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Departments of 1 Cancer Biology, 2 Urology, and 3 Genitourinary Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX
Requests for reprints: David McConkey, Department of Cancer Biology, Unit 173, U.T. M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-8591; Fax: (713) 792-8747. E-mail: dmcconke{at}mdanderson.org
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 26S proteasome is a large, multicatalytic structure responsible for degrading a number of different polypeptides important for cell cycle progression and cell survival (1–3). The subunits of the proteasome are structurally conserved through evolution (4), and complete loss-of-function mutations in the subunits are lethal, indicating that normal proteasome function plays a requisite role in normal cell physiology. Interestingly, however, recent studies demonstrated that proteasome inhibitors are much more active in tumor cells compared to their normal counterparts (5, 6), and in some cases, proteasome inhibitors actually delay or prevent programmed cell death (7, 8). These observations stimulated the development of bortezomib (Velcade, previously PS-341) for cancer therapy in humans (9–12). Bortezomib exhibited excellent activity in the National Cancer Institute's panel of 60 cell lines, with a mean IC50 across the panel of 7 nM (9). More importantly, its activity was unlike that of any other class of compounds examined in the screen (n > 100,000) (9), strongly suggesting that it interacted with a unique biological target. Preclinical studies confirmed that the drug was active in tumor xenografts (9, 13–16), and bortezomib is currently being evaluated in a series of Phase I–III clinical trials in patients with a variety of solid and hematological malignancies (11). Importantly, there is significant interest in developing bortezomib for the therapy of androgen-independent prostate cancer because of promising activity observed in the studies conducted to date.
Here we examined the effects of bortezomib on the growth of in vivo-selected derivatives of two popular human prostate cancer cell lines (LNCaP and PC-3). LNCaP-Pro5 was selected by orthotopic "recycling" for aggressive local growth within the prostate and retains many features of early-stage disease (androgen-sensitive, wild-type p53, nonmetastatic) (17). In contrast, PC3M-Pro4 was derived from the highly metastatic PC-3M subline, and it is not only locally aggressive but also highly metastatic (17). PC3M-Pro4 is androgen receptor- and p53-negative and is completely androgen independent in vitro and in vivo. Our overall goal was to evaluate the activity of bortezomib in disparate prostate cancer models and to characterize the biological mechanisms involved in whatever tumor growth inhibition was observed.
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Materials and Methods |
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MTT Assays
Cells were cultured in 96-well plates at a concentration of 10,000 cells/well and left to recover (LNCaP-Pro5: 48 h, PC3M-Pro4: 24 h). Cells were exposed to bortezomib for 48 h, drug was removed, and cells were cultured in drug-free medium overnight. Cells were then incubated with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide in PBS at 5 mg/ml, Sigma Chemical Co., St. Louis, MO) (10 µl/well) for 2 h (37°C). The medium was replaced with DMSO (40 µl/well) and MTT precipitates were dissolved for 1 h before quantification of optical densities (570 nm).
Propidium Iodide and Fluorescence-activated Cell Sorting Analysis
Cells were incubated with bortezomib for 48 h and harvested by trypsinization. The trypsinized cells, media, and PBS wash were mixed and centrifuged. Supernatants were removed and the cells were resuspended in 400 µl of a cold (4°C) propidium iodide solution (50 µg/ml propidium iodide, 0.1% Triton X-100, and 0.1% sodium citrate, in PBS). Cells were incubated in the solution for at least 2 h at 4°C before analysis by flow cytometry. Cells with a subdiploid DNA content were scored as apoptotic (% sub-G0/G1) as described previously (18).
Caspase 3 Activation
Cells were split to 10-cm plates and left to recover for 48 h before treatment. After treatment, cells were harvested by trypsinization, and the trypsinized cells, the media, and the PBS washes were added together and collected by centrifugation. The cells were washed in cold PBS, resuspended in 500 µl of a 2% solution of paraformaldehyde in PBS, and left on ice for 20 min. The cells were then pelleted and resuspended in a 0.2% solution of Triton X-100 in PBS and left on ice for 5 min. One milliliter of cold fluorescence-activated cell sorting (FACS) buffer was then added (1% sodium azide, 2% fetal bovine serum in PBS). The cells were then pelleted and washed again in FACS buffer. The cells were then resuspended in 50 µl of FACS buffer containing 20 µl phycoerythrin (PE)-conjugated anti-active caspase 3 antibody (BD Biosciences, Palo Alto, CA), and samples were incubated for 30 min on ice. Samples were washed twice in FACS buffer, resuspended in 500 µl FACS buffer, and analyzed by flow cytometry. The proportion of cells staining for PE above the levels of a nonspecific PE-conjugated control antibody (PE-conjugated anti-mouse Ig, 
light chain, PharMingen, Palo Alto, CA) was quantified.
