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1 Division of Urologic Surgery and 2 Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cytotoxic effects on normal cells frequently limit systemic therapies. A relatively new proapoptotic agent, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, also known as Apo2L; refs. 2, 3, 4 ), has been used effectively in systemic animal trials and has the unique feature of inducing apoptosis in cancer cells and sparing normal cells (5, 6, 7) . Therefore, TRAIL produces limited cytotoxicity when used systemically. Other apoptotic inducing ligands, like tumor necrosis factor (TNF) and Fas ligand (FasL), also induce apoptosis by using signaling pathways that are similar to TRAIL signaling. However, TNF and FasL have severe systemic cytotoxic effects, limiting their use as systemic agents.
TRAIL interacts with specific death domain receptors, DR4 and DR5 (8, 9, 10, 11) , to induce intracellular cytoplasmic formation of the death-inducing signaling complex (12, 13, 14, 15) . Following formation of death-inducing signaling complex at the intracellular plasma membrane, proapoptotic signals are initiated by caspase-8, which can further activate downstream proapoptotic molecules and subsequent programmed cell death, which also may activate the mitochondrial-mediated proapoptotic pathways (16) .
Regulation of apoptosis in cancer cells after treatment with TRAIL has been correlated with expression of FLICE inhibitory protein (c-FLIP) in melanoma and other cancer types (17 , 18) . However, since the beginning of its discovery, the role of c-FLIP has been controversial (19, 20, 21, 22, 23, 24, 25, 26) . c-FLIP is a human cellular homologue of viral FLICE-inhibitory proteins (27) and is homologous to procaspase-8 and procaspase-10 (28) . c-FLIP(Long) and c-FLIP(short) isoforms, c-FLIP(L) and c-FLIP(s), can bind to the DED domains of Fas-associated death domain (FADD) and caspase-8 and regulate apoptosis through their interference with recruitment of caspase-8 to FADD; however, binding of c-FLIP to caspase-8 may be different between different c-FLIP isoforms (29) . Most published reports involving ectopic expression of c-FLIP(L) suggest that c-FLIP(L) has an antiapoptotic role. Moreover, c-FLIP–/– mouse embryonic fibroblasts have been shown to be more sensitive to FasL-induced apoptosis (30) , which strongly suggests that c-FLIP(L) has an antiapoptotic function. Furthermore, two recent reports have proposed that c-FLIP(L) may have a dual function, a proapoptotic function at low physiologic concentrations and an antiapoptotic function at high cellular concentrations (31 , 32) .
Although many cancers undergo TRAIL-induced apoptosis, some develop resistance, making TRAIL ineffective as an anticancer agent. Expression of certain apoptotic mediating genes has been suggested to regulate sensitivity of cancer cells to TRAIL-mediated apoptosis, including nuclear factor 
B (NFB; refs. 33
, 34
), Akt (35, 36, 37)
, Bcl-2 (38)
, Bax (39)
, and c-FLIP (35
, 37 , 40)
. Considering that there are numerous ways to regulate TRAIL-mediated apoptosis, this study focused on the role of c-FLIP(L) in mediating resistance to TRAIL-induced apoptosis. Although expression of c-FLIP(L) has been correlated with TRAIL resistance in select cancer models, the direct functional role of c-FLIP(L) in TRAIL-mediated apoptosis in prostate cancer has not been well studied. In this study, we show that persistent expression of c-FLIP(L) is necessary and sufficient to maintain resistance to apoptotic pathways induced by TRAIL, whereas silencing c-FLIP(L) expression converts TRAIL-resistant prostate cancer cells to a TRAIL-sensitive phenotype. Therefore, regulation of c-FLIP(L) is sufficient to overcome the necessary threshold to modulate TRAIL-mediated apoptosis in prostate cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Tubulin antibody and anti-FLAG antibody were purchased from Sigma (St. Louis, MO).
Cell Culture.
All of the cell culture materials were from Cellgro (Herndon, VA), and plasticware was from Becton Dickinson Labware (Bedford, MA). PC3, DU145, and LNCaP prostate cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). PC3-TR was a TRAIL-resistant subline established from parental PC3 cells by TRAIL treatment selection. Briefly, PC3 cells were treated with TRAIL (100 ng/mL). After 24 hours, by removing TRAIL and replenishing the cells with full medium, viable cells were rescued. When the plates reached 80% confluency, the cells again were treated with TRAIL (100 ng/mL) for 24 hours. The cycle was repeated, and PC3-TR cells were generated after 2 months and maintained in medium with TRAIL. PC3-TR cells were released from TRAIL at least one passage before use. All of the cells were cultured in RPMI 1640 tissue culture medium supplemented with 2 mmol/L L-glutamine, 10% FCS, and 1% penicillin-streptomycin (each at 50 µg/mL) at 37°C with 5% CO2.
