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Experimental Therapeutics |
Department of Surgery, University of South Florida, Tampa, Florida 33612 [M. H., A. S. R.]; Department of Surgical Oncology, Ohio State University, Columbus, Ohio 43210 [E. E. Z.]; and James A. Haley Veterans Affairs, Tampa, Florida 33612 [A. S.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The impact of conventional chemotherapy, such as 5-fluorouracil, on pancreatic cancer has been minimal (2) . Approved by the United States Food and Drug Administration only several years ago, gemcitabine has begun to play a fundamental role in the chemotherapeutic approach to pancreatic cancer (3) . Nonetheless, results with chemotherapy, including gemcitabine, are not significantly remarkable (4) .
Pancreatic cancers express a relatively high concentration of MMPs2 (5) . These enzymes participate in basement membrane and peritumoral stromal degradation and angiogenesis, critical aspects of cancer growth and invasion (6, 7, 8) . Trial studies with the MMP inhibitor have suggested that MMP inhibition prolongs survival in a murine model of human pancreatic cancer, and these inhibitors are well tolerated by humans with pancreatic cancer (9 , 10) . Additional studies have shed light on the pathways and implications of MMP inhibition in pancreatic cancer (11) and further promote this treatment approach.
This study was undertaken in an established murine orthotopic model of human pancreatic cancer to confirm that MMP inhibition prolongs survival. This study was also designed to compare MMP inhibition with gemcitabine therapy and determine the impact of combination therapy involving both gemcitabine and MMP inhibition. Our hypotheses in undertaking this study were that MMP inhibition using BB-94 in a murine model of human pancreatic cancer would improve survival, would be superior to gemcitabine therapy, and would augment gemcitabine therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gemcitabine (chemically, difluorodeoxycytidine) is a nucleoside analogue with a wide antitumor activity in murine models of solid tumors (13) . After intracellular phosphorylation, the drug competes with deoxycytidine triphosphate for incorporation into DNA (14 , 15) , which in turn inhibits DNA synthesis (16) . The drug also appears to inhibit DNA synthesis by inhibition of ribonucleotide reductase (16) . Gemcitabine (Gemzar; Eli Lilly & Co., Indianapolis, IN) came in a suspension (1 g of gemcitabine hydrochloride, 1 g of manitol, and 62.5 mg of sodium acetate). Hydrochloric acid and/or sodium hydroxide may have been used to adjust the pH value. Gemcitabine was administered at 120 mg/kg after dilution with 0.9% sodium chloride (2.4 mg/mouse/injection i.p.) every third day from 7 days after orthotopic injection until death or sacrifice.
Pancreatic Cancer Cell Line.
HPAC (ATCC CRL-2119) is a moderately differentiated human
adenocarcinoma of ductal origin (17)
. HPAC cells were
grown in DMEM:F12–4F5, which is a mixture of DMEM and Ham’s F-12
nutrient medium (1:1) containing 1.2 g/liter
NaHCO3 and 15 nM HEPES (pH 7.3; Life
Technologies, Inc., Gaithersburg, MD). This culture medium is
supplemented with six factors (5% fetal bovine serum, 2 µg/ml
insulin, 5 µg/ml transferrin, 40 ng/ml hydrocortisone, 10 ng/ml
epidermal growth factor, and 1x antibiotic/antimycotic mixture). Cell
cultures were incubated at 37°C in a humidified atmosphere of 5%
CO2-enriched air, and medium was exchanged every
3 days according to the protocol.
Xenografts.
Ten million HPAC cells were injected at celiotomy into the head of
pancreata of BALB/c nu/nu mice (Life Sciences, Inc., St.
Petersburg, FL) as described previously (9)
. All mice were
maintained under identical conditions. Seven days after celiotomy and
intrapancreatic tumor injection, the mice were randomly assigned to one
of four treatment groups: (a) vehicle control
(n = 26); (b) BB-94
(n = 27); (c) gemcitabine
(n = 28); or (d) combination
therapy involving gemcitabine and BB-94 (n =
24). Preterminal mice and mice alive at 84 days were sacrificed, and
their tumors were harvested, weighed, and snap-frozen for MMP analysis.
Protein Extraction.
Tumor tissue (100 mg) from each mouse was individually homogenized at
4°C in 0.1 M Tris buffer (pH 8.1) containing protease
inhibitors (100 µg/ml aprotinin, 200 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin; Sigma). The
homogenate was sonicated for 1 min at 4°C and then spun in an
ultracentrifuge at 100,000 x g for 45 min at
4°C. The supernatant was collected, and protein concentrations were
determined using the Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules,
CA).
Affinity Chromatography.
