Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome. We examined the antitumor activity of combination therapy with bortezomib + docetaxel in two human pancreatic cancer cell lines (MiaPaCa-2 and L3.6pl) selected for their divergent responses to bortezomib alone. Bortezomib blocked docetaxel-induced apoptosis in the MiaPaCa-2 cells and failed to enhance docetaxel-induced apoptosis in L3.6pl cells in vitro but did interact positively with docetaxel to inhibit clonogenic survival. These effects were associated with decreased accumulation of cells in M phase, stabilization of the cyclin-dependent kinase inhibitors, p21 and p27, and inhibition of cdk2 and cdc2 activities. In orthotopic xenografts, combination therapy produced significant reductions in tumor weight and volume in both models associated with accumulation of p21, inhibition of proliferation, and increased apoptosis. Combination therapy also reduced tumor microvessel densities, effects that were associated with reductions in tumor cell production of vascular endothelial growth factor and increased levels of apoptosis in tumor-associated endothelial cells. Together, our results suggest that bortezomib enhances the antitumoral activity of taxanes by enforcing cell growth arrest and inhibiting angiogenesis.

Pancreatic carcinoma is the fourth most common cause of cancer-related death in the United States (1). Strategies for early detection of this cancer have not been developed; consequently, most pancreatic carcinomas present with metastatic or locally advanced disease at the time of diagnosis (2). Chemotherapy and irradiation are largely ineffective, and metastatic disease frequently develops even after potentially curative surgery. Most patients with metastatic disease possess survival rates of only 3–6 months (3). Therefore, new and more effective therapies are clearly needed for treatment of this disease.

Although little progress has been made in identifying more effective therapies for pancreatic cancer, recent investigation has made significant inroads into defining the molecular alterations that mediate pancreatic cancer progression (4). Among the most common genetic alterations observed in well-differentiated tumors are activating mutations in K-ras and inactivating mutations in p16 (4), with inactivation of p53 and DPC4/Smad-4 occurring later (5–7). These events are associated with a variety of different epigenetic events that probably cooperate with them to enhance tumor invasion, metastasis, and drug resistance (4, 8). With regard to the latter, recent work indicates that the nuclear factor κB (NFκB) transcription factor is constitutively activated in most human pancreatic cancer cell lines and primary tumor specimens (9). Furthermore, conventional cancer chemotherapeutic agents can activate NFκB, which limits drug efficacy via inhibition of apoptosis (10–14).

Bortezomib is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome (15). Preclinical analysis of bortezomib's growth inhibitory activity against the National Cancer Institute's panel of 60 tumor cell lines demonstrated an average GI50 of 7 nm, placing the compound among the top 10 agents with respect to potency tested to date in the screen (15). By stabilizing cytoplasmic IκB, bortezomib inhibits the activity of NFκB in several models (16–19). These features make bortezomib an attractive candidate for use in the therapy of pancreatic cancer.

We recently demonstrated that bortezomib was effective in blocking the growth of established orthotopic L3.6pl pancreatic tumor xenografts, effects that were associated with direct induction of tumor cell death and indirect effects on the vasculature (16). Another group has confirmed that bortezomib possessed both direct cytotoxic and antiangiogenic effects in a preclinical model of squamous cell carcinoma (17). Like bortezomib, taxanes possess both cytotoxic and antiangiogenic properties (20, 21) but act via different biological mechanisms. Therefore, we examined the in vitro and in vivo antitumoral effects of combination therapy with bortezomib + docetaxel in two pancreatic cancer cell lines, one that is highly sensitive to bortezomib as a single agent (L3.6pl) and another that is not (MiaPaCa-2; Ref. 16).

Animals, Cell Lines, and Antibodies

Male nude mice (BALB/c background) were purchased from the Animal Production Area of the National Cancer Institute Frederick Cancer Research and Development Center (Frederick, MD). L3.6pl human pancreatic cancer cells were established from COLO-357 fast-growing cells by injecting them into the pancreas of nude mice. Hepatic metastases were harvested and reinjected into the pancreas. This process was repeated six times, resulting in the isolation of the L3.6pl cell line, which produce significantly higher incidence and number of lymph node and liver metastases than parental cells (22). The human pancreatic cancer cell line MiaPaCa-2 was obtained from the American Type Culture Collection (Rockville, MD). Cell lines were maintained in MEM supplemented with 10% fetal bovine serum (FBS) along with sodium pyruvate, nonessential amino acids, l-glutamine, vitamins, and antibiotics under conditions of 5% CO2 in air at 37°C. Antibodies were obtained from the following commercial sources: anti-p21 and p27 (Transduction Laboratories, San Diego, CA), anti-cdc2, cdk2, and vascular endothelial growth factor (VEGF; Santa Cruz Biotechnology, Santa Cruz, CA), CD31-platelet/endothelial cell adhesion molecule 1 (PharMingen, San Diego, CA), anti-actin (Sigma Chemical Co., St. Louis, MO), anti-proliferating cell nuclear antigen (PCNA; Dako, Glostrup, Denmark), peroxidase-conjugated F(ab′)2 goat anti-rabbit IgG, and peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, CA).