Cell Viability and Apoptosis Assays.
Cell viability was determined by MTT method in accordance with the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN). In brief, 5 x 104 PC3, DU145 cells and 7.5 x 104 LNCaP cells were seeded in 96-well plates and cultured for 24 hours before treatment. Cells were then treated with various concentrations of TRAIL for 24 hours. MTT was added, followed by solubilization buffer 4 hours later. Absorbance was measured at 590 nm (630 nm was the reference wavelength) using a microtiter plate reader. Viability of untreated cells was set at 100%, and absorbance of wells with medium and without cells was set as zero. Flow cytometry was used to assess the sub-G1 DNA population of cells undergoing apoptosis. Cells were treated with BrdUrd (10 µmol/L) for 2 hours before being released from plates and fixed with 70% ethanol. Analysis of the sub-G1 DNA content was performed on a flow cytometer (using 488 nm for excitation and 515 nm for detection) according to the manufacturer’s protocol (Roche Diagnostics, Indianapolis, IN) with BrdUrd and propidium iodide (PI; 50 ìg/mL) staining. All of the results were from at least triplicate experiments.
Western Blot Analysis.
Cell lysates were prepared in RIPA buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% deoxycholate, 1.0% NP40, and 0.1% SDS] supplemented with a protease inhibitor mixture stock solution (Roche Molecular Biochemicals, Mannheim, Germany). After sonication for 10 seconds, cell debris was removed by centrifugation at 12,000 x g for 10 minutes at 4°C, and the protein concentration was determined by BCA protein assay reagent (Pierce, Rockford, IL). Equivalent amounts of proteins, as verified by Ponceau S staining and by immunoblot analysis against anti–-tubulin, were resolved by 10 to 12% SDS-PAGE and transferred to nitrocellulose membranes by electroblot analysis. Nitrocellulose blots were blocked with 5% (w/v) nonfat dry milk or 3% BSA in Tris-buffered saline/Tween buffer and incubated with the indicated primary antibody in Tris-buffered saline/Tween containing 2% milk or 1% BSA overnight at 4°C. The blots then were stained with the appropriate horseradish peroxidase-conjugated secondary antibody. Immunostained proteins were visualized on X-ray film using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).
Real-Time Quantitative Reverse Transcription-PCR.
Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA). The yield and quality of RNA was evaluated by measuring its absorbance at A260/A280 and gel electrophoresis. A total of 0.3 µg of each sample was included in a 50-µL reaction containing 25 µL of 2x TaqMan universal PCR master mix, 1.25 µL of 40x Multiscribe Reverse Transcriptase/RNase inhibitor mix (Applied Biosystems, Foster City, CA), forward and reverse primers (900 nmol/L), fluorogenic TaqMan probe (200 nmol/L), and RNase/DNase free water. The thermal cycling conditions were 30 minutes at 48°C, a 10-minute initial denaturation step at 95°C, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Thermal cycling was performed on an ABI Prism 7700 Sequence Detector (Applied Biosystems). The sequence of the custom primers was c-FLIP(L) forward primer, 5'-TCT CAC AGC TCA CCA TCC CTG-3'; and reverse primer, 5'-CAG GAG TGG GCG TTT TCT TTC-3'. Each sample was run in duplicate for c-FLIP(L), negative control of c-FLIP(L), and glyceraldehyde-3-phosphate dehydrogenase–positive and –negative controls. Results were from at least three independent experiments and normalized to the glyceraldehyde-3-phosphate dehydrogenase control amplification.
Transfection of c-FLIP(L) and Small Interfering RNA.
