfunction and apoptosis. We therefore identify Akt as a constitutively active kinase that promotes survival of prostate cancer cells and demonstrate that modulation of Akt activity, by pharmacological or genetic approaches, alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to treat prostate cancer.
Prostate cancer (PC) 1 is the most common malignancy and the second leading cause of cancer death in men (1). It is believed that PC, like other neoplastic diseases, develops and progresses through an accumulation of genetic alterations. The exact molecular mechanisms underlying the onset and progression of PC are unknown. Unfortunately, there are limited treatment options available for this disease because chemo-and radiotherapy are largely ineffective, and metastatic disease frequently develops even after surgery (2)(3)(4). Thus, new ways of treating prostate cancer must be developed. Recently, TRAIL (for tumor necrosis factor-related apoptosis-inducing ligand)/ Apo-2L has been shown to be a potential candidate for cancer therapy (5).
Several groups, including ours, have shown that TRAIL induces apoptosis in many cancer and transformed cells (6 -11). However, its pro-apoptotic effects are minimal in normal cells (12)(13)(14). Recently, based on in vitro experiments, some TRAILresistant cancer cell lines have been discovered. The reason for the TRAIL resistance is unknown, but is not regulated solely by the differential expression of the known TRAIL receptors DR4 and DR5 (5). Instead, it appears that intracellular inhibitor(s) acting downstream of TRAIL receptors renders some cells insensitive to TRAIL (15). Furthermore, resistance of many types of cancer cells to TRAIL can be reversed by treatment with RNA synthesis inhibitors (7,16), protein synthesis inhibitors (15,17,18), or chemotherapeutic agents (19,20). The study of the intracellular mechanisms that control TRAIL resistance may enhance our knowledge of death receptor-mediated signaling and help to develop TRAIL-based approaches for cancer treatment (5).
The binding of TRAIL to its receptors DR4 and DR5 leads to the cleavage and activation of caspase-8 (5,11,19,21), which in turn activates downstream effector caspases such as caspase-3 and caspase-7 (11,22). Activation of caspase-8 by TRAIL may also cleave BID (a Bcl-2 inhibitory protein) to tBID (truncated BID), which triggers mitochondrial depolarization (decrease in ⌬⌿ m ) and subsequent release of cytochrome c from the mitochondria (11,23). Once released into the cytosol, cytochrome c binds to apoptotic protease-activating factor 1 (Apaf-1) and, in the presence of dATP, recruits and activates procaspase-9 to form the apoptosome (24). Activated caspases cleave several downstream death substrates and activate endonucleases resulting in the apoptosis (25,26).
Akt/PKB is activated in response to activation by many different growth factors, including insulin-like growth factor-I, epidermal growth factor, basic fibroblast growth factor, insulin, interleukin-3, interleukin-6, and macrophage-colony stimulating factor (27). Akt is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses including inhibiting apoptosis and stimulating cellular growth. There are three mammalian isoforms of this enzymes, Akt1, Akt2, and Akt3 (28 -30). Akt1 is the cellular homologue of a viral oncogene (v-akt) (28 -30). Activation of all three isoforms is similar in that phosphorylation of two sites, one in the activation domain and other in the COOH-terminal hydrophobic motif, are necessary for full activity. For Akt, phosphorylation of Thr-308 in the activation domain by PDK1 is dependent on the products of PI3K, phosphatidylinositol 4,5bisphosphate and phosphatidylinositol 3,4,5-triphosphate (PIP 3 ). Phosphatidylinositol 4,5-bisphosphate and PIP 3 bind to the pleckstrin homology domains of Akt and PDK1, which relieves steric hindrance, fully activates PDK1, and translocates Akt to the plasma membrane (31). The mechanism of Ser-473 phosphorylation is less clear. Kinases potentially responsible for Ser-473 phosphorylation include PDK1 (32), integrin-linked kinase (33,34), and Akt itself (35). Akt activation may also be achieved through PI3K-independent means, either through phosphorylation of Akt by kinases such as cAMP-dependent protein kinase (36) or Ca 2ϩ /calmodulin-dependent protein kinase kinase (37), or under conditions of cellular stress (38 -40). Interestingly enough, activation of Akt by cAMP-dependent protein kinase or Ca 2ϩ /calmodulin-dependent protein kinase kinase does not appear to require phosphorylation of Ser-473. The relative importance of PI3K-independent and -dependent means of Akt activation in vivo is unclear. However, once activated, Akt exerts antiapoptotic effects through phosphorylation of substrates such as Bad (41,42) or caspase 9 (43), which directly regulates the apoptotic machinery or substrates such as the human telomerase reverse transcriptase subunit (44), forkhead transcription family members (45,46), or IB kinases (47) that indirectly inhibit apoptosis (48).
Previous studies have demonstrated that Akt plays an important role in survival when cells are exposed to different apoptotic stimuli such as growth factor withdrawal, UV irradiation, matrix detachment, cell cycle discordance, DNA damage, and administration of anti-Fas antibody, transforming growth factor-␤, glutamate, or bile acids (49 -61). Furthermore, Akt was found to be overexpressed in some gastric adenocarcinomas and in breast, ovarian, prostate, and pancreatic cancers (38,62). However, the role of Akt in tumor cell survival and resistance to cancer therapy has not been well studied in any tumor system.
The PTEN tumor suppressor gene is inactivated by mutations in many types of tumors including endometrium, brain, and prostate (63)(64)(65)(66)(67)(68). PTEN is a lipid phosphatase that can dephosphorylate PIP 3 (69). Through direct regulation of PIP 3 levels, PTEN negatively regulates the PI3K signaling pathway, which transduces extracellular growth regulatory signals to intracellular mediators of growth and cell survival (70). Accordingly, inactivation of PTEN due to mutations in tumors led to increased activity of Akt (71,72), whereas reintroduction of PTEN suppressed Akt activity (72)(73)(74). Finally, much of the ability of PTEN to regulate the cell cycle and induce apoptosis appears to be mediated via its ability to regulate Akt enzymatic activity (72)(73)(74).
The aim of this study was to examine the intracellular mechanisms of differential sensitivity of prostate cancer cells to TRAIL. We demonstrate that Akt is activated in the LNCap cell line. LNCap cells use Akt for survival because when PI3K inhibitors are added or kinase-dead Akt transfected, LNCap cells undergo apoptosis. Manipulating Akt activity alters sen-sitivity to TRAIL, transfecting constitutively active Akt into PC-3M cells that have low endogenous Akt activity increases resistance to TRAIL; alternatively, adding a PI3K inhibitor or transfecting kinase-dead Akt into cells with high levels of Akt activity results in dramatic sensitization to these modalities. These data show that targeting a specific kinase that promotes survival such as Akt can change the apoptotic potential of prostate cancer cells resulting in greater efficacy of TRAIL in vitro.
Preparation of Recombinant TRAIL-The expression construct used to produce TRAIL was described elsewhere (pet 15b-HIS-TRAIL) (75). Polyhistidine-tagged TRAIL was purified from Escherichia coli by using standard nickel affinity chromatography as per manufacturer's instructions (Novagen, Inc.). Preparations were made endotoxin-free by sequential detergent extraction (Triton X-114) (76,77). Detergent was removed from the final preparation by using Bio-Rad SM-2 beads (77). Endotoxin testing was done by using the Limulus amebocyte lysate assay (Bio-Whittaker) (77).
XTT Assay-Cells (1 ϫ 10 4 in 200 l of culture medium/well) were seeded in a 96-well plate (flat bottom), treated with or without drugs, and incubated for various time points at 37°C and 5% CO 2 . Before the end of the experiment, 50 l of XTT labeling mixture (final concentration: 125 M sodium XTT and 25 M phenazine methosulfate) per well was added and plates were incubated for another 4 h at 37°C and 5% CO 2 . The spectrophotometric absorbance of the sample was measured using a microtiter plate (ELISA) reader. The wavelength to measure absorbance of the formazon product was 450 nm, and the reference wavelength was 650 nm.
