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[Cancer Research 62, 7343-7349, December 15, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Simultaneous Reduction in Cancer Pain, Bone Destruction, and Tumor Growth by Selective Inhibition of Cyclooxygenase-21

Mary Ann C. Sabino, Joseph R. Ghilardi, Joost L. M. Jongen, Cathy P. Keyser, Nancy M. Luger, David B. Mach, Christopher M. Peters, Scott D. Rogers, Matthew J. Schwei, Carmen de Felipe and Patrick W. Mantyh2

Neurosystems Center and Departments of Preventive Sciences, Psychiatry, Neuroscience, and Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455 and VA Medical Center, Minneapolis, Minnesota 55417 [M. A. C. S., J. R. G., J. L. M. J., C. P. K., N. M. L., D. B. M., C. M. P., S. D. R., M. J. S., P. W. M.], and Instituto de Neurociencias, Universidad Miguel Hernandez-Consejo Superior de Investigaciones Cientificas, Elehe 03202, Spain [C. d. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
More than half of all chronic cancer pain arises from metastases to bone, and bone cancer pain is one of the most difficult of all persistent pain states to fully control. Several tumor types including sarcomas and breast, prostate, and lung carcinomas grow in or preferentially metastasize to the skeleton where they proliferate, and induce significant bone remodeling, bone destruction, and cancer pain. Many of these tumors express the isoenzyme cycloxygenase-2 (COX-2), which is involved in the synthesis of prostaglandins. To begin to define the role COX-2 plays in driving bone cancer pain, we used an in vivo model where murine osteolytic 2472 sarcoma cells were injected and confined to the intramedullary space of the femur in male C3HHeJ mice. After tumor implantation, mice develop ongoing and movement-evoked bone cancer pain-related behaviors, extensive tumor-induced bone resorption, infiltration of the marrow space by tumor cells, and stereotypic neurochemical alterations in the spinal cord reflective of a persistent pain state. Thus, after injection of tumor cells, bone destruction is first evident at day 6, and pain-related behaviors are maximal at day 14. A selective COX-2 inhibitor was administered either acutely [NS398; 100 mg/kg, i.p.] on day 14 or chronically in chow {MF. tricyclic; 0.015%, p.o.} from day 6 to day 14 after tumor implantation. Acute administration of a selective COX-2 inhibitor attenuated both ongoing and movement-evoked bone cancer pain, whereas chronic inhibition of COX-2 significantly reduced ongoing and movement-evoked pain behaviors, and reduced tumor burden, osteoclastogenesis, and bone destruction by >50%. The present results suggest that chronic administration of a COX-2 inhibitor blocks prostaglandin synthesis at multiple sites, and may have significant clinical utility in the management of bone cancer and bone cancer pain.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metastasis to bone is a common sequel of malignant tumors, and is associated with significant complications including severe pain, skeletal fractures, bone marrow suppression, hypercalcemia, and an overall reduced quality of life (1) . Bone metastases are the most common indication that breast, prostate, or lung cancers have spread beyond the primary organ, and metastases of these tumors to bone are generally associated with a poorer prognosis (2) . Pain is the most frequent presenting symptom indicating tumor metastasis to bone (3) . In general, there are two types of pain in patients with bone cancer. The first type is known as ongoing pain and is usually described as a dull aching or throbbing pain that increases in severity over time (3) . A second type of bone cancer pain, known as movement-evoked, breakthrough, or episodic pain, emerges frequently over time, is more acute in nature, and often occurs as spontaneous and intermittent exacerbations of pain or by movement of the cancerous bone (4) . Breakthrough pain represents one of the most serious and highly debilitating complications of cancer, as it frequently can be difficult to effectively manage.

The mechanisms that underlie tumor-induced bone cancer pain have only begun to be elucidated. We developed previously an animal model of bone cancer pain by injecting osteolytic murine sarcoma cells into the mouse femur (5) . Using this model we demonstrated that blocking tumor-induced bone resorption resulted in a reduction in ongoing and movement-evoked pain behaviors and spinal neurochemical changes reflecting both peripheral and central sensitization (6 , 7) . Whereas this and other work suggest that tumor-induced bone resorption plays a role in driving bone cancer pain, other mechanisms, such as the release of pronociceptive compounds (e.g., prostaglandins) by tumor and/or inflammatory cells may also be involved (8) .

To test this hypothesis, we have focused on prostaglandins, as they have been implicated in a number of biological and pathological processes including mediating pain and inflammation (9, 10, 11) , bone homeostasis (12, 13, 14) , and tumorigenesis (15, 16, 17, 18) . Prostaglandins are lipid-derived eicosanoids that are synthesized from arachidonic acid by COX3 isoenzymes COX-1 and COX-2. NSAIDs inhibit both COX-1 and COX-2, and whereas they are clinically effective in attenuating acute nonmalignant skeletal pain, NSAIDs are generally not indicated for extended use in cancer patients as they have significant side effects such as gastrointestinal ulceration, neutropenia, enhanced bleeding, and disruptions in renal function (19 , 20) . As selective COX-2 inhibitors have significantly fewer side effects than mixed NSAIDs, and thus could potentially be used for extended periods in patients with cancer, a critical question is what effects selective COX-2 inhibitors have in an in vivo model of bone cancer pain.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Induction of Bone Cancer.
Experiments were performed on 39 adult male C3H mice (The Jackson Laboratory, Bar Harbor, Maine), weighing 18–20 g. The mice were housed in accordance with NIH guidelines, and all of the procedures were approved by the Animal Care and Use Committees at the University of Minnesota. Injection of tumor cells were performed as described previously (5, 6, 7 , 21) . After induction of general anesthesia with sodium pentobarbital (50 mg/kg, i.p.), an arthrotomy was performed exposing the condyles of the distal femur. HBSS (Sigma, St. Louis, MO; sham, n = 8) or medium containing 105 osteolytic murine sarcoma cells (20 µl, NCTC 2472; American Type Culture Collection, Rockville, MD; tumor, n = 16) was injected into the intramedullary space of the mouse femur and the injection site sealed with dental amalgam.

Treatment with Selective COX-2 Inhibitor.
Mice were randomly placed into two treatment groups receiving either regular diet (sham + vehicle: n = 4; sarcoma + vehicle: n = 9) or diet containing MF tricyclic (0.015% or ~30 mg/kg/day; Merck and Co., Kirkland, Quebec, Canada; sham + MF tricyclic: n = 4; sarcoma + MF tricyclic: n = 9) beginning 6 days after injection. MF tricyclic is a selective COX-2 inhibitor, which demonstrates >3000-fold selectivity for COX-2 than COX-1 (22) and is more selective than either celecoxib or rofecoxib (23) . An established dose-response relationship for MF tricyclic and inhibition of tumorigenesis has been described previously (22 , 24) , and the dose we used was without observable side effects (23 , 25) . Initiation of treatment was based on the time at which observable bone destruction begins (6) and was terminated at 14 days after injection when there was significant bone destruction. Terminal bleeds were performed, and plasma was sent for analysis (Merck and Co.). To test the effects of acute COX-2 inhibition on bone cancer pain, sham (n = 6) or sarcoma-injected mice (n = 9) were administered the selective COX-2 inhibitor NS-398 (100 mg/kg, p.o.; Sigma) or vehicle (DMSO, p.o.; Sigma) 14 days after tumor or vehicle injection, and then behaviorally tested 30 min later. NS-398 was used in acute studies because of the unavailability of MF tricyclic in powdered, nonchow form at that time.

Pain-related Behaviors.
All of the mice were tested for pain-related behaviors 14 days after sham or tumor injections. Ongoing and movement-evoked pain behaviors were analyzed as described previously (7) . Quantification of spontaneous and palpation-induced flinches over a 2-min observation period were used to measure ongoing and palpation-evoked pain, respectively.

Pain because of ambulation was evaluated using tests validated previously (7) . Normal limb use during normal ambulation was scored on a scale of 4 to 0: (4) normal use and (0) complete lack of limb use. Forced ambulatory guarