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Departments of Pathology (C.C., D.C., M.A.J., C.S.) and Urology (B.C., F.J.T.), Hospitales Universitarios Virgen del Rocío, Seville 41013, Spain
Address all correspondence and requests for reprints to: Carmen Sáez, M.D., Department of Pathology, Hospitales Universitarios Virgen del Rocío, Avenida Manuel Siurot s/n, Seville 41013, Spain. E-mail: csaez{at}cica.es.
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Introduction |
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The Bcl-2 family proteins have an essential role in the mitochondrial pathway of apoptosis. This family includes both proapoptotic and antiapoptotic members. In fact, the interactions between these subpopulations determine, at least in part, the susceptibility of cells to a death signal. The Bcl-2 family proteins fall into three groups that share at least one of four conserved Bcl-2 homology domains (BH1–4). Bcl-2, Bcl-xL, Bcl-w, A1, and Mcl-1 bear three or four BH domains and promote cell survival. Bcl-xL is a potent antiapoptotic factor that mediates resistance to staurosporin-induced apoptosis in PC3 cells and apoptosis can be restored when Bcl-xL is down-regulated (7, 8). Bcl-xL may neutralize the release of cytochrome c (9), but the exact mechanism by which Bcl-xL exerts its antiapoptotic effect remains controversial (10). Multidomain proapoptotic proteins Bax, Bak, and Bok share three domains (BH1–3) with Bcl-2 and are essential mediators in the apoptotic process. In mouse embryonic fibroblasts the absence of Bax and Bak is necessary for the effectiveness of the apoptotic blockade (11). Bax overexpression resulted in apoptotic cell death in prostate cancer cells such as PC3 and DU145, which are known to offer resistance to a variety of chemical proapoptotic agents (8, 12). The third group is constituted by the diverse group of proapoptotic BH-3-only proteins, including Bad, Bim, Bid, Puma, and Noxa, which have in common only the small BH3 interaction domain (13). These proteins control the activation of multidomain proapoptotic proteins, but the exact mechanism remains unclear. After an apoptotic signal, BH3-only proteins could promote multidomain proapoptotic proteins activation via their ability to inactivate the Bcl-2-like proteins. Alternatively, Bax may be activated via direct engagement by certain BH3-only proteins, the best documented being the active truncated form of Bid, tBid (14, 15, 16).
These models can explain how multidomain proapoptotic proteins are activated on a death stimulus; however, the mechanism by which Bax and Bak are kept in an inactive state in untreated cells remains a matter of discussion. In this regard, it has been published the interaction between Bak with Bcl-xL in untreated HeLa (17) and PC3 cells (18). The case of Bax is more controversial. Several authors state that Bax is predominantly a cytosolic monomer in untreated cells (19); during apoptosis it undergoes conformational changes, translocates to the outer mitochondrial membrane, and forms oligomers that cause mitochondrial dysfunction and apoptotic cell death (20, 21, 22, 23). On the other hand, the interaction between Bax and Bcl-xL in untreated T24 (24) and KB-3 cells (25) has been published recently. Also, it has been proposed that several unrelated proteins modulate Bax activity via direct interaction in untreated cells, such as Bif-1, 14–3-3
, Hsp60, and Ku70 (26, 27, 28, 29). Thus, whether the antiapoptotic proteins restrain Bax activation directly or indirectly remains uncertain (30) because it seems to be highly dependent on the cellular type.
Our aim was first to investigate whether proteins with suppressor activity on proapoptotic Bax and Bak, like Bcl-xL, were overexpressed in prostate cancer tissues and study their association with hormone-resistant, high-grade phenotype. Moreover, we intended to elucidate whether Bcl-xL actually interact with proapoptotic Bax and Bak in prostate cancer cell lines and tissues and thereby support that their blockade by Bcl-xL has a role in the prevention of apoptosis and the progression of hormone-resistant prostate cancer.
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Materials and Methods |
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Cell culture
Human prostate cancer cell lines PC3 and LNCaP-FNC were obtained from the Interlab Cell Line Collection (Genoa, Italy) and routinely grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM glutamine in a 37 C, humidified incubator under 5% CO2. Subconfluent cell cultures were harvested by trypsinization.