Cells and Culture Conditions-Human prostate cancer LNCap, PC-3, PC-3M, TSU-Pr1, and DU-145 cells were obtained from the American Type Culture Collection (Rockville, MD). Human prostate normal PrEC cells were purchased from the Clonetics (San Diego, CA). DU-145 cells contain one wild-type PTEN allele and a second variant allele (M134L). PC-3 cells have sustained a homozygous deletion of PTEN. LNCap cells have a deletion of one allele and a mutation of the other PTEN allele. LNCap, PC-3, TSU-Pr1, and PC-3M cells were grown in RPMI 1640 supplemented with D-glucose, HEPES buffer, 2 mM L-glutamine, 1% penicillin-streptomycin mixture, and 10% fetal bovine serum. DU-145 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 1% penicillin-streptomycin, and 10% fetal bovine serum. Human prostate normal PrEC cells were grown in PrEGM medium (Clonetics). All cells were grown on tissue culture dishes at 37°C with 5% CO 2 . Cells were transfected with pSFFVneo-Bcl-2, pSFFVneo-Bcl-X L , or pSFFV-neo plasmids as we described elsewhere (11,26,78).
Measurement of Mitochondrial Energization-Retention of DiOC 6 (3) was used as a measure of mitochondrial energization (26). Cells (5 ϫ 10 5 in 500 l of complete RPMI 1640 medium) were loaded with 40 nM DiOC 6 (3) during the last 30 min of treatment, the cells were isolated at 700 ϫ g for 10 min, and the cell pellet was resuspended and washed in phosphate-buffered saline two times. Cells were lysed by addition of 600 l of deionized water followed by mechanical homogenization. The concentration of retained DiOC 6 (3) was determined on a fluorescence spectrometer at 480 nm excitation and 510 nm emission (26).
Unlysed cells and nuclei were removed by centrifugation at 750 ϫ g for 10 min. The supernatant was spun at 10,000 ϫ g for 25 min, and the mitochondrial pellet resuspended in buffer A. The supernatant was spun at 100,000 ϫ g for 1 h. The supernatant from this final centrifugation represented the S100 fraction.
Western Blot Analysis-Lysis of cells was done in a buffer containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA, 1% SDS, 1 mM sodium orthovanadate, and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin A, 2 g/ml aprotinin). Lysates were sonicated for 10 s, centrifuged for 20 min at 12,000 ϫ g, and stored at Ϫ70°C. Equal amounts of lysate protein were run on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose. Nitrocellulose blots were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 buffer (20 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 0.01% Tween 20) and incubated with primary antibody in Tris-buffered saline with Tween 20 containing 5% bovine serum albumin overnight at 4°C. Immunoreactivity was detected by sequential incubation with horseradish peroxidase-conjugated secondary antibody and ECL reagents.
Nucleosome ELISA Assay-Cells (2 ϫ 10 6 ) were seeded into 24-well plates in the presence or absence of various drugs for 36 h (see figure legends). Cells were harvested for nucleosome ELISA assay according to the manufacturer's directions (Oncogene Research Products, Cambridge, MA). Briefly, anti-histone 3 biotin-labeled antibody binds to the histone component of captured nucleosomes and is detected by following incubation with streptavidin-linked horseradish peroxidase conjugate. Horseradish peroxidase catalyzes the conversion of colorless tetramethylbenzidine to blue, and addition of a stop solution changes the color to yellow, the intensity of which is proportional to the number of nucleosomes in the sample. The nucleosome ELISA allows quantitation of apoptotic cells in vitro by DNA affinity-mediated capture of free nucleosomes followed by their anti-histone-facilitated detection. In this assay, mono-and oligo-nucleosomes are captured on pre-coated DNA-binding proteins.

Comparison of Cell Viability of Human Prostate Normal and
Cancer Cells in Response to TRAIL Treatment-Cytotoxic effects of TRAIL on human prostate normal (PrEC) and cancer cell lines (LNCap, PC-3, TSU-Pr1, PC-3M, and DU-145) were compared by XTT assay (Fig. 1). Cell viability assays demonstrated that DU-145 and PC-3M cells were very sensitive to TRAIL; PC-3 and TSU-Pr1 revealed moderate sensitivity in a dose-and time-dependent manner, whereas resistance to TRAIL was seen in prostate cancer LNCap and normal PrEC cells (Fig. 1, A and B). Prostate normal PrEC cells do not express death receptor DR4 and DR5, whereas LNCap cells express both receptors (data not shown). Nucleosome ELISA assay was used to confirm whether cell death occurred via apoptosis. Similar to viability data, the results further confirmed that LNCap and PrEC cells were resistant to apoptosis by TRAIL; PC-3 and TSU-Pr1 cells were moderately sensitive, whereas PC-3M and DU-145 were very sensitive (data not shown).