Quantification of DNA Fragmentation

DNA fragmentation was measured by propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) analysis as described previously (23). Cells were plated in six-well plates (1 × 104 cells/well). Although high, the concentration of bortezomib used for the in vitro studies (10 μm) was selected to ensure maximal target inhibition, and previous studies indicate that the drug retains selectivity for the proteasome at high micromolar concentrations (J. Adams and P. Elliott, personal communication). Note that this concentration did not induce appreciable levels of apoptosis in the MiaPaCa-2 cells (Fig. 1), consistent with our previous observations (16). Following incubation with 10 μm bortezomib, 1 μm docetaxel, bortezomib + docetaxel, 1 μm staurosporine, or 10 μm roscovitine in vitro, cells were harvested, pelleted by centrifugation, and resuspended in PBS containing 50 μg/ml PI, 0.1% Triton X-100, and 0.1% sodium citrate. Cells were incubated with the PI solution for 16 h, and flow cytometric analysis of stained cells was performed with a FACScan (Becton Dickinson, Mountain View, CA).

Figure 1.

Effects of bortezomib and docetaxel on inducing apoptosis in human pancreatic cancer cells in vitro. A, effects on DNA fragmentation. Cell lines were incubated with 10 μm bortezomib, 1 μm docetaxel, bortezomib + docetaxel, or 1 μm staurosporine for 24 h. DNA fragmentation was measured by PI staining and FACS analysis as described in Materials and Methods. Columns, mean (n = 3); bars, SD. B, effects on caspase activity. Cells were incubated for 24 h with 10 μm bortezomib, 1 μm docetaxel, bortezomib + docetaxel, or 1 μm staurosporine. Caspase-3-like activity was measured in cytosolic extracts using a fluorogenic substrate (Asp-Glu-Val-Asp-AMC) as described in Materials and Methods. Columns, mean (n = 3); bars, SD. *, significantly different from control. **, significant difference between docetaxel and bortezomib + docetaxel treatments.

Figure 1.

Effects of bortezomib and docetaxel on inducing apoptosis in human pancreatic cancer cells in vitro. A, effects on DNA fragmentation. Cell lines were incubated with 10 μm bortezomib, 1 μm docetaxel, bortezomib + docetaxel, or 1 μm staurosporine for 24 h. DNA fragmentation was measured by PI staining and FACS analysis as described in Materials and Methods. Columns, mean (n = 3); bars, SD. B, effects on caspase activity. Cells were incubated for 24 h with 10 μm bortezomib, 1 μm docetaxel, bortezomib + docetaxel, or 1 μm staurosporine. Caspase-3-like activity was measured in cytosolic extracts using a fluorogenic substrate (Asp-Glu-Val-Asp-AMC) as described in Materials and Methods. Columns, mean (n = 3); bars, SD. *, significantly different from control. **, significant difference between docetaxel and bortezomib + docetaxel treatments.

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Caspase Assay

Cells (1 × 105) were plated in 10-cm dishes with 10% FBS-MEM and were allowed to attach for 24 h. Cells were treated with 10 μm bortezomib, 1 μm docetaxel, combination, or 1 μm staurosporine for 24 h. Following treatment, cells were lysed at 4°C with 200 μl caspase lysis buffer [100 mm HEPES (pH 7.4), 1% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, and 1 mm EDTA] containing a Complete Mini Protease Inhibitor Tablet (Boehringer Mannheim, Indianapolis, IN). Lysate was added to 800 μl of caspase buffer and 2 μl of 25 mm amino-4-methylcoumarin (AMC) fluorogenic substrate (Enzyme Systems Products, Livermore, CA) and incubated for 1 h at 37°C. After incubation, 1 ml of caspase buffer was added to dilute the mix and the samples were read on a Shimadzu (Columbia, MD) spectrophotometer (Model RF-1501).

Clonogenic Survival Assays

Pilot studies were performed to define the concentrations of bortezomib and docetaxel required to produce incomplete inhibition of clonogenic survival. Cells were then treated with 10 nm bortezomib, 1 nm docetaxel, or drug combination for 12 h. After drug treatment, 300 cells/well were plated into six-well plates with fresh medium for 14 days. The colonies were washed with PBS, fixed with methanol, and stained with crystal violet. The colonies were counted using a gel documentation system (AlphaInnotech, San Leandro, CA). The surviving fraction was determined by dividing the number of surviving colonies in the treated wells by the number of colonies in the nontreated control groups.

Immunoblotting

Cells (1 × 105) were incubated with 10 μm bortezomib, 1 μm docetaxel, or combination of the two drugs for 24 h. Cells were collected using a cell scraper at 4°C and were then lysed as previously described (24). Approximately 20 μg of total cellular protein from each sample were subjected to SDS-PAGE, proteins were transferred to nitrocellulose membranes, and the membranes were blocked with 5% nonfat milk in a Tris-buffered saline solution containing 0.1% Tween 20 for 1 h. The blots were then probed overnight with relevant antibodies, washed, and probed with species-specific secondary antibodies coupled to horseradish peroxidase. Immunoreactive material was detected by enhanced chemiluminescence (West Pico, Pierce Chemical Co., Rockville, IL). Quantification of band intensity was performed using UN-SCAN-IT gel software (Silk Scientific, Orem, UT).

Kinase Assay

Cells (1 × 105) were incubated on 100 × 20-mm plates with 10 μm bortezomib, 1 μm docetaxel, or combination for 24 h. Cells were lysed on the plates at 4°C with a 1% Triton X-100 lysis buffer containing 150 mm NaCl, 25 mm Tris (pH 7.5), 1 mm NaF, 1 mm sodium orthovanadate, 10 mm β-glycerophosphate, and a Complete Mini Protease Inhibitor Tablet. After centrifugation, the clarified supernatant was incubated with anti-cdc2 or anti-cdk2 antibody for 1 h followed by incubation with protein A/G-Sepharose beads for 12 h at 4°C. The immunocomplex was washed thrice with lysis buffer and washed twice with reaction buffer containing 25 mm Tris (pH 7.2) and 10 mm MgCl2. The cdc2 or cdk2 immunoprecipitates were incubated with 1 μg of histone H1, 150 μm ATP, and 20 μCi of [γ-32P]ATP in 50 μl of reaction buffer for 15 min at 30°C. The reaction was terminated by adding SDS sample buffer, and the mixture was loaded onto a 12% SDS-PAGE gel after boiling at 95°C for 5 min. The gel was stained with Coomassie blue, dried, and autoradiographed. Quantification of band intensity was done using UN-SCAN-IT gel software.

Quantification of VEGF Secretion by ELISA

To evaluate VEGF expression after treatment with bortezomib and docetaxel, cells (1 × 104) were plated in six-well plates with 2 ml of MEM with 1% FBS. After 24 h, cells were exposed to 100 nm bortezomib, 100 nm docetaxel, or drug combination for 12 h. These concentrations and time point were selected because they did not produce significant toxicity in the L3.6pl cells. Supernatants were collected and VEGF protein levels were determined using Quantikine ELISA kits (R&D Systems, Inc., Minneapolis, MN). Cell numbers were equivalent in control and treated samples.

Orthotopic Implantation of Tumor Cells

Intrapancreatic human tumors were established as described previously (22). Cells were harvested from culture flasks after brief trypsinization and transferred to serum-free HBSS. Only single-cell suspensions of >90% viability determined by trypan blue exclusion were used. Male nude mice were anesthetized with methoxyflurane, a small left abdominal flank incision was made and the spleen was exteriorized, and tumor cells (1 × 106 cells) were injected into the subcapsular region of the pancreas using a 30-gauge needle and a calibrated push button-controlled dispensing device (Hamilton Syringe Company, Reno, NV). A successful subcapsular intrapancreatic injection of tumor cells was indicated by the appearance of a fluid bubble without i.p. leakage. To prevent leakage, a cotton swab was held cautiously for 1 min over the site of injection. The abdominal wound was closed in one layer with wound clips (Autoclip, Clay Adams, Parsippany, NJ).

Treatment Schedule

Tumors were established for 14 days. Preliminary studies were conducted to identify a dose of docetaxel that would yield significant but incomplete inhibition of L3.6pl tumor growth; the dose of bortezomib was its maximum tolerated dose. Animals were then injected i.p. with bortezomib in saline at a dose of 1.0 mg/kg, with 5 mg/kg docetaxel, or with both every 72 h for 14 days. Mice were killed by cervical dislocation and primary tumors in the pancreas were excised and weighed. Tumor volumes were calculated according to the following formula: volume = (l) (w2 / 2), where l = length and w = width. For immunohistochemistry, one part of the tumor tissue was formalin fixed and paraffin embedded and another part was embedded in OCT compound (Miles Inc., Elkhart, IN), rapidly frozen in liquid nitrogen, and stored at −70°C.

Quantification of Apoptosis in Situ

Analysis of DNA fragmentation by fluorescent terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was performed using a commercial kit (Promega, Madison, WI). Percentages of positive cells were then determined using a laser scanning cytometer (LSC) as described previously (16, 25, 26). For each group (control, bortezomib, docetaxel, and combination), four independent fields were selected at random from different tumors so that the comparison among groups would involve roughly equivalent numbers of cells. For CD31 TUNEL staining, frozen sections were stained using an anti-CD31 primary antibody followed by a Texas red- or Cy5-conjugated secondary antibody. TUNEL analysis was then performed as described above.

Quantification of Tumor Microvessel Density and VEGF, p21, and PCNA Expression

Tumors were characterized using colorimetric immunohistochemistry for microvessel densities (MVD) and VEGF, p21, or PCNA expression. Briefly, paraffin sections (4–6 μm thick) were mounted on positively charged Superfrost slides (Fisher Scientific, Houston, TX) and dried overnight. Sections were deparaffinized in xylene, treated with a graded series of alcohol [100%, 95%, and 80% ethanol/double-distilled H2O (v/v)] and rehydrated in PBS (pH 7.5), treated with pepsin (Biomeda, Foster City, CA) for 15 min at 37°C, and washed with PBS. Immunohistochemical procedures were performed as described previously (27). Positive reactions were 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. Staining intensity was quantified by densitometric analysis of five random high-power fields containing viable tumor cells, and results correspond to the average absorbance.

Statistical Analyses

Statistical significance of differences observed in drug-treated and control samples was determined using the Tukey-Kramer Comparison Test. Differences were considered significant in all experiments at P < 0.05 (*, Significantly different from untreated controls. **, Significantly different from bortezomib and docetaxel single treatment tumors, unless otherwise stated).

Effects of Bortezomib and Docetaxel on Cell Growth in Vitro

We previously reported that human pancreatic cancer cell lines display marked heterogeneity in their responses to bortezomib-induced apoptosis in vitro and in vivo (16). Therefore, we selected representative bortezomib-sensitive (L3.6pl) and bortezomib-resistant (MiaPaCa-2) cell lines to evaluate the effects of bortezomib + docetaxel on cell growth in vitro. We exposed cells to bortezomib, docetaxel, combination, or staurosporine (a positive control for apoptosis) for 48 h and measured apoptosis by PI staining and FACS analysis (to quantify DNA fragmentation) and assessment of caspase-like protease activity (via hydrolysis of the fluorogenic dye Asp-Glu-Val-Asp-AMC). Both assays confirmed that the MiaPaCa-2 cell line was resistant to bortezomib-induced apoptosis, but the cells were sensitive to either docetaxel or staurosporine (Fig. 1, A and B). In contrast, the L3.6pl cell line was equally sensitive to bortezomib, docetaxel, or bortezomib + docetaxel (Fig. 1, A and B). Combined treatment with bortezomib + docetaxel produced lower levels of DNA fragmentation and caspase activation than were observed with single-agent docetaxel in the MiaPaCa-2 cell line, and levels of apoptosis were similar in cells treated with bortezomib versus bortezomib + docetaxel in the L3.6pl cell line (Fig. 1, A and B).