Gelatin-Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) was washed
three times with equilibration buffer [50 mM Tris, 150
mM NaCl2, 0.02% Tween 20, 0.7% Brij
35, and 10 mM EDTA (pH 7.5)]. Twenty µl of equilibrated
gelatin-Sepharose 4B were added to 1600 µg of tumor protein extracts
and diluted to a final volume of 1 ml with PBS. Samples were then
placed on an end-over-end shaker overnight at 4°C to allow the
binding of gelatinases to the gelatin-Sepharose. After overnight
shaking, nonspecific bound proteins were washed from gelatin-Sepharose
with salted equilibration buffer (50 mM Tris, 200
mM NaCl, 5 mM CaCl2,
0.02% Tween 20, 0.07% Brij 35, and 10 mM EDTA). MMPs were
eluted from the washed gelatin-Sepharose by adding 15 µl of
nonreducing Laemmli solution.
Gelatin Zymography.
The affinity-purified samples (diluted 1:4) were electrophoretically
separated on an 8% SDS-PAGE gel impregnated with gelatin (1 mg/ml).
After incubation, the gels were rinsed twice in 2.5% Triton X-100 and
three times in double-distilled H2O. The gels
were then incubated at 37°C for 4 h in buffer [200
mM NaCl, 10 mM CaCl2,
0.07% Brij 35, and 50 mM Tris-HCl (pH 7.4)]. The
gels were stained with 0.05% Coomassie Brilliant Blue and destained in
10% acetic acid in H2O. Gelatinolytic enzymes
were detected as transparent bands on the background of the Coomassie
Blue-stained gel. Relative enzyme activity was quantified by
densitometric analysis of the negatively stained bands. Latent MMP-2
(Mr 72,000), activated MMP-2
(Mr 62,000), latent MMP-9
(Mr 92,000), and activated MMP-9
(Mr 86,000) were identified by
comparing them with known gelatinolytic activities from conditioned
media of HT-1080 cells (18)
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ELISA.
Active levels of serum MMP-2 were determined by using an ELISA
technique (Amersham Pharmacia Biotech). With aseptic precaution, blood
was collected by cardiac puncture at the time of nude mice necropsy.
Clear serum was collected after centrifugation at 3000 rpm for 10 min
at 4°C. The experiment was done according to the recommended
procedure suggested by the company. One hundred µl of standards and
serum samples from the mice were incubated in 96-well microtiter plates
precoated with anti-MMP-2 antibody. Any MMP-2 present would be bound to
the wells, with other components of the sample being removed by washing
and aspiration. Endogenous free active MMP-2 was detected through
activation of the modified prodetection enzyme and the subsequent
cleavage of its chromogenic peptide substrate. The resultant color was
read at 405 nm wavelength for absorbance by a microtiter plate
spectrophotometer (Dynatech Laboratories Inc.). The concentration of
active MMP-2 in a given sample was determined by interpolation from a
standard curve.
Data and Statistical Analyses.
Relative band densities were determined using the UVP GDS 8000 gel
documentation system (UV Products, Upland, CA). Peak areas were
determined using GDS Image Analysis Software (UV Products) and compared
using the ANOVA test. Implantation rates and survival curves in mice
were compared using contingency tables, the log likelihood ratio test,
and the Wilcoxon test. Animal weights and tumor weights were averaged
for each group and compared using the Mann-Whitney U test.
MMP levels in tumors and serum were compared by using an ANOVA test of
repeated measures. Statistical analysis was undertaken using
TRUE-EPISTAT (Epistat Services, Richardson, TX). Data are reported as
the mean ± STD when appropriate. Significance was
accepted with 95% confidence.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Implantation of HPAC occurred in only 5 of 24 (21%) mice receiving
combination therapy of BB-94 and gemcitabine, significantly below the
implantation rates seen with either therapy alone
(P < 0.0001) or the implantation rates seen
with the other therapy groups combined (P <
0.0001, log likelihood ratio test; Fig. 1
).
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).
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).
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). Active MMP-9 levels were low, and
changes with therapy were small, although therapy did reduce the levels
(Fig. 4, A and B
). There was a significant
reduction in active form MMP-2 and latent form MMP-2 with therapy
(P < 0.05). Among mice receiving therapy,
differences in MMP levels were not significant (Fig. 4, A and B
). Similar previous results were confirmed by
Western blot from our laboratory (19)
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). Active form MMP-2 levels were lowest in mice receiving combination
therapy (Gem+BB-94).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The murine orthotopic model of human pancreatic cancer used in this study is well established for in vivo pancreatic tumor growth (9 , 20) . The BALB/c nude mice used were genetically inbred and have been widely used for xenograft studies. HPAC, the human pancreatic cancer cell line used in this series of experiments, has been studied for many years and is well established (7) . The MMP inhibitor BB-94 has been used for years in mice at the same dose (30 mg/kg) and route (i.p.) (12) that we used, as has gemcitabine in humans (21) , without species-specific complications or idiosyncrasies. Random assignment of therapy 7 days after orthotopic injection of HPAC cells removed potential treatment bias.