Effects of PI3K Inhibition on Akt Activity and Apoptosis on Prostate Cancer Cells-Pro-survival function of growth factors signaling occurs through PI3K/Akt pathway (73,80,81). Since Akt is an important regulator of various intracellular events in prostate tumor progression (82,83), we explored whether increased Akt activity may be involved in TRAIL resistance. To elucidate the mechanisms controlling the resistance to LNCap cells to the cytotoxic effects of TRAIL, Akt activity was meas-ured by immunoblotting using phosphospecific Ser-473 Akt antibody (80). The data revealed the highest expression of constitutively active Akt in LNCap cells, moderate expression in PC-3 and TSU-Pr1, and the lower expression in PC-3M and DU-145 ( Fig. 2A). By comparison, normal prostate cells do not express constitutively active Akt. Total Akt levels in LNCap, PC-3, PC-3M, TSU-Pr1, DU-145, and PrEC did not change. We further confirmed the constitutive activation of Akt by kinase assay (Fig. 2B). The kinase assay revealed the differential expression of constitutively active Akt with LNCap cells expressing the highest and DU-145 cells expressing the lowest activity (Fig. 2B), similar to observations revealed by the Western blot analysis ( Fig. 2A).
We next examined whether inhibition of PI3K by LY-294002 and wortmannin, or protein synthesis by cycloheximide can inhibit constitutively active Akt (Fig. 2C). Treatment of LNCap cells with LY-294002 (20 M), wortmannin (200 nM), or cycloheximide (10 M) for 8 h reversed the high constitutive activity of Akt (Fig. 2C). Since LNCap cells possess a high constitutively active Akt, we sought to examine whether down-regulation of constitutively active Akt makes LNCap cells sensitive to TRAIL. LNCap and DU-145 cells were pre-treated with LY-294002 (20 M), wortmannin (200 nM), or cycloheximide (10 M) for 45 min, followed by treatment with TRAIL (50 ng/ml), and apoptotic nuclei were then counted by DAPI staining (Fig. 2D). TRAIL alone induced apoptosis in DU-145 cells, but had no effect on LNCap cells. Pretreatment of LNCap cells with the inhibitors of PI3K (wortmannin or LY-294002) or protein synthesis (cycloheximide) sensitizes LNCap cells to TRAIL. Wortmannin, LY-294002, and cycloheximide further enhanced the effects of TRAIL on apoptosis in DU-145 cells, which express low levels of constitutively active Akt. Thus, a high constitutively active Akt level in LNCap cells renders them resistant to TRAIL.
TRAIL-mediated Apoptosis Occurs at the Level of BID Cleavage following Caspase-8 Activation in LNCap Cells-Our earlier work has demonstrated that the formation of death-induc- ing signaling complex (DISC) is essential for TRAIL-induced signaling, which require an adaptor protein FADD (10,11). Furthermore, formation of DISC is essential for activation of caspase-8 (5,11). To rule out the possibility of defects in caspase-8, we compared the caspase-8 activity between LNCap and DU-145 cells (Fig. 3). There were no difference in TRAILinduced caspase-8 activity between LNCap and DU-145 cells. Pre-incubation of cells with wortmannin or LY-294002 did not affect TRAIL-induced caspase-8 activity. These data suggest that resistance of LNCap cells to TRAIL is not due to defects in caspase-8 activation.
Treatment of cells with TRAIL results in BID (a BH3-domain-only molecule) cleavage at its amino terminus as demonstrated previously by our laboratory (11). Cleavage of cytosolic p22 BID by caspase-8 generates a p15 tBID fragment that translocates to the mitochondria. Immunodepletion of tBID from subcellular fractions argues that tBID is required for cytochrome c release from the mitochondria. We therefore examined the effects of TRAIL on BID cleavage in LNCap and DU-145 cells (Fig. 3B). Although ineffective alone, TRAIL along with wortmannin or LY-294002 cleaved BID in both LNCap and DU-145 cells.