Antibodies
Rabbit polyclonal anti-Bax and anti-Bak, and mouse monoclonal anti-Bcl-xL antibodies were available from BD PharMingen (San Diego, CA), a second mouse monoclonal anti-Bcl-xL antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse monoclonal anti-ß-actin from Sigma (St. Louis, MO). Dilutions used in Western blots were anti-Bax (1:1000; anti-Bak (1:2000); anti-Bcl-xL (1:100; Santa Cruz Biotechnology); and anti-ß-actin (1:5000). For immunohistochemical procedures, dilutions were anti-Bax (1:2000) and anti-Bcl-xL (BD PharMingen) (1:1000).
Western blotting
Forty micrograms (from cell lines) or 100 µg (from tissues with tumor cell content over 75%) of total protein, as determined by using BCA protein assay kit (Pierce, Rockford, IL), were separated by SDS-PAGE on 4–20% gradient polyacrylamide gels (Invitrogen, Carlsbad, CA). Gels were electroblotted onto nitrocellulose membranes (Amersham, Little Chalfont, UK). For immunodetection, blots were blocked in 1% blocking reagent (Roche, Mannheim, Germany) in 0.05% Tween 20-PBS for 1 h and incubated with primary antibody overnight at 4 C diluted in blocking buffer. Blots were then washed in 0.05% Tween 20-PBS and incubated with either goat antimouse (1:10,000; Amersham) or goat antirabbit (1:20,000; Amersham) peroxidase-labeled antibodies in blocking buffer for 1 h. Enhanced chemoluminescent system was applied according to the manufacturer’s protocol (Amersham). Scanning densitometry was performed with Scan Analysis software (Biosoft, Cambridge, UK). Arbitrary densitometric units of the protein of interest were corrected for those of ß-actin.
Immunohistochemistry
Five-micrometer-thick tissue sections from paraffin blocks were dewaxed and rehydrated. Sections were immersed in 3% H2O2 aqueous solution for 30 min to exhaust endogenous peroxidase activity and then covered with 10% normal swine serum in Tris-buffered saline to block nonspecific binding sites. Antigen retrieval was performed with a pressure cooker, using citrate buffer (pH 6.0 for Bax) or 10 mM Tris and 1 mM EDTA (pH 9.0 for Bcl-xL). Bak immunohistochemistry was performed without antigen retrieval. Sections were incubated with primary antibodies overnight at 4 C. Peroxidase-labeled secondary antibodies and 3,3'-diaminobenzidine were applied to develop immunoreactivity, according to manufacturer’s protocol (EnVision; Dako, Glostrup, Denmark). Slides were then counterstained with hematoxylin and mounted in DPX (BDH Laboratories, Poole, UK). Sections in which primary antibody was omitted were used as negative controls. Immunostaining was evaluated independently by two observers on at least 10 microscopic fields at magnification x200 and scored as follows: 2+, greater than 50%; +, greater than 5%; and –, 1–5% of the carcinoma cells immunostained.
Immunoprecipitations
LNCaP and PC3 cells or tumor tissues were lysed in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) buffer [20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 0.25 M sucrose, and 2% CHAPS], containing protease inhibitors (1:100 dilution of protease inhibitors cocktail, catalog no. P8340; Sigma). One to two milligrams of total lysate were incubated with either 80 µl protein A-Sepharose beads (Amersham) for polyclonal antibodies or 80 µl of protein G-Sepharose beads (Amersham) for monoclonal Bcl-xL antibody at 4 C for 3 h. After centrifugation, beads were discarded and supernatants were incubated with normal rabbit serum for polyclonal antibodies or mouse IgG1 for anti-Bcl-xL overnight at 4 C, followed by protein A or G-Sepharose beads for 2 h. After centrifugation, beads were discarded and supernatants were incubated at 4 C for 1 h in a rotator with anti-Bax polyclonal antibody, anti-Bak polyclonal antibody, anti-Bcl-xL monoclonal antibody, and normal rabbit serum or mouse IgG1 as negative controls. Immunocomplexes were precipitated with 40 µl of protein A or G-Sepharose beads for 1 h at 4 C. After eight washes in CHAPS buffer, beads were boiled in 15 µl Laemmli buffer and subjected to SDS-PAGE. Normal rabbit serum and mouse IgG1 were from Dako.