Analysis of Phosphatidylserine Exposure by Annexin V Staining
Annexin V staining was conducted with the use of a kit (Annexin V-PE apoptosis detection kit I, BD Biosciences). Cells were split to 10-cm plates and left to recover for 24 (PC3M-Pro4) or 48 (LNCaP-Pro5) h before exposure to bortezomib. After treatment, cells were harvested by trypsinization, and the trypsinized cells, the media, and the PBS wash were added together and collected. The cells were washed twice in cold PBS and resuspended in 100 µl of binding buffer (supplied by the vendor) containing 5 µl PE-conjugated annexin V. Cells were incubated for 15 min on ice, and 400 µl of binding buffer was added to each sample before analysis by flow cytometry.
Secretion of Angiogenic Factors in Vitro
Cells were incubated in a conventional incubator or in a hypoxia chamber (1% O2) for the times indicated in the absence or presence of bortezomib. Conditioned media were collected and frozen at -20°C. Factor production in the supernatants was quantified by ELISA using commercial kits specific for vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), or interleukin-8 (IL-8) (R&D Systems, Minneapolis, MN) following product protocols. The colorimetric readings at 450 nm were normalized to the cell counts.
Tumor Xenografts
PC3M-Pro4 and LNCaP-Pro5 cells were mixed with Matrigel (Becton Dickinson Labware, Bedford, MA) (1 x 106 cells in 200 µl) and injected into each flank of 6-week-old male nude mice (BALB/c background, purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD). Tumors were established for 2 weeks before the start of treatment. Mice were subjected to a course of six tail vein injections of 1 mg/kg bortezomib in 20 µl of saline or 20 µl saline alone (controls) every 72 h. Tumor measurements (the longest and shortest tumor diameters) were performed at the time of each injection. Tumor volumes were calculated according to the following formula: volume = (l)(w2/2), where l = length and w = width. Mice were euthanized by cervical dislocation 24 h after the last injection. The tumors were harvested and either fixed in formaldehyde and embedded in paraffin or immersed in OCT compound (Optimal Cutting Temperature, Sakura Finetechnical Co., Torrance, CA) and frozen in liquid nitrogen.
Immunofluorescence Analyses
Tumor sections on charged slides were deparafinized and subjected to staining for cellular morphology with H&E, and for proliferating cell nuclear antigen (PCNA) (primary antibody, mouse monoclonal, DAKO, Carpinteria, CA); secondary antibody, Alexa 488-conjugated rabbit polyclonal, Molecular Probes, Eugene, OR), VEGF (primary antibody, rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA; secondary antibody, FITC-conjugated goat polyclonal, The Jackson Laboratory, Bar Harbor, ME), bFGF (primary antibody, rabbit polyclonal, Sigma; secondary antibody: FITC-conjugated goat polyclonal, The Jackson Laboratory), and IL-8 (primary antibody, rabbit polyclonal, Biosource International, Camarillo, CA; secondary antibody, FITC-conjugated goat polyclonal, The Jackson Laboratory). Tissues were washed twice in PBS and circled with a pap pen before the slides were transferred to humidity chambers. Slides were incubated in protein blocking solution (5% normal horse serum, 1% goat serum in PBS) for 20 min before the addition of the primary antibody (diluted in blocking solution), which was left on the tissues overnight at 4°C. Tissues were then washed 3x in PBS and blocked for 10 min before incubation with secondary antibodies in blocking solution for 1 h. This and subsequent steps were conducted in the dark. Sections were washed twice in PBS before they were counterstained with 1 µg/ml propidium iodide in PBS for 10 min. Sections were washed 3x, and Prolong antifade mounting agent (Molecular Probes) and a cover slide were added to each. Images were captured with an Axioplan 2 microscope, a Hamamatsu color chilled 3CCD camera, and Optimus software (Bioscan, Edmonds, WA).