Mitochondria appear to play a key role in apoptosis (11,84). During apoptotic cell death, the early events that occur are mitochondrial depolarization and the loss of cytochrome c from the mitochondrial intermembrane space (24,84). The fluorescent dye DiOC 6 (3) localizes to the mitochondria, and the mitochondrial permeability transition reduces the accumulation of DiOC 6 (3) as a consequence of the loss in mitochondrial membrane potential (⌬ m ) (11,26). If the high constitutive activity of Akt causes TRAIL resistance in LNCap cells, then Akt may block apoptosis either upstream (41,85)  lease from the mitochondria. Since TRAIL-induced BID cleavage in LNCap cells occurs in the presence of wortmannin or LY-294002, we sought to investigate mitochondrial dysfunction by measuring ⌬ m . Incubation of cells with TRAIL, cycloheximide, wortmannin, or LY-294002 alone had no effect on ⌬ m in LNCap cells (Fig. 3C). However, TRAIL in combination with cycloheximide, wortmannin, or LY-294002 caused a rapid decrease in ⌬ m in LNCap cells (Fig. 3C). In contrast, treatment of DU-145 cells with TRAIL alone caused a significant drop in ⌬ m (data not shown). Since opening of the mitochondrial permeability transition pores causes releases of cytochrome c from mitochondria, we sought to examine the levels of cytochrome c retained in mitochondria in LNCap cells treated with various drug combinations (Fig. 3D). TRAIL, cycloheximide, wortmannin, or LY-294002 alone had no effect on cytochrome c release from mitochondria to cytosol in LNCap cells (Fig. 3D). However, the combination of TRAIL with cycloheximide, wortmannin, or LY-294002 resulted in loss of cytochrome c from the mitochondria (Fig. 3D).
TRAIL Induced Activation of Caspase-9 and Caspase-3 in DU-145 Cells but Not in LNCap Cells-Mitochondrial dysfunction appears to be essential for the formation of apoptosomes (a complex consisting of cytochrome c, Apaf-1, and ATP), which in turn activate caspase-9 and caspase-3 (24,84). We therefore sought to examine the activation of caspase-9 and caspase-3 in cells treated with TRAIL in the presence and absence of wortmannin or LY-294002. Treatment of LNCap and DU-145 cells with wortmannin or LY-294002 had no effect on caspase-9 and caspase-3 activity (Fig. 4, A and B). As expected, TRAIL-induced caspase-9 and caspase-3 activity in DU-145 cells but not in LNCap calls. By comparison, TRAIL further enhanced the activation of caspase-9 and caspase-3 when combined with LY-294002 or wortmannin (Fig. 4, A and B).
Attenuation of Constitutively Active Akt by Dominant Negative Akt (DN-Akt) or PTEN Sensitizes LNCap Cells to TRAIL-If Akt is the only signaling molecule for TRAIL resistance in LNCap cells, then down-regulation of Akt by dominant negative Akt (DN-Akt) will sensitize cells to TRAIL. We, therefore, used the genetic approach to down-regulate Akt activity in LNCap cells with dominant negative Akt and examined the effects of TRAIL on apoptosis and BID cleavage (Fig. 5, A and  B). LNCap cells were transiently transfected with empty vector, WT-Akt, CA-Akt, or DN-Akt and treated with or without TRAIL (50 nM). Transfection of LNCap cells with empty vector, WT-Akt, or CA-Akt had no effect on apoptosis, whereas transfection of cells with DN-Akt makes LNCap cells sensitive to TRAIL (Fig. 5A). By comparison to LNCap cells, transfection of empty vector or WT-Akt in DU-145 cells had no significant difference on TRAIL-induced apoptosis, whereas transfection of CA-Akt abrogated TRAIL-induced apoptosis (Fig. 5A). In addition, transfection of DN-Akt in DU-145 cells slightly enhanced TRAIL-induced apoptosis compared with empty vector or WT-Akt-transfected cells. Furthermore, down-regulation of Akt activity by DN-Akt in LNCap cells results in BID cleavage in response to TRAIL treatment (Fig. 5B), suggesting inhibition of constitutively active Akt in LNCap cells is sufficient to induce apoptosis by TRAIL via BID cleavage.