In a second series of experiments, we assessed the effects of bortezomib + docetaxel treatment on cell survival using clonogenic assays, which more accurately measured long-term effects of drugs on all forms of permanent cell growth arrest and cell death. We first conducted pilot dose-response studies to identify concentrations of bortezomib (Fig. 2A) and docetaxel (Fig. 2B) that produced about 50–70% inhibition of clonogenic growth. We then cultured cells with 10 nm bortezomib, 1 ng/ml (1.25 nm) docetaxel, or both for 12 h, replated cells in fresh medium, and cultured them for 14 days before quantifying colony numbers by crystal violet staining (Fig. 2C). Exposure to either drug alone resulted in a significant reduction in colony formation, and exposure to the drug combination led to even greater inhibition of cell growth (Fig. 2D).

Figure 2.

Effects of bortezomib + docetaxel on clonogenic survival. A, concentration-dependent effects of bortezomib on clonogenic growth. MiaPaCa-2 and L3.6pl cells were treated with indicated concentrations of bortezomib for 12 h and replated as outlined in Materials and Methods. Cells were cultured for 14 days, and colonies were detected by staining with crystal violet. Columns, mean (n = 3); bars, SD. B, concentration-dependent effects of docetaxel on clonogenic growth. Cells were exposed to the indicated concentrations of docetaxel for 12 h and replated, and colony formation was measured after 14 days by crystal violet staining. Columns, mean (n = 3); bars, SD. C, representative colonies obtained after therapy. MiaPaCa-2 and L3.6pl cells were treated with 10 nm bortezomib, 1 ng/ml docetaxel, or both agents for 12 h and replated as outlined in Materials and Methods. Cells were cultured for 14 days, and colonies were stained with crystal violet. D, quantitative analysis of the results. Data are expressed as percentage of colonies formed by treated cells compared with untreated cells. Columns, mean (n = 3); bars, SD. *, significantly different from control. **, significant difference between docetaxel or bortezomib and bortezomib + docetaxel.

Figure 2.

Effects of bortezomib + docetaxel on clonogenic survival. A, concentration-dependent effects of bortezomib on clonogenic growth. MiaPaCa-2 and L3.6pl cells were treated with indicated concentrations of bortezomib for 12 h and replated as outlined in Materials and Methods. Cells were cultured for 14 days, and colonies were detected by staining with crystal violet. Columns, mean (n = 3); bars, SD. B, concentration-dependent effects of docetaxel on clonogenic growth. Cells were exposed to the indicated concentrations of docetaxel for 12 h and replated, and colony formation was measured after 14 days by crystal violet staining. Columns, mean (n = 3); bars, SD. C, representative colonies obtained after therapy. MiaPaCa-2 and L3.6pl cells were treated with 10 nm bortezomib, 1 ng/ml docetaxel, or both agents for 12 h and replated as outlined in Materials and Methods. Cells were cultured for 14 days, and colonies were stained with crystal violet. D, quantitative analysis of the results. Data are expressed as percentage of colonies formed by treated cells compared with untreated cells. Columns, mean (n = 3); bars, SD. *, significantly different from control. **, significant difference between docetaxel or bortezomib and bortezomib + docetaxel.

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Bortezomib Inhibits Cell Cycle Progression

In the process of measuring the effects of bortezomib and docetaxel on DNA fragmentation, we observed that bortezomib blocked docetaxel-induced accumulation of cells in the M phase of the cell cycle (Fig. 3A). These effects were associated with accumulation of the cyclin-dependent kinase (cdk) inhibitors p21 and p27 (Fig. 3B) and inhibition of baseline and docetaxel-induced activation of cdk2 and cdc2 (Fig. 3C). To confirm that the observed cdk inhibition was sufficient to explain bortezomib's interference with docetaxel-induced apoptosis, we treated cells with docetaxel in the presence or absence of a chemical cdk inhibitor, roscovitine (10 μm), and measured G2-M-phase arrest and apoptosis by PI staining and FACS analysis. This concentration of roscovitine was the maximum that could be used without toxicity. As we had observed in cells treated with bortezomib, roscovitine inhibited docetaxel-induced M-phase accumulation and apoptosis (Fig. 3A). A higher concentration of roscovitine (25 μm) that produced some direct cytotoxicity (15% sub-G0/G1 population) in both cell lines completely blocked the M arrest induced by docetaxel (data not shown). Together, these data strongly suggest that bortezomib's ability to promote cell cycle arrest before M phase (most likely via stabilization of cdk inhibitors) is responsible for the observed interference with docetaxel-induced apoptosis.

Figure 3.

Effects of bortezomib and docetaxel on the cell cycle. A, effects of drug treatment on cell cycle phase distribution. Cell lines were treated with 10 μm bortezomib, 1 μg/ml docetaxel, 10 μm roscovitine, or the indicated combinations of drugs for 24 h. Cell cycle analysis was performed by PI staining and FACS analysis as described in Materials and Methods. Gates and percentages, sub-G0/G1 population of cells (apoptotic). Results of one experiment representative of three independent replicates are shown. B, effects of bortezomib and docetaxel on p21 and p27 expression. Cells were incubated with 10 μm bortezomib, 1 μg/ml docetaxel, or combination for 24 h. Cells were harvested and lysed, and levels of p21, p27, and actin were measured by immunoblotting. Results are representative of three independent replicates. The relative band intensities of p21 and p27 were measured as described in Materials and Methods and are indicated below each lane. C, effects of bortezomib and docetaxel on cdk2 and cdc2 kinase activity. Cells were incubated with 10 μm bortezomib, 1 μg/ml docetaxel, or both drugs for 24 h. Cell lysates were then subjected to immunoprecipitation using antibodies against cdk2 or cdc2 as described in Materials and Methods. Immunocomplexes were subjected to kinase assay using histone H1 as the substrate. Results are representative of three independent experiments. The relative intensity of each band is indicated below each lane.