Apoptosis induction and assays
Stock solution of camptothecin (Sigma) was prepared at 100 mM and stored frozen. Subconfluent monolayers of cells were treated with 3 µM camptothecin in culture medium for 18 and 36 h. One million cells were washed in cold PBS and suspended in 100 µl Annexin V binding buffer (R&D Systems, Minneapolis, MN) containing 5 µg/ml propidium iodide and 0.5 µg/ml annexin V-fluorescein isothiocyanate, incubated for 15 min at room temperature in the dark, and diluted in 400 µl Annexin V binding buffer. Fluorescence was measured on a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA) within 1 h. The collected events were gated on the forward and side scatter plots to exclude cellular debris. Three discrete cell populations (viable, early apoptotic, and late apoptotic) were identified measuring fluorescence on FL1 and FL2 channels. Annexin V binding assays were repeated three times in independent experiments. Caspase-mediated cleavage of poly(ADP-ribose) polymerase (PARP) was assessed by Western blot using a monoclonal antihuman PARP (BD PharMingen) at 1:250 dilution.
Statistics
Association of Bcl-xL with clinicopathological variables was analyzed by Fisher’s exact test. Hormone-refractory, disease-free survival curves were calculated by the method of Kaplan and Meier. Comparison of survival curves was done by the log-rank test of Mantel and Haenszel. Calculations were performed using Prism 4.0 (GraphPad, San Diego, CA).
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Results |
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, A–C. Well-differentiated, low Gleason-grade prostatic carcinomas showed very low levels of Bcl-xL, in contrast to tumors of moderate to high Gleason grade that exhibited increasing amounts of Bcl-xL. The appearance of Bcl-xL immunoreactivity was coarsely granular, cytoplasmic staining. Some benign hyperplastic glands contained few immunoreactive cells, but the staining was far less intense and finely granular. Stromal cells did not show immunoreactivity for Bcl-xL. Immunostaining for Bax and Bak was detected in all prostatic carcinomas with no major differences according to Gleason grade (data not shown).
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, top). In tissues from prostatic carcinomas, the levels of Bcl-xL protein expression increased with the Gleason grade because the signal intensity of the detected 30-kDa band was low in grades 3–6 and high in grades 7–9. In the same tissues, the expression levels of Bak and Bax proteins were similar in all samples tested and were not dependent on Gleason grade. Bax protein was detected as a double band in all prostatic carcinoma tissues. Average densitometric values of normalized Bcl-xL protein expression levels showed 4.5-fold increase in the group of tumors of Gleason grades 7–9, compared with that of Gleason grades 3–6. The group of carcinomas of higher Gleason grades had slightly lower levels of Bak and Bax than carcinomas of lower grades (Fig. 1D, bottom). In addition, we performed a statistical analysis of immunohistochemical Bcl-xL expression that showed a significant association with Gleason grade (P < 0.0001) (Table 1). Most (85.1%) low Gleason-grade tumors (grades 2–4) showed low levels of Bcl-xL expression. Intermediate Gleason grade tumors (grades 5–7) had low or moderate levels in 65.6% of tumors and high in 34.4% of tumors. Most tumors of Gleason grades 8–9 (93.8%) expressed high levels of Bcl-xL. Immunohistochemical expression of Bcl-xL was also associated with the presence of regional lymph node involvement, distant metastasis, and initial serum PSA levels (Table 1).
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). Survival curves were significantly different (P < 0.0001 from Mantel and Haenszel log-rank test; 
2 = 21.2; degrees of freedom = 1) between both groups with a median survival time of 14 months and a hazard ratio of 9.71 (3.72–26.16; 95% confidence interval) for the high Bcl-xL-expressing tumors.