The tumor suppressor gene PTEN is quite often inactivated in primary human prostate cancers, particularly in the more advanced cancers (87), and in human prostate cancer xenografts and cell lines including PC-3 and LNCap (63,88,89). These studies suggest that downstream target of the PI3K pathway, such as AKT that is negatively regulated by PTEN (72,73,90), may be increasingly activated with prostate tumor progression. We, therefore, transfected LNCap cells with either empty vector or PTEN cDNA and incubated in the presence or absence of TRAIL (Fig. 5C). Transfection of LNCap cells with PTEN cDNA resulted in induction of apoptosis (Fig. 5C) and cleavage of BID by TRAIL (Fig. 5D). These data confirmed our previous findings that constitutively active Akt is involved in the resistance of LNCap cells to TRAIL.

Inhibition of Wortmannin/LY-294002 plus TRAIL-induced Drop in Mitochondrial Membrane Potential and Apoptosis in
LNCap Cells by Bcl-2 or Bcl-X L -Our and other laboratories have shown previously that Bcl-2 and Bcl-X L did not inhibit TRAIL-induced apoptosis in lymphoid cells, whereas they inhibited TRAIL-induced apoptosis in non-lymphoid cells (5,10,11,(91)(92)(93). Cells of lymphoid origin are considered as "type I" cells, where FasL/TRAIL can induce apoptosis independent of mitochondrial participation, whereas prostate cancer cells appear to be "type II" cells, where mitochondrial dysfunctions are observed during apoptosis. In type II cells, the release of mitochondrial cytochrome c serves as an amplification loop that potentiates the activation of caspases-3. We therefore pursued these studies in LNCap cells to examine whether wortmannin/LY-294002/cycloheximide plus TRAIL-induced apoptosis can be blocked by Bcl-2 or Bcl-X L . We transfected LNCap cells with Bcl-2 (LNCap/Bcl-2), Bcl-X L (LNCap/Bcl-X L ), or empty vector (LNCap/Neo). It was observed that overexpression of Bcl-2 or Bcl-X L in LNCap cells attenuated apoptosis induced by wortmannin plus TRAIL, LY-294002 plus TRAIL, or Cycl plus TRAIL as measured by nucleosome ELISA assay (Fig. 6A). We next examined whether Bcl-2 or Bcl-X L inhibits apoptosis at the level of mitochondria by measuring mitochondrial membrane potential (Fig. 6B). Interestingly, Bcl-2 or Bcl-X L inhibited the drop in mitochondrial membrane potential induced by wortmannin plus TRAIL, LY-plus TRAIL or Cycl plus TRAIL. Therefore, the inhibition of apoptosis induced by wortmannin plus TRAIL, LY-294002 plus TRAIL, or Cycl plus TRAIL by Bcl-2 or Bcl-X L was correlated with inhibition of ⌬ m . Although wortmannin plus TRAIL, LY-294002 plus TRAIL, or Cycl plus TRAIL failed to kill Bcl-2/Bcl-X L -overexpressing LNCap cells, leukemic cells overexpressing Bcl-2/Bcl-X L remain highly sensitive to treatment with TRAIL alone (11,92).
Enforced Expression of Constitutively Active Akt in PC-3M Cells Confers TRAIL Resistance-Since TRAIL treatment alone can kill PC-3M cells owing to low levels of constitutively active Akt, we wished to investigate the effects of the overexpression of constitutively active Akt on TRAIL-induced drop in mitochondrial membrane potential and apoptosis. Transfection of empty vector, WT-Akt, CA-Akt, and DN-Akt was performed in PC-3M cells, and Akt activity was measured by kinase assay (Fig. 7A). As expected, group transfected with CA-Akt cDNA expressed active form of Akt, whereas activation (phosphorylation) of Akt was not observed in groups transfected with empty vector, WT-Akt, and DN-Akt. We next examined the effects of WT-Akt, CA-Akt, and DN-Akt on TRAIL-induced drop in ⌬⌿ m . TRAIL-induced drop in ⌬⌿ m in PC-3M cells transfected with empty vector, WT-Akt or DN-Akt (Fig. 7B). In contrast, transfection of CA-Akt cDNA in PC-3M cells inhibited TRAIL-induced drop in ⌬⌿ m . We next examined the effects of WT-Akt, CA-Akt, and DN-Akt on TRAIL-induced apoptosis as measured by the nucleosome ELISA assay (Fig. 7C). PC-3M cells were transfected with empty vector, WT-Akt, CA-Akt, and DN-Akt and treated with or without TRAIL for 24 h. TRAIL promoted apoptosis in cells transfected with empty vector, WT-Akt, and DN-Akt. In contrast, transfection of CA-Akt cDNA in PC-3M cells inhibited TRAIL-induced apoptosis. These data suggest that an increased Akt activity due to overexpression of CA-Akt in PC-3M cells makes them resistant to TRAIL. This effect is similar to LNCap cells, which express high levels of constitutively active Akt and are resistant to TRAIL. DISCUSSION In this report, we demonstrate that PI3K/AKT signaling pathway modulates sensitivity of prostate cancer cells to TRAIL and that high constitutively active Akt levels directly correlate with cell survival and resistance to TRAIL. Recent studies suggest that constitutively active Akt in prostate cancer cells may cause drug resistance. Akt is the kinase that enhances cell proliferation and inhibits apoptosis in cancer cells. Attenuation of Akt activity by DN-Akt or PI3K inhibitors, LY-294002 and wortmannin, renders LNCap cells sensitive to TRAIL. On the other hand, up-regulation of Akt activity in PC-3M cells, which express very low constitutively active Akt, restores TRAIL resistance. Thus, constitutively active Akt in prostate cancer cells is an important regulator of TRAIL sensitivity.