Figure 3.

Effects of bortezomib and docetaxel on the cell cycle. A, effects of drug treatment on cell cycle phase distribution. Cell lines were treated with 10 μm bortezomib, 1 μg/ml docetaxel, 10 μm roscovitine, or the indicated combinations of drugs for 24 h. Cell cycle analysis was performed by PI staining and FACS analysis as described in Materials and Methods. Gates and percentages, sub-G0/G1 population of cells (apoptotic). Results of one experiment representative of three independent replicates are shown. B, effects of bortezomib and docetaxel on p21 and p27 expression. Cells were incubated with 10 μm bortezomib, 1 μg/ml docetaxel, or combination for 24 h. Cells were harvested and lysed, and levels of p21, p27, and actin were measured by immunoblotting. Results are representative of three independent replicates. The relative band intensities of p21 and p27 were measured as described in Materials and Methods and are indicated below each lane. C, effects of bortezomib and docetaxel on cdk2 and cdc2 kinase activity. Cells were incubated with 10 μm bortezomib, 1 μg/ml docetaxel, or both drugs for 24 h. Cell lysates were then subjected to immunoprecipitation using antibodies against cdk2 or cdc2 as described in Materials and Methods. Immunocomplexes were subjected to kinase assay using histone H1 as the substrate. Results are representative of three independent experiments. The relative intensity of each band is indicated below each lane.

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Effects of Bortezomib and Docetaxel on Orthotopic Tumors

We next examined the effects of the bortezomib + docetaxel on the growth of human pancreatic tumor xenografts in vivo. MiaPaCa-2 and L3.6pl cells were placed orthotopically in the pancreas of nude mice and tumors were allowed to grow unperturbed for 14 days. We delivered bortezomib, docetaxel, or combination by i.p. injection every 72 h because previous work has shown that 20S proteasome activity recovers to control levels by 72 h (15). Preliminary studies indicated that the maximum tolerated dose for bortezomib was 1 mg/kg (16). We selected 5 mg/kg (approximately half of the LD10 in this model; data not shown) as our dose of docetaxel because it produced ∼50% inhibition of tumor growth in the L3.6pl xenografts (Fig. 4). We observed no toxicity in any of the treatment arms of this study. Tumor growth was significantly inhibited by single agent bortezomib or docetaxel in the L3.6pl tumors (Fig. 4). We observed liver metastases in three of six untreated mice bearing L3.6pl tumors but detected none in any of the treatment groups. Single-agent therapy also appeared to inhibit the growth of the MiaPaCa-2 tumors, although the differences observed were not statistically significant (Fig. 4). No metastases were detected in any of the mice bearing MiaPaCa-2 tumors. In both tumor types, combination therapy with bortezomib + docetaxel produced significant decreases in tumor weights and volumes compared with those observed in any of the other arms of the study (Fig. 4). Tumors were collected at the end point for all of the histological studies described below.

Figure 4.

Effects of bortezomib and docetaxel on orthotopic human pancreatic tumors. Orthotopic tumors were generated by injecting 1 × 106 cells into the pancreas glands of nude mice. Tumors were established for 14 days before therapy. Tumors from control animals or from animals treated with 1 mg/kg bortezomib, 5 mg/kg docetaxel, or combination every 72 h were harvested after 14 days. Tumor burden was assessed by measuring weights and volumes. Columns, mean (n = 6); bars, SD.

Figure 4.

Effects of bortezomib and docetaxel on orthotopic human pancreatic tumors. Orthotopic tumors were generated by injecting 1 × 106 cells into the pancreas glands of nude mice. Tumors were established for 14 days before therapy. Tumors from control animals or from animals treated with 1 mg/kg bortezomib, 5 mg/kg docetaxel, or combination every 72 h were harvested after 14 days. Tumor burden was assessed by measuring weights and volumes. Columns, mean (n = 6); bars, SD.

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To investigate the role of apoptosis in tumor growth inhibition, we stained tumor sections collected at the end of therapy using fluorescent TUNEL (to detect apoptosis-associated DNA fragmentation; Fig. 5A) and quantified levels of apoptosis using a LSC (Fig. 5B). This time point was selected to complement data obtained in a previous study (16), where we studied the effects of bortezomib on apoptosis at earlier time points (24–28 h). Consistent with those results (16), therapy with single-agent bortezomib significantly increased levels of apoptosis in the L3.6pl tumors but not in the MiaPaCa-2 tumors (Fig. 5B). Nonetheless, there did appear to be a trend toward increased apoptosis in the MiaPaCa-2 tumors that may have been caused by indirect effects of bortezomib on the tumor stroma. Similar results were obtained with tumors isolated from animals receiving single-agent docetaxel (Fig. 5B). Combination therapy with bortezomib + docetaxel produced significant increases in apoptosis in both tumor types, although overall levels of cell death were still higher in the L3.6pl tumors as compared with the MiaPaCa-2 tumors (Fig. 5B).

Figure 5.