Full-length human c-FLIP(L) cDNA was cloned into a FLAG-tagged vector by using reverse transcription-PCR product from MDA–MB-435 breast cancer cells. Amplification was performed with the following primers: forward primer, 5'-GCA GAT ATC ATG GAT TAC AAA GAC GAT GAC GAT AAA TCT GCT GAA GTC ATC CAT CAG-3'; and reverse primer, 5'-CCG CTC GAG TTA TGT GTA GGA GAG G-3'. The amplified PCR products was extracted and subcloned into the pcDNA3/Zeo(+) vector (Invitrogen, Carlsbad, CA) between EcoRV and XhoI restriction sites and was confirmed by sequencing. PC3 and PC3-TR cells were plated at 105 cells/well in 24-well plates the day before transfection. The plasmids were transfected with Lipofectamine 2000 (Invitrogen), and small interfering RNA (siRNA) was transfected into cells by TransMessenger Transfection Reagent (Qiagen) according to the manufacturer’s instructions. After 48 hours, the cells were seeded in 96-well plates for cell viability testing, or protein was extracted for Western blot analysis. The siRNA gene target sequence of c-FLIP(L) is 5'-AAT TCA AGG CTC AGA AGC GAG-3', and the siRNA gene target sequence of c-FLIP(s) is 5'-AAC ACC CTA TGC CCA TTG TCC-3', which were designed and highly purified by Qiagen. Control siRNA to lamin A/C was used as positive control for these experiments (Qiagen).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cell viability assays showed that LNCaP cells were resistant to TRAIL, DU145 cells were moderately sensitive to TRAIL, and PC3 cells were sensitive to TRAIL (Fig. 1A)
, a finding consistent with others (36)
. In accordance with the cell viability results (Fig. 1A)
, phase microscopy (Fig. 1B)
showed that LNCaP cells were viable when treated with TRAIL (100 ng/mL), whereas the majority of TRAIL-sensitive PC3 cells had undergone cell shrinkage and pyknosis, morphologic findings consistent with cell death. Although the results shown herein represent cells that had been treated with TRAIL for 24 hours, numerous apoptotic-appearing PC3 cells could be found even after 4 hours of TRAIL treatment (data not shown). To ensure that the TRAIL-sensitive cells had undergone apoptosis, we further confirmed our cell viability (Fig. 1A)
and morphologic findings (Fig. 1B)
by assessing the sub-G1 apoptotic cell population of LNCaP, DU145, and PC3 cells (Fig. 1C)
. Prostate cancer cells were treated with TRAIL (100 ng/mL) for 4 hours, and the sub-G1 DNA content was assessed by flow cytometry. Nearly 50% of PC3 cells were apoptotic, whereas the LNCaP and DU145 cells showed much lower percentages of cell death after treatment with TRAIL (Fig. 1C)
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c-FLIP(L) has been shown to be an important mediator of cell death signaling downstream of the DR4/DR5 receptors and FADD complexes. Therefore, we postulated that expression of c-FLIP(L) might play a critical role in regulating TRAIL sensitivity in prostate cancer. Basal c-FLIP(L) expression was equivalent in DU145, PC3, and LNCaP cells. However, treatment with TRAIL led to decreased c-FLIP(L) protein expression in PC3 cells, a moderate reduction of c-FLIP(L) in DU145 cells, and no change in c-FLIP(L) expression in LNCaP cells (Fig. 2A)
. As for c-FLIP(s), it could not be detected in LNCaP or PC3 cells. In contrast, DU145 cells expressed a relatively high level of c-FLIP(s), which was decreased after TRAIL treatment (data not shown). These results show that persistent expression of c-FLIP(L) correlates with TRAIL resistance, whereas expression of c-FLIP(s) does not correlate with TRAIL sensitivity.
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. Because the cell viability results (Fig. 1A)
showed that 
80% of PC3 cells died when treated with 10 ng/mL of TRAIL for 24 hours, we chose the dose of 10 ng/mL for the time course experiments. LNCaP cells showed no significant changes in c-FLIP(L) protein expression, and DU145 cells showed a moderate reduction in the level of c-FLIP(L). In contrast, PC3 cells exhibited a gradual reduction in the level of c-FLIP(L) following TRAIL treatment. These results correlated with the degree of TRAIL sensitivity in LNCaP, DU145, and PC3 prostate cancer cell lines (Fig. 1)
. Interestingly, PC3 cells treated with 10 ng/mL of TRAIL for 24 hours reexpressed c-FLIP(L) protein. Reexpression of c-FLIP(L) occurred when PC3 cells were treated at low concentrations of TRAIL, whereas higher TRAIL concentrations (50 or 100 ng/mL) for 24 hours did not lead to c-FLIP(L) reexpression. This finding may be associated with the survival of a subpopulation of PC3 cells that are resistant to TRAIL-mediated apoptosis (see below).