The PTEN/MMAC tumor suppressor gene is frequently inactivated in primary human prostate cancers particularly in the more advanced cancers (87), and in human prostate cancer xenografts and cell lines including PC-3 and LNCap (63,88,89). These studies suggest that components of the PI3K pathway that are negatively regulated by PTEN, such as the key cell survival kinase Akt (72,73,90), may be increasingly activated with prostate tumor progression. Indeed, activated Akt regulates a number of intracellular targets implicated in cell growth and apoptosis. For instance, Akt may phosphorylate (and inactivate) the pro-apoptotic proteins such as BAD (41) and caspase-9 (43), and activate anti-apoptotic NFB-mediated transcriptional pathway (47,48). Additionally, Akt may enhance cell cycle progression by suppressing AFX/Forkhead transcription factor activity (45,94,95), which would result in diminished expression of AFX target genes such as the cell cycle inhibitor p27 Kip1 (96). Furthermore, based on this study, Akt can inhibit BID cleavage as a mechanism through which PI3K and Akt block apoptotic signals.
Growth factors signaling through the PI3K/Akt pathway promote cell survival. The constitutively active Akt corresponds to increased activity of integrin-linked kinase, the kinase putatively responsible for the activation of Akt by phosphorylation at Ser-473 (80), and markedly reduced expression of the cell cycle inhibitor p27 Kip1 (96). The increased Akt activity and the reduction in p27 Kip1 expression, which has been routinely associated with prostate tumor progression, may be important markers for human tumor progression. Akt inhibits the activity of DEVD-targeted caspases without changing the steady-state levels of Bcl-2 and Bcl-X L . Akt inhibits apoptosis and the processing of procaspases to their active forms by delaying mitochondrial changes in a caspase-independent manner. Akt activation is sufficient to inhibit the release of cytochrome c from mitochondria and the alterations in the inner mitochondrial membrane potential. However, Akt cannot inhibit apoptosis induced by microinjection of cytochrome c. Akt inhibits apoptosis and cytochrome c release induced by several pro-apoptotic Bcl-2 family members. Akt-and Bcl-2/Bcl-X L -induced resistance to TRAIL appears to maintain mitochondrial homeostasis and block apoptotic response. However, direct connection between cellular resistance caused by Akt and Bcl-2/Bcl-X L has not been reported. Taken together, Akt promotes cell survival by intervening in the apoptosis cascade before cytochrome c release and caspase activation via a mechanism that is distinct from Bad phosphorylation.