Effects of bortezomib on apoptosis in orthotopic human pancreatic tumors. A, representative TUNEL-stained sections. Established tumors were treated i.p. with 1 mg/kg bortezomib, 5 mg/kg docetaxel, or both drugs every 72 h for 14 days. Apoptosis was measured on frozen sections by fluorescein-based (green) TUNEL staining as described in the Materials and Methods. Tissues were counterstained with PI (red) to detect all cell nuclei. B, quantification of TUNEL staining by LSC. Percentages of TUNEL-positive cells were quantified in four independent fields as described in Materials and Methods. Columns, mean; bars, SD.

Figure 5.

Effects of bortezomib on apoptosis in orthotopic human pancreatic tumors. A, representative TUNEL-stained sections. Established tumors were treated i.p. with 1 mg/kg bortezomib, 5 mg/kg docetaxel, or both drugs every 72 h for 14 days. Apoptosis was measured on frozen sections by fluorescein-based (green) TUNEL staining as described in the Materials and Methods. Tissues were counterstained with PI (red) to detect all cell nuclei. B, quantification of TUNEL staining by LSC. Percentages of TUNEL-positive cells were quantified in four independent fields as described in Materials and Methods. Columns, mean; bars, SD.

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Because bortezomib inhibited cell cycle progression in vitro, we next measured the effects of bortezomib, docetaxel, or both agents on tumor cell proliferation by staining tumor sections with an antibody to PCNA (Fig. 6A). Quantitative analysis of the results confirmed that the drug combination was more effective at blocking cell proliferation than was therapy with either bortezomib or docetaxel alone (Fig. 6B). To determine whether the reductions in tumor cell proliferation were associated with stabilization of cdk inhibitor, we stained tumor sections with an antibody to p21 (Fig. 6C) and measured the levels of the protein by densitometry (Fig. 6D). Consistent with our in vitro findings, bortezomib caused marked accumulation of p21, whereas docetaxel did not (Fig. 6, C and D). Levels of p21 in tumors treated with the drug combination were identical to those observed in tumors treated with bortezomib alone (Fig. 6, C and D).

Figure 6.

Effects of bortezomib and docetaxel on tumor cell proliferation. Tumors were harvested within 12 h of the last treatment on the last day of therapy (28 days postimplantation). A, immunohistochemical analysis of tumor PCNA levels. Sections were stained with an antibody to PCNA as described in Materials and Methods. Representative images are shown. B, densitometric quantification of PCNA levels. Optical densities from random fields were analyzed using Optimas software as described previously (30). Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel. C, immunohistochemical analysis of tumor p21 levels. Paraffin sections were stained with an anti-p21 antibody as described in Materials and Methods. Representative sections are shown. D, densitometric quantification of p21 levels. Optical densities from random fields were analyzed using Optimas software. Columns, mean (n = 5); bars, SD. *, significantly different from control.

Figure 6.

Effects of bortezomib and docetaxel on tumor cell proliferation. Tumors were harvested within 12 h of the last treatment on the last day of therapy (28 days postimplantation). A, immunohistochemical analysis of tumor PCNA levels. Sections were stained with an antibody to PCNA as described in Materials and Methods. Representative images are shown. B, densitometric quantification of PCNA levels. Optical densities from random fields were analyzed using Optimas software as described previously (30). Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel. C, immunohistochemical analysis of tumor p21 levels. Paraffin sections were stained with an anti-p21 antibody as described in Materials and Methods. Representative sections are shown. D, densitometric quantification of p21 levels. Optical densities from random fields were analyzed using Optimas software. Columns, mean (n = 5); bars, SD. *, significantly different from control.

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Effects of Bortezomib and Docetaxel on Tumor Vasculature

The apparent discrepancy between the effects of bortezomib + docetaxel on tumor cell apoptosis in vitro versus in vivo prompted us to investigate its effects on the tumor vasculature. In a first series of experiments, we quantified tumor MVDs in tumors harvested from control and drug-treated animals by staining them with an antibody to CD31 (Fig. 7A). Consistent with previous reports, therapy with either bortezomib or docetaxel alone resulted in significant decreases in MVD (Fig. 7B). However, combination therapy with bortezomib + docetaxel resulted in stronger decreases in MVD as compared with the results obtained with either single agent (Fig. 7B).

Figure 7.

Effects of bortezomib and docetaxel on tumor vasculature. Tumors were harvested within 12 h of the last treatment on the last day of therapy (28 days postimplantation). A, sections from control or treated tumors were stained with an antibody to CD31 to visualize tumor microvessels. Representative images are shown. B, quantification of tumor MVD. Numbers of CD31-positive microvessels were counted manually in five independent fields. Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel. C, analysis of endothelial cell death. Frozen sections were stained with an antibody to an endothelial cell marker (anti-CD31; red) followed by fluorescent TUNEL (green) to visualize apoptotic endothelial cells. Representative images are shown. D, quantification of TUNEL staining by LSC. Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel.

Figure 7.

Effects of bortezomib and docetaxel on tumor vasculature. Tumors were harvested within 12 h of the last treatment on the last day of therapy (28 days postimplantation). A, sections from control or treated tumors were stained with an antibody to CD31 to visualize tumor microvessels. Representative images are shown. B, quantification of tumor MVD. Numbers of CD31-positive microvessels were counted manually in five independent fields. Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel. C, analysis of endothelial cell death. Frozen sections were stained with an antibody to an endothelial cell marker (anti-CD31; red) followed by fluorescent TUNEL (green) to visualize apoptotic endothelial cells. Representative images are shown. D, quantification of TUNEL staining by LSC. Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel.