Detection of Tumor Microvessels
Frozen tissue sections were fixed in acetone, washed in PBS, and blocked for endogenous peroxidase activity with a 3% hydrogen peroxide solution in methanol for 12 min. Sections were then washed in PBS and incubated in protein blocking solution for 20 min. Slides were incubated overnight at 4°C with anti-CD31 primary antibody (rat polyclonal, Pharmacia, Piscataway, NJ) in protein blocking solution. The primary antibody was removed, sections were washed in PBS and blocked for 10 min, and tissues were incubated for 1 h at room temperature with the secondary antibody (goat polyclonal horseradish peroxidase-conjugated, The Jackson Laboratory). Sections were washed in PBS, and positive staining was visualized by incubating the slides with stable 3,3'-diaminobenzidine for 10–20 min. The sections were rinsed with distilled water, counterstained with Gill's hematoxylin (colorimetric development), and mounted with Universal Mount (Research Genetics, Huntsville, AL). Control samples exposed to secondary antibody alone showed no specific staining.
In Vivo Measurement of Apoptosis
Frozen tissue sections were fixed in acetone, washed in PBS, and blocked for 20 min before they were incubated with anti-CD31 primary antibody (rat polyclonal, Pharmacia) in blocking solution overnight at 4°C. Tissues were washed in PBS, blocked for 10 min, and incubated with the secondary antibody (Cy5-conjugated goat polyclonal, The Jackson Laboratory) for 1 h. This and subsequent steps were conducted in the dark. Sections were then washed with PBS and treated with a 4% formaldehyde solution in PBS for 10 min. Slides were washed, incubated with 0.2% Triton X-100 for 15 min, and washed again. DNA fragmentation was visualized by staining tissue sections with a commercial kit (DeadEnd fluorometric TUNEL system, Promega, Madison, WI). Tissues were incubated in equilibration buffer for 10 min before the addition of the reaction cocktail containing terminal dNTP transferase and FITC-conjugated nucleotides. Slides were incubated for 1 h at 37°C, and reactions were terminated by incubation in 2x SSC for 15 min. Tissues were washed in PBS and counterstained with a 1 µg/ml solution of propidium iodide in PBS for 10 min. After a final round of washes, slides were mounted with Prolong (Molecular Probes) and cover slides. Images were captured with an Axioplan 2 microscope, a Hamamatsu color chilled 3CCD camera, and Optimus software (Bioscan). TUNEL-positive cells were quantified by laser scanning cytometry (LSC; Compucyte, Cambridge, MA) (15, 19).
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Discussion |
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The promising results obtained with bortezomib in Phase I prompted us to evaluate its antitumoral activity and mechanisms of action in two human prostate tumor xenografts. Our group developed variants of the popular LNCaP and PC-3M prostate adenocarcinoma lines selected for aggressive growth in vivo (17), and we selected these cells for analysis here. In addition, we attempted to generate tumor xenografts using a third human prostate cancer line (DU-145), but the cells were not nearly as tumorigenic as the other lines and the results obtained were impossible to interpret. The results of our MTT experiments demonstrate that bortezomib induces strong growth arrest at clinically relevant concentrations in vitro (
10 nM), and tumor xenografts treated with i.v. bortezomib display substantial inhibition of proliferation (measured by PCNA staining) in vivo. However, other aspects of bortezomib's mechanisms of action differ in the two models. In PC3M-Pro4 cells, clinically relevant concentrations of bortezomib induced DNA fragmentation characteristic of apoptosis, whereas higher levels of the drug (1 µM) were required to stimulate apoptosis in the LNCaP-Pro5 cells. Similar patterns were observed in the xenografts, where bortezomib induced higher levels of TUNEL staining in the PC3M-Pro4 tumors as compared with the levels observed in LNCaP-Pro5 tumors. These results were somewhat surprising given that the PC3M-Pro4 cells are considered much more aggressive than the LNCaP-Pro5 cells. Because proteasome inhibitors appear to preferentially kill cycling cells (6), it is possible that the PC3M-Pro4 cells possess cell cycle checkpoint defect(s) that prevent them from undergoing sustained growth arrest, whereas LNCaP-Pro5 cells do so more efficiently because they possess wild-type p53 and p21. We are currently exploring the role of proteasome inhibitor-mediated cell cycle arrest in resistance to therapy-induced apoptosis in the model systems described here as well as in others. These effects may be especially important when bortezomib is combined with conventional agents (nucleoside analogues, anthracyclines, taxanes) that preferentially target cells at specific stages of the cell cycle.