Both gemcitabine and BB-94 reduced rates of cancer implantation after injection at celiotomy, although neither reduction was significant. Combined therapy of BB-94 and gemcitabine dramatically reduced implantation, although therapy was initiated 7 days after tumor cell injection. This reduction was so profound that only a small number of mice receiving combination therapy were noted to have pancreatic cancer at necropsy after sacrifice. The implications of this seem profound, suggesting a role for combination therapy as an adjuvant therapy after pancreatic resections of curative intent, when the potential tumor burden should be very low.
The impact of therapy on survival was determined by analyzing only the survival of mice that were subsequently noted to have cancer at necropsy. Compared with vehicle control, both gemcitabine and BB-94 prolonged survival and improved the configuration of the survival curves, but not significantly. Conversely, combination therapy of gemcitabine and BB-94 significantly and dramatically impacted survival, regardless of how it was quantified or described. Consistent with improved survival, mice receiving combination therapy enjoyed an improved quality of life as evidenced by subjective measures such as shinier coats, increased energy and activity, and an overall healthier appearance and objective measures such as more optimal body weights and reduced tumor weights.
Of the 15 recognized MMPs, MMP-2 and MMP-9 are felt to be the most important in determining the aggressiveness of pancreatic cancer. We have shown that inhibition of MMP-2 seems to be most important in prolonging survival (20) . With this specific cancer cell line in this model, tumor MMP-2 levels were greater than MMP-9 levels. Changes in active form MMP-9 levels with therapy were small, although all three therapies reduced tumor MMP-9 levels compared with the vehicle control. This reduction was more evident with latent form MMP-9. Tumor MMP-2 levels were relatively higher than tumor MMP-9 levels, and there were significant reductions in both active and latent forms of MMP-2 with all therapies, but combination therapy seemed to be the best at reducing active MMP-2 levels.
Because tumor MMP levels were determined per standard weight of tumor, the smallest tumors had the least amount of MMP. Because combination therapy resulted in the smallest tumors, a broad assimilation of the data conveys that combination therapy with gemcitabine and BB-94 unquestionably produced the most notable reduction in total MMP levels. Consistent with this observation are the serum levels of active form MMP-2 among the four treatment groups. Combination therapy with gemcitabine and BB-94 resulted in the lowest serum level of active form MMP-2.
Compared with the vehicle control, gemcitabine reduces MMP levels by decreasing tumor growth kinetics and tumor size and thereby indirectly reduces tumor and serum MMP levels and activity commensurately. BB-94 directly inhibits MMP activity and zymogen activation (19) and thereby reduces tumor growth, size, and kinetic activity. Combined therapy with gemcitabine and BB-94 inhibits MMP activity and reduces tumor growth kinetics, thereby limiting tumor growth through two diverse mechanisms, resulting in the lowest MMP levels and the best outcomes. Moreover, similar results have been reported previously in an ovarian carcinoma xenograft when BB-94 was administered in adjuvant therapy with a different cytotoxic agent, namely, cisplatin (22) .
Gemcitabine has become well established in the treatment of pancreatic cancer (4) . MMP inhibition, however, remains to be established, although early trials suggest a clinical role (23 , 24) that remains to be defined. From this preclinical study, it seems that combination therapy of MMP inhibition and cytotoxic therapy holds promise in the treatment of pancreatic cancer. This seems especially true under the circumstance of relatively minimal tumor burden. A clinical trial using marimastat to compare MMP inhibition with gemcitabine in patients with unresectable pancreatic cancer has completed enrollment, and the results will soon be forthcoming. A trial comparing gemcitabine and marimastat with gemcitabine alone in patients with unresectable pancreatic cancer has completed enrollment, and follow-up continues. Results from this clinical trial may be known within 1 year.
This preclinical study gives us hope that MMP inhibition will significantly prolong the survival of pancreatic cancer patients as compared with that seen in patients treated with cytotoxic therapy alone and begins to elucidate mechanisms that may result in prolonged survival.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Surgery, University of South Florida,
P. O. Box 1289, Room F145, Tampa General Hospital, Tampa, Florida
33601. Phone: (813) 251-7393; Fax: (813) 253-1920. 
2 The abbreviation used is: MMP, matrix
metalloproteinase.
Received 9/20/99. Accepted 4/10/00.
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,
implantation; 
, no implantation. There was a significant reduction
of implantation in mice treated with combination therapy
(Gem+BB-94; P < 0.0001,
Wilcoxon test). Gem, gemcitabine. Control group
(n = 26), implantation seen in 21 of 26
mice; BB-94 (n = 27), implantation seen
in 19 of 27 mice; gemcitabine (n = 28),
implantation seen in 21 of 28 mice; gemcitabine + BB-94
(n = 24), implantation seen in 5 of 24
mice.