Bcl-xL forms complexes with Bax and Bak in androgen-independent PC3 cells
We used cell lysates from androgen-dependent LNCaP cells and androgen-independent PC3 cells to elucidate the mechanism by which proapoptotic potential of Bax and Bak are diminished in relation to hormone-resistant disease. Interactions of Bcl-xL, with proapoptotic Bax and Bak were analyzed in cell lysates obtained with CHAPS buffer. Figure 2A shows the basal levels of Bcl-xL, Bak, and Bax in PC3 and LNCaP cells. Bcl-xL protein was more abundant in PC3 than LNCaP cells. Conversely, Bax protein levels were higher in LNCaP cells. Immunoprecipitation experiments with these cell lysates showed that Bcl-xL was coimmunoprecipitated with both anti-Bax and anti-Bak polyclonal antibodies in PC3 but only with anti-Bak in LNCaP cells (Fig. 2B). Figure 2C shows a Western blot of immunoprecipitated Bax and Bak that demonstrates the specificity and efficiency of immunoprecipitation experiments. The reciprocal immunoprecipitation for Bcl-xL confirms the interaction between Bcl-xL and Bax in PC3 cells (Fig. 2D). These results suggest that Bax and Bak are subject to a blockade by Bcl-xL in the androgen-independent PC3 cells. To further investigate whether interactions between Bcl-xL and proapoptotic Bax and Bak are potential mechanisms involved in prostate cancer progression, we performed immunoprecipitation experiments with prostatic carcinoma tissue lysates. Figure 2E shows these experiments in two lysates from prostatic carcinoma tissues of moderate and high Gleason grades (Gleason 5 and 9), respectively. Bcl-xL was coimmunoprecipitated with both anti-Bax and anti-Bak antisera in the lysate from prostatic carcinoma of high Gleason grade. In contrast, those interactions were not seen when using the lysate from moderately differentiated Gleason-grade prostatic carcinoma. These findings support the proposed mechanism of a blockade of Bax and Bak by Bcl-xL that might be involved in progression to hormone-refractory, high-grade prostate cancer.
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). In LNCaP cells treated with 3 µM camptothecin, the 85-kDa cleavage product appears at both 18 and 36 h, whereas PC3 cells show an intact PARP 36 h after the same treatment. Flow cytometry experiments with cells labeled with annexin V-fluorescein isothiocyanate and propidium iodide (Fig. 3B) confirm that PC3 cells are camptothecin insensitive. Conversely, LNCaP cells are camptothecin sensitive and undergo apoptosis upon the treatment. In this context, we examined the basal levels of Bcl-xL, Bak, and Bax in both cell lines, as well as the interactions between these proteins after treatment with 3 µM camptothecin, to assess changes in the status of the interactions observed in untreated cells. In PC3 cells exposed to camptothecin during 36 h, Bak protein level is elevated, whereas the levels of Bcl-xL and Bax proteins are slightly diminished, compared with control cells (Fig. 4A). Remarkably, these cells still maintain the interactions between Bcl-xL with Bax and Bak (Fig. 4B). In LNCaP cells exposed to the same stimulus, Bax and Bak protein levels are elevated; however, Bcl-xL protein expression is strongly down-regulated at 18 h and is almost undetectable at 36 h (Fig. 4A). According to the kinetics of Bcl-xL expression, the interaction between Bcl-xL and Bak is diminished at 18 h and have almost disappeared at 36 h in camptothecin-treated LNCaP cells (Fig. 4B); thus, Bak would be gradually free from Bcl-xL to exert its proapoptotic function.
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Discussion |
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We used two antibodies that recognize specifically the antiapoptotic Bcl-xL isoform to study a large series of patients by immunohistochemistry. We investigated the association of Bcl-xL overexpression with selected clinicopathological variables and the appearance of resistance to hormone therapy. We observed that Bcl-xL is significantly more abundant in high and intermediate Gleason-grade prostatic carcinomas than in more differentiated low-grade tumors. Bax and Bak were not differentially expressed in prostatic carcinomas of various Gleason grades, a fact that has been previously noted in the case of Bax (5). We also observed a statistically significant association with lymph node metastasis, distant metastasis, and initial serum PSA level. In the group of patients treated by complete androgen ablation, we found a significant association between the expression of Bcl-xL and the onset of hormone-refractory disease.
Actually, different models try to explain the activation mechanism of multidomain proapoptotic proteins (30); however, it remains unclear how these proteins are maintained in an inactive state in the absence of any apoptotic stimulus. Willis et al. (17) demonstrated that Bak interacts with Bcl-xL and Mcl-1 in untreated HeLa cells; overexpressed Noxa binds Mcl-1 tightly and displaces Bak. The authors propose that other BH3-only proteins must be activated, provided that Bak-mediated apoptosis requires the neutralization of both Mcl-1 and Bcl-xL. Similarly, Shiau et al. (18) demonstrated recently that Bak interacts with Bcl-xL in untreated but not 
-tocopheryl succinate-treated PC3 cells. In these cases, Bak activation would be restrained by antiapoptotic Bcl-2 family members via direct interaction. In relation to Bax protein, several authors reported interactions of Bax with Bcl-xL (24, 25) and proteins unrelated to the Bcl-2 family (26, 27, 28, 29) in untreated cells that kept Bax in an inactive state. Moreover, in vitro studies documented that multidomain proapoptotic proteins are executors of the mitochondrial pathway of apoptosis whose activation can be prevented by antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xL (41, 42, 43, 44). However, whether this inhibition is via direct interaction between Bax and antiapoptotic proteins or not is still a matter of discussion because in vivo interactions seem to be dependent on the cellular model examined (23). The physiological significance of these interactions as well as the possible implication of the interactions status in untreated and apoptotic cells in the resistance or sensitivity to apoptosis remains to be fully understood.