Because protein expression of c-FLIP(L) seems to be correlated with resistance to TRAIL-mediated apoptosis, we wished to determine whether differential expression of c-FLIP(L) after TRAIL treatment is regulated at the transcriptional level. Therefore, we assessed the relative mRNA levels of c-FLIP(L) in prostate cancer cells after treatment with TRAIL (Fig. 2C)
. c-FLIP(L) mRNA levels were reduced in all of the prostate cancer cell lines following 4 hours of treatment with TRAIL. However, after 24 hours c-FLIP(L) transcription was increased to threefold above baseline in the TRAIL-resistant LNCaP cells. In contrast, the c-FLIP(L) mRNA levels were not altered significantly in the DU145 and PC3 cells following 24 hours of TRAIL treatment (Fig. 2C)
. Collectively, these data suggest that expression of c-FLIP(L) is correlated with resistance to TRAIL-mediated apoptosis, and steady expression of c-FLIP(L) protein in LNCaP cells (Fig. 2A)
is correlated with increased c-FLIP(L) mRNA levels 24 hours after TRAIL treatment (Fig. 2C)
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Ectopic Expression of c-FLIP(L) Increased TRAIL Resistance in PC3 Cells.
Because expression of c-FLIP(L) correlated with TRAIL resistance in prostate cancer cells, we wished to determine whether c-FLIP(L) is directly responsible for inducing resistance to TRAIL-mediated apoptosis. c-FLIP(L) was overexpressed in TRAIL-sensitive PC3 cells using a FLAG-tagged vector (Fig. 3A)
. Transient overexpression of c-FLIP(L) changed the phenotype of PC3 cells from TRAIL sensitive to TRAIL resistant (Fig. 3B)
. Therefore, these data indicate that c-FLIP(L) expression is sufficient to cultivate resistance to TRAIL-mediated apoptosis (Figs. 2
and 3B)
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; see Materials and Methods for details). Evaluating the genetic components that have been altered between PC3 and PC3-TR cells should serve as a better model to study TRAIL sensitivity. After exposure to TRAIL, PC3-TR cells had normal-appearing morphology (Fig. 4B)
and underwent apoptosis at low rates compared with the parental PC3 cells (Fig. 4C)
. The relative mRNA levels of c-FLIP(L) in PC3 cells were reduced after TRAIL treatment, whereas PC3-TR cells showed equivalent (4-hour treatment) or increased (24-hour treatment) mRNA levels following treatment with TRAIL (Fig. 4D)
. In accordance with our earlier findings, protein expression of c-FLIP(L) was reduced after treatment with TRAIL in PC3 cells, whereas TRAIL-resistant PC3-TR cells had a higher baseline level of c-FLIP(L) expression and maintained a steady expression of c-FLIP(L) protein after TRAIL treatment (Fig. 4E)
. Maintained protein expression of c-FLIP(L) after treatment with TRAIL can be partially explained by the increased transcription of c-FLIP(L) mRNA in PC3-TR cells (Fig. 4D)
. These results are consistent with our findings in the TRAIL-resistant LNCaP cells, which exhibited increased c-FLIP(L) mRNA levels 24 hours after TRAIL treatment and concurrent steady c-FLIP(L) protein expression (Fig. 2B and C)
.
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. As shown in Fig. 5A
, c-FLIP(L) protein was knocked down after siRNA transfection. Silencing the c-FLIP(L) isoform, but not the c-FLIP(s) isoform, followed by treatment with TRAIL led to a significant reduction in cell viability and apoptosis in PC3-TR cells (Fig. 5B)
. c-FLIP(s) siRNA served as a negative control for the aforementioned experiments and successfully reduced the expression of c-FLIP(s) protein in DU145 cells, which was the only prostate cancer cell line that expressed c-FLIP(s) in our studies (data not shown). These results suggest that silencing c-FLIP(L) protein can convert TRAIL-resistant prostate cancer cells to become TRAIL sensitive, whereas maintained expression of c-FLIP(L) is necessary and sufficient to render prostate cancer cells resistant to TRAIL-mediated apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Processing and signal transductions associated with TNF may involve two sequential signaling complexes: a cell survival promoting complex I and a cell death promoting complex II (44)
. It has been postulated that complex I, the initial plasma membrane bound complex, rapidly activates NFB and promotes cell survival. In contrast, complex II promotes cell death through a mechanism involving TNF-R1–associated death domain and RIP1, which associate with FADD and caspase-8 and -10. The first complex activates NFB, which stimulates its target genes, including c-FLIP(L) (45
, 46)
. c-FLIP(L), in turn, becomes incorporated into complex II and inhibits its function, thus promoting cell survival. In the absence of c-FLIP(L), the second complex is activated and leads to programmed cell death (44)
. It is presently unknown whether TRAIL-induced apoptosis involves formation of complex I and complex II in a manner similar to TNF (44)
or whether the initial TRAIL-induced apoptotic events are solely mediated through the death-inducing signaling complex plasma membrane complex (8, 9, 10, 11, 12, 13, 14, 15)
. Nonetheless, our results regarding TRAIL treatment are consistent with the findings of Micheau and Tschopp (44)
because we have shown that persistent c-FLIP(L) protein expression in prostate cancer cells leads to cell survival in correlative and functional studies.