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In previous studies, we demonstrated that the growth inhibitory effects of antiangiogenic agents are associated with increased apoptosis in tumor-associated endothelial cells as measured by two-color fluorescent CD31 TUNEL analysis (Fig. 7C; 25, 28–31). To determine the role of endothelial cell apoptosis in the antiangiogenic effects of bortezomib + docetaxel, we performed similar staining and quantified levels of endothelial cell death by LSC. The results mirrored those obtained in the MVD measurements where therapy with either agent produced significant endothelial cell death but combination therapy was significantly more effective (Fig. 7D).

We recently demonstrated that bortezomib's effects on angiogenesis are associated with inhibition of tumor cell production of VEGF (not basic fibroblast growth factor or interleukin-8; 32). Therefore, in a final series of experiments, we measured the effects of bortezomib, docetaxel, or drug combination on VEGF production by immunohistochemistry (Fig. 8A). The results revealed that therapy with either bortezomib or docetaxel resulted in significant inhibition of VEGF production in both tumor models in vivo (Fig. 8, A and B). Furthermore, combination therapy resulted in stronger suppression of VEGF production than was observed with either single agent (Fig. 8B). Because only viable regions of the tumors were analyzed, the reduction of VEGF was not a secondary effect of induction of tumor cell apoptosis. To investigate whether direct effects of the drugs on tumor cell secretion of VEGF could account for the effects observed, we exposed L3.6pl and MiaPaCa-2 cells to bortezomib, docetaxel, or both drugs and measured VEGF levels in conditioned media by ELISA. Consistent with the in vivo findings, the drug combination reduced VEGF levels more effectively than single-agent treatment (Fig. 8C). Because VEGF is a survival factor for endothelial cells (31), the observed reductions in VEGF secretion probably contributed to the increased endothelial cell apoptosis and reduced MVDs observed in the tumors exposed to the bortezomib + docetaxel drug combination in vivo.

Figure 8.

Effects of bortezomib and docetaxel on VEGF production. Tumors were harvested within 12 h of the last treatment on the last day of therapy (28 days postimplantation). A, immunohistochemical analysis of tumor VEGF levels. Paraffin sections were stained with an anti-VEGF antibody as described in Materials and Methods. Representative images are shown. B, densitometric quantification of VEGF levels. Densitometry was performed using Optimas imaging software as described previously (30). Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel. C, VEGF secretion measured by ELISA. Cells were incubated for 12 h in the presence of 100 nm bortezomib, 100 ng/ml docetaxel, or combination, and VEGF production was measured by ELISA. Columns, mean (n = 3); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel.

Figure 8.

Effects of bortezomib and docetaxel on VEGF production. Tumors were harvested within 12 h of the last treatment on the last day of therapy (28 days postimplantation). A, immunohistochemical analysis of tumor VEGF levels. Paraffin sections were stained with an anti-VEGF antibody as described in Materials and Methods. Representative images are shown. B, densitometric quantification of VEGF levels. Densitometry was performed using Optimas imaging software as described previously (30). Columns, mean (n = 5); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel. C, VEGF secretion measured by ELISA. Cells were incubated for 12 h in the presence of 100 nm bortezomib, 100 ng/ml docetaxel, or combination, and VEGF production was measured by ELISA. Columns, mean (n = 3); bars, SD. *, significantly different from control. **, significantly different from bortezomib or docetaxel.

Close modal

The proteasome mediates the degradation of many proteins involved in cell cycle progression and apoptosis, making it an attractive target for pharmacological inhibition in cancer. Bortezomib is a potent and selective proteasome inhibitor that has displayed marginal toxicity (mostly peripheral neuropathy in patients who have received platinum analogues or other agents linked to this toxicity) in phase I clinical trials in a variety of different tumor types (33), and the drug recently received Food and Drug Administration approval for use in the therapy of multiple myeloma. Among its cell survival-associated molecular targets, the proteasome is known to play a critical role in the activation of the transcription factor, NFκB (14), which is constitutively activated in most human pancreatic cancer cell lines and primary tumors (9). In vitro and in vivo studies by our laboratory and others have demonstrated that bortezomib has antitumor activity in pancreatic cancer models (34, 35) and that it sensitizes some pancreatic cancer cells to apoptosis induced by taxanes in vitro via a mechanism that involves inhibition of NFκB (36). The observation that NFκB inhibitors sensitize tumor cells to conventional chemotherapeutic agents has been reported extensively (reviewed in Refs. 12, 14). There is interest in using taxanes for pancreatic cancer therapy within our clinical translational research team (J. L. Abbruzzese, personal communication) but they have not been evaluated extensively either in preclinical models of pancreatic cancer or in clinical trials. These factors prompted us to conduct the present study.

In a first series of experiments, we evaluated the effects of bortezomib + docetaxel therapy on cell growth and apoptosis in vitro. We selected the L3.6pl and MiaPaCa-2 cell lines for these experiments because we had previously shown that the L3.6pl cells are highly sensitive to bortezomib whereas the MiaPaCa-2 cells are highly resistant (16). We found that the drug combination inhibited docetaxel-induced DNA fragmentation and caspase activation in the MiaPaCa-2 cell line and had no additive effect in the L3.6pl cell line. The apparent antagonism in cells treated with bortezomib + docetaxel appeared to be caused by inhibition of cdk activation via stabilization of p21 and p27. Our results are consistent with recent work demonstrating that ErbB2-mediated up-regulation of p21 and phosphorylation of cdc2 on Tyr15 blocked taxol-induced cdc2 activation and apoptosis (37, 38). However, our findings contrast with those obtained in another recent study that concluded that bortezomib sensitized human ASPC1 pancreatic cancer cells to paclitaxel-induced apoptosis via a mechanism involving down-regulation of NFκB and consequent reductions in Bcl-XL expression (36). In our models, we detected no change in the level of Bcl-XL protein expression following exposure to bortezomib (data not shown) and the apparent discrepancy between our results and those obtained previously probably has to do with the fact that we used different cell lines. Nonetheless, when cell growth inhibition was measured using clonogenic assays, the bortezomib + docetaxel was significantly more effective than either single agent alone, consistent with the overall conclusion reached in the previous study (36). Clonogenic assays take into account all forms of permanent cell growth arrest (apoptosis, necrosis, senescence, etc.) and probably more closely mirror the conditions present in tumor xenografts. Our observation that bortezomib is a potent inhibitor of cdk activity, cell cycle progression, and S phase in both MiaPaCa-2 and L3.6pl pancreatic cancer cells is consistent with our observations in other human tumor cells (prostate cancer cells; 32) and bladder cancer cells (Kamat et al., in press).

We next evaluated the effects of the bortezomib + docetaxel on the growth of established orthotopic L3.6pl and MiaPaCa-2 xenografts in vivo. Because the L3.6pl cell line has been cycled through mice to produce larger and more metastatic tumors (22), the L3.6pl tumors grow more rapidly than the MiaPaCa-2 tumors but they are also more sensitive to bortezomib alone (16). We treated tumors with bortezomib, docetaxel, or both agents for 14 days and measured various parameters of cell growth arrest and apoptosis using highly quantitative immunohistochemical and immunofluorescence-based assays. Consistent with the results obtained in the clonogenic assays, the drug combination produced significant reductions in tumor size in both xenograft models compared with controls or single-agent therapy. Combination therapy also stimulated significant increases in tumor cell apoptosis and significant decreases in proliferation. In vivo and in vitro exposure to bortezomib (but not docetaxel) resulted in marked accumulation of p21, indicating that the drugs inhibit tumor cell proliferation via complementary mechanisms that appear to act at different points in the cell cycle. It is important to point out that the differences in bortezomib's effects on apoptosis in the L3.6pl versus MiaPaCa-2 tumors were probably due in part to differences in growth rates and tumor volumes at the initiation of therapy.

In previous studies, our laboratory and another have demonstrated that bortezomib possesses strong antiangiogenic properties (16, 17, 32). It has also been reported that docetaxel inhibits angiogenesis and is synergistic with other antiangiogenic agents (20). Considering this common mechanism, we evaluated the effects of the bortezomib + docetaxel on various parameters of angiogenesis in the L3.6pl and MiaPaCa-2 xenografts. Single-agent therapy with either bortezomib or docetaxel produced reductions in tumor MVDs, and combination therapy reduced MVDs even further. In previous studies, we have demonstrated that angiogenesis inhibition is often associated with increases in tumor endothelial cell death that can be observed as early as 3 days or as late as 8 weeks after the initiation of therapy and remain elevated until the experimental end point (25, 28). Two-color immunofluorescence/LSC analysis of tumor endothelial cell death demonstrated that combination therapy also produced significant increases in steady-state levels of apoptosis compared with the levels observed in control tumors or in tumors treated with either bortezomib or docetaxel alone. In the future, we plan to conduct careful kinetic analyses with bortezomib and docetaxel to determine whether these increases in endothelial cell death occur as a direct result of drug exposure or are related to indirect effects on angiogenic factor production because angiogenic cytokines act as survival factors for tumor-associated endothelial cells. Consistent with the possible involvement of the latter, analysis of tumor angiogenic factor production revealed that the combination therapy produced strong reductions in VEGF expression. Down-regulation of VEGF probably resulted from direct effects of the drugs on tumor cells because exposure of L3.6pl or MiaPaCa-2 cells to bortezomib + docetaxel in vitro also resulted in significant inhibition of VEGF secretion. The combined antiangiogenic properties of bortezomib and docetaxel may explain the discrepancies observed between our in vitro and in vivo tumor cell apoptosis data because the death of endothelial cells and reduction in MVD should trigger increased apoptosis in the tumor cells (28). VEGF expression has been associated with an increase in tumor vasculature and a decrease in pancreatic cancer patient survival (39, 40). Therefore, the effects of the bortezomib + docetaxel on VEGF-mediated angiogenesis could prove to be beneficial in patients with pancreatic cancer.

Several different agents, such as flavopiridol, bryostatin-1, and cisplatin, cause cell cycle-mediated drug resistance when administered to tumor cells before or concurrently with paclitaxel treatment (41–44). Similar to bortezomib, flavopiridol inhibits cdk2 and cdc2, preventing the cells from progressing into M phase where the taxanes are active. However, flavopiridol enhanced caspase activation and apoptosis when it was administered after paclitaxel treatment (41). In our study, bortezomib and docetaxel were given simultaneously, but we have also conducted preliminary in vitro studies to determine the effects of schedule on bortezomib-mediated interference with docetaxel in vitro (data not shown). When cells were treated with bortezomib prior to docetaxel, bortezomib blocked docetaxel-induced M-phase arrest and apoptosis in a manner analogous to what we had observed with concomitant administration of the drugs. In contrast, administration of bortezomib after docetaxel overcame the cell cycle interference but still failed to increase levels of tumor cell apoptosis beyond what was observed with docetaxel alone. Because bortezomib has complex effects on cell proliferation, apoptosis, and angiogenesis, adoption of a different schedule would produce stronger in vivo antitumoral activity than was observed in the present study. Considering the strong effects of the bortezomib + docetaxel on tumor growth and angiogenesis inhibition, our data support planned clinical trials to evaluate the efficacy of bortezomib/taxane combination chemotherapy in patients with pancreatic cancer and other solid malignancies.

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.

Grant support: National Cancer Institute U54 CA 090810 and P50 CA101936.

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