The anti-apoptotic effects of Bcl-2 and Bcl-X L have been analyzed extensively. Bcl-2 inhibits apoptosis against various toxic stresses through stabilization of ⌬ m (24,84), blocking the release of apoptosis inducer proteins such as cytochrome c (26,97) and AIF (98) from mitochondria to cytoplasm, and thus inhibits the subsequent apoptosis-executing signaling events. Because most clinically administered chemotherapeutic drugs mediate apoptosis mainly by inducing mitochondrial dysfunction, tumor cell resistance is likely to include overexpression of Bcl-2 and Bcl-X L . We and others have shown that Bcl-2 and Bcl-X L cannot inhibit TRAIL-induced apoptosis in leukemic cells (10,11,92), suggesting tumor cells that have already acquired resistance to chemotherapeutic drugs by Bcl-2 and Bcl-X L can be killed by TRAIL. In contrast, Bcl-2/Bcl-X L abro- FIG. 7. Overexpression of constitutively active Akt inhibits TRAIL-induced drop in mitochondrial membrane potential and apoptosis. A, PC-3M cells were transiently transfected with wild type Akt, dominant negative Akt (DN-Akt), constitutively active Akt (CA-Akt), or empty vector. Akt activity was measured by kinase assay as described under "Materials and Methods." B, TRAIL causes mitochondrial depolarization that is not prevented by overexpression of CA-Akt. PC-3M cells were transfected with empty vector, WT-Akt, CA-Akt, or DN-Akt for 24 h. Cells were treated with or without TRAIL (50 ng/ml) for another 24 h, and apoptosis was measured. During the last 30 min of treatment, DiOC 6 (3) was added. An aliquot of cells was used for the determination of cell-associated DiOC 6 (3) fluorescence. The data represent mean Ϯ S.D. of three independent experiments. C, overexpression of constitutively active Akt makes PC-3M cells resistant to TRAILinduced apoptosis. PC-3M cells were transiently transfected with empty vector, WT-Akt, CA-Akt, or DN-Akt. Cells were treated with or without TRAIL (50 ng/ml) for another 24 h, and apoptosis was measured by nucleosome ELISA assay. The data represent mean Ϯ S.D. of three independent experiments. gated TRAIL-induced mitochondrial dysfunction and apoptosis in non-lymphoid cells (91,93), suggesting a differential regulation of apoptosis by Bcl-2/Bcl-X L .
Death receptors trigger caspase activation through formation of DISC. Engagement of TRAIL to its receptors recruits adaptor protein FADD, which in turn recruits procaspases-8 (11). The role of FADD in TRAIL receptor DISC has recently been confirmed by many laboratories including ours (8,11,99,100). Our data suggest that caspase-8 activation is essential for TRAIL-induced apoptosis. In addition, caspase-3 can be activated either directly by caspase-8 or mitochondrial events such as cytochrome c release, which lead to cell death. The active caspases efficiently cleave procaspase-3 (and other executioner caspases), and apoptosis proceeds. Death receptor-mediated signaling appears to be cell type-specific (type I and type II cells). In type I cells, death receptor signaling is not blocked by Bcl-2/Bcl-X L , whereas in type II cells it is (101). In type II cells, activation of caspases-8 may not be sufficient to induce apoptosis; therefore, amplification signal by mitochondrial cytochrome c is required for activation of effector caspases. Caspase-8 activation results in cleavage of BID to tBID, which translocates to mitochondria to trigger Bax oligomerization and cytochrome c release (24,102). Furthermore, Bcl-2 and Bcl-X L appear to maintain mitochondrial homeostasis in type II cells by blocking the release of mitochondrial proteins.
Analysis of TRAIL-induced apoptosis in vitro has demonstrated that there are some TRAIL-resistant human melanoma and colon carcinoma cell lines (7,18). It appears that intracellular inhibitors acting downstream of the TRAIL receptors render specific transformed cell lines resistance to TRAIL. Addition of protein synthesis inhibitors to TRAILresistant LNCap cells rendered them sensitive to TRAIL, indicating that the presence of intracellular apoptosis inhibitors may mediate resistance to TRAIL. Expression of FLIP was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas (15). In HeLa and Kym-1 cells, death induction by TRAIL was dependent on the presence of cycloheximide (18). Interestingly, cycloheximide down-regulated endogenous cellular FLICE inhibitory protein (cFLIP), and overexpression of cFLIP inhibited death receptor-induced NFB activation (18), suggesting a novel functional role of cFLIP as a negative regulator of gene induction and apoptosis.
The cytotoxic effects of TRAIL on prostate cancer cells vary significantly and inversely correlate with the levels of constitutively active Akt. Cells having higher constitutively active Akt were more resistant to undergo apoptosis by TRAIL. Down-regulation of constitutively active Akt by pharmacological or genetic approach alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that downregulate Akt activity can be used to kill TRAIL-resistant prostate cancer cells.