The exact function of c-FLIP(L) in the regulation of apoptosis mechanisms remains controversial. Although many studies have shown that c-FLIP(L) has an antiapoptotic function (17
, 19, 20, 21, 22)
, others have shown c-FLIP(L) functions as a proapoptotic molecule at low concentrations and an antiapoptotic molecule only at high concentrations (31)
. Our data indicated that c-FLIP(L) transcript and protein levels were higher in TRAIL-resistant LNCaP and PC3-TR cells than in TRAIL-sensitive cells 24 hours after treatment with TRAIL. Moreover, c-FLIP(L) protein and mRNA levels were decreased in TRAIL-sensitive PC3 cells after TRAIL treatment, thus showing a strong correlation between expression of c-FLIP(L) and TRAIL sensitivity (Figs. 2
and 4)
. In direct experiments focusing on c-FLIP(L) function, we showed that ectopic expression of c-FLIP(L) in TRAIL-sensitive PC3 cells and silencing of c-FLIP(L) in TRAIL-resistant PC3-TR cells resulted in conversion of TRAIL sensitivity (Figs. 3
and 5)
. These results strongly indicate that c-FLIP(L) is necessary and sufficient to promote TRAIL resistance in prostate cancer cells and that c-FLIP(L) expression may be partially regulated at the transcriptional level.
Persistent expression of c-FLIP(L) also may play a critical role in mediating the activation of cytoplasmic and mitochondrial caspase-mediated apoptotic pathways (47 , 48) . In additional studies, we have found that persistent expression of c-FLIP(L) inversely correlates with activation of the apical cytoplasmic caspases (caspase-8 and -10), which mediate TRAIL-induced apoptosis, whereas secondary activation of mitochondrial-mediated caspase-9 was less crucial. We are actively pursuing these findings in greater depth.
The antiapoptotic role of c-FLIP(L) in TRAIL-induced apoptosis has been supported in an erythroid model system, wherein c-FLIP(L) was down-regulated during differentiation (49)
. Further cytoplasmic apoptotic pathways also may regulate the activity of caspase-mediated pathways, thus affecting TRAIL sensitivity and potentially the expression and function of c-FLIP(L) as well. For example, Akt protects LNCaP cells from TRAIL-induced apoptosis, and suppression of Akt causes LNCaP cells to become sensitive to TRAIL (36
, 50) . Expression of c-FLIP(L) seems to depend on the activity of the phosphatidylinositol 3'-kinase/Akt pathway (37)
. Akt may inhibit apoptosis in multiple other ways, including modulation of the proapoptotic proteins BAD and caspase-9, activation of NFB pathway, inhibition of FOXO transcription factors, and inhibition of BID protein (51)
. In conclusion, although there seems to be multiple mechanisms by which to regulate TRAIL sensitivity, regulation of c-FLIP(L) was sufficient to overcome the necessary threshold to modulate TRAIL-mediated apoptosis in prostate cancer.
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FOOTNOTES |
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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.
Requests for reprints: Aria F. Olumi, 330 Brookline Avenue, Division of Urological Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215. Phone: 617-667-4075; Fax: 617-975-5570; E-mail: aolumi{at}bidmc.harvard.edu or Roya Khosravi-Far, Department of Pathology, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Boston, MA 02215; Phone: 617-667-8526; Fax: 617-667-3524; E-mail: rkhosrav{at}bidmc.harvard.edu
Received 4/28/04. Revised 7/22/04. Accepted 7/28/04.
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S. M. Pulukuri, C. S. Gondi, S. S. Lakka, A. Jutla, N. Estes, M. Gujrati, and J. S. Rao RNA Interference-directed Knockdown of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Inhibits Prostate Cancer Cell Invasion, Survival, and Tumorigenicity in Vivo J. Biol. Chem., October 28, 2005; 280(43): 36529 - 36540. [Abstract] [Full Text] [PDF]
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 J. J. Lum, G. Bren, R. McClure, and A. D. Badley Elimination of Senescent Neutrophils by TNF-Related Apotosis-Inducing Ligand J. Immunol., July 15, 2005; 175(2): 1232 - 1238. [Abstract] [Full Text] [PDF]
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D. A. Sharp, D. A. Lawrence, and A. Ashkenazi Selective Knockdown of the Long Variant of Cellular FLICE Inhibitory Protein Augments Death Receptor-mediated Caspase-8 Activation and Apoptosis J. Biol. Chem., May 13, 2005; 280(19): 19401 - 19409. [Abstract] [Full Text] [PDF]
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