Although bortezomib induced higher levels of apoptosis in the PC3M-Pro4 cells, bortezomib produced stronger growth inhibition in LNCaP-Pro5 tumors (>70%) as compared to PC3M-Pro4 tumors (approximately 50%). One important mechanism of bortezomib-induced tumor growth inhibition identified in our previous studies and work conducted by others is angiogenesis inhibition. Specifically, we recently showed that bortezomib inhibited VEGF secretion and lowered MVDs in human L3.6pl and Mia PaCa-2 pancreatic tumor xenografts (15), and another group reported similar findings in a model of human squamous cell carcinoma (14). Recent studies indicate that tumors can vary markedly with respect to their dependence on angiogenesis, effects that are linked in part to p53 status. Because p53 appears to mediate hypoxia-induced apoptosis (28), p53-deficient tumors may require fewer blood vessels per unit area to survive and expand (29). As noted above, the LNCaP-Pro5 cells retain wild-type p53, whereas the PC3M-Pro4 cells do not.
Here we demonstrate that the LNCaP-Pro5 cells constitutively secrete very high levels of VEGF that can be increased further by exposing the cells to hypoxia. In contrast, PC3M-Pro4 tumors secrete very low levels of VEGF at baseline, and hypoxia has no effect on VEGF production. Consistent with these in vitro findings, LNCaP-Pro5 tumors produce high levels of VEGF and display high MVDs in vivo, whereas PC3M-Pro4 tumors produce lower levels of VEGF and display lower MVDs. Bortezomib inhibited baseline and hypoxia-induced VEGF production in LNCaP-Pro5 cells in vitro and reduced tumor VEGF expression and MVDs in LNCaP-Pro5 tumors in vivo, whereas its effects on angiogenesis in PC3M-Pro4 tumors were much less remarkable. Together, our results strongly suggest that inhibition of tumor angiogenesis plays an important role in the inhibition of LNCaP-Pro5 but not PC3M-Pro4 tumor growth. Nonetheless, our data should not be interpreted to mean that VEGF plays no role in tumor growth and angiogenesis in PC3M-Pro4 tumors. In fact, direct targeting of VEGF-R2/KDR using a blocking monoclonal antibody (DC-101) (30) or small molecule inhibitors (31, 32) attenuated the growth of established PC-3 tumors by more than 70%, presumably because some VEGF signaling is absolutely required for endothelial cell differentiation and survival. We are in the process of directly defining the role of p53 in this dichotomy using LNCaP-Pro5 cells that stably express the p53-inhibitory human papillomavirus E6 protein. We consider it likely that a large fraction of the tumor cell death observed in the bortezomib-treated LNCaP-Pro5 tumors resulted from indirect effects of the drug on the tumor vasculature.
Despite recent progress in defining the molecular alterations that contribute to prostate cancer progression and metastasis, there is general consensus that current therapeutic strategies are relatively inactive in patients with androgen-independent cancer. The results of this study and others demonstrate that bortezomib interferes with tumor cell proliferation and angiogenesis and induces apoptosis in tumors at clinically achievable concentrations of the drug. Bortezomib affects a number of different pathways important for tumor progression, including p532 and the transcription factor, NFB (13). Dose escalation in the Phase I clinical trials conducted to date has been guided by biochemical assays of 20S proteasome activity in patent blood samples, and surprisingly little toxicity has been observed at doses of the drug that achieve target inhibition of 70–80% (11). As noted earlier, promising clinical activity has been observed in Phase I trials of bortezomib in androgen-independent prostate cancer, including reductions in serum PSA and IL-6 and radiological evidence of response. These properties make bortezomib an attractive candidate for combination with conventional agents for the therapy of advanced prostate cancer.
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Footnotes |
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Grant support: A grant from the Department of Defense Prostate Cancer Research Program and developmental funds from the M.D. Anderson Prostate Cancer SPORE.
1 C. Papandreou and C. Logothetis, unpublished observations. 
2 S. Williams and D. J. McConkey. The proteasome inhibitor bortezomib stabilizes a novel active form of p53 in human LNCaP-Pro5 prostate cancer cells, submitted for publication.
Received 5/ 5/03; revised 6/17/03; accepted 6/24/03.
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  K.-F. Chen, P.-Y. Yeh, K.-H. Yeh, Y.-S. Lu, S.-Y. Huang, and A.-L. Cheng Down-regulation of Phospho-Akt Is a Major Molecular Determinant of Bortezomib-Induced Apoptosis in Hepatocellular Carcinoma Cells Cancer Res., August 15, 2008; 68(16): 6698 - 6707. [Abstract] [Full Text] [PDF]
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Q. Xu, M. Farah, J. M. |