Thus, we sought to investigate in vivo interactions of Bax and Bak with Bcl-xL in the prostate cancer cell lines LNCaP (androgen responsive) and PC3 (androgen unresponsive) by coimmunoprecipitation assays. We used CHAPS as the only detergent in the coimmunoprecipitation procedures, according to previous reports that demonstrate that immunoprecipitations with nonionic detergents may form artifactual complexes (45). We observed that both Bax and Bak coimmunoprecipitate with Bcl-xL in PC3 cells, but only Bak coimmunoprecipitates with Bcl-xL in LNCaP cells. In these cells, the interaction between Bax and Bcl-xL would occur to a much lesser extent, if any, than in PC3 cells. We also performed the immunoprecipitation with the anti-Bax antibody in the absence of any detergent and the interaction between Bax and Bcl-xL was again evidenced in PC3 but not LNCaP cells (data not shown). To assess whether these interactions occurred in primary prostate cancer, we also performed immunoprecipitation assays in prostatic carcinoma tissues of high and intermediate Gleason grades and found that Bcl-xL interacts with Bax and Bak only in the high-grade carcinoma. This finding supports that the described interactions may be of physiological relevance. As far as we know, this is the first time that in vivo interaction between Bax and Bcl-xL has been demonstrated in the prostate cancer PC3 cell line as well as in primary prostatic tissues.
To further investigate whether Bcl-xL represents a blockade over proapoptotic multidomain proteins that has to be removed for the effectiveness of the apoptotic process, we treated PC3 and LNCaP cells with the topoisomerase I inhibitor camptothecin. In camptothecin-treated LNCaP cells, the level of Bcl-xL protein diminished gradually; at 36 h, when the majority of cells were apoptotic, Bcl-xL protein was almost undetectable, and thus, the interaction between Bcl-xL and Bak was absent. These findings support a model in which Bcl-xL would exert an inhibiting effect over Bak via heterodimerization. In unstimulated LNCaP cells, Bcl-xL would keep Bak in an inactive state; upon an apoptotic stimulus such as camptothecin treatment, it takes place a gradual loss of the interaction between these proteins that would allow Bak to exert its proapoptotic function. As previously reported (46), camptothecin did not induce apoptosis in PC3 cells, although it does inhibit cell growth, suggesting that the drug is reaching its target in the cell. Notably, the interactions between Bcl-xL with Bax and Bak were still present after 36 h of treatment. Thus, these observations suggest that the maintenance of the interactions between Bcl-xL with Bax and Bak in PC3 cells after apoptotic damage could be of relevance in the resistance to apoptosis, although the elucidation of this point would require further investigation. In this regard, the disappearance of the interaction between Bcl-xL and Bak during -tocopheryl succinate-induced apoptosis in PC3 cells supports this notion (18).
In this context, heterodimerization of Bcl-xL with Bax or Bak in high-grade prostatic carcinoma and camptothecin-treated PC3 cells may represent a mechanism of apoptosis blockade that facilitates tumor progression and androgen resistance, and that is absent in either carcinomas of moderate grade or LNCaP cells.
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Acknowledgments |
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Footnotes |
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The authors declare no conflict of interest.
First Published Online June 22, 2006
Abbreviations: CHAPS, 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate; PARP, poly(ADP-ribose) polymerase; PSA, prostate-specific antigen.
Received April 18, 2006.
Accepted for publication June 14, 2006.
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References |
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-Tocopheryl succinate induces apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 function. J Biol Chem 281:11819–11825This article has been cited by other articles:
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J. Witham, M. R. Valenti, A. K. De-Haven-Brandon, S. Vidot, S. A. Eccles, S. B. Kaye, and A. Richardson The Bcl-2/Bcl-XL Family Inhibitor ABT-737 Sensitizes Ovarian Cancer Cells to Carboplatin Clin. Cancer Res., December 1, 2007; 13(23): 7191 - 7198. [Abstract] [Full Text] [PDF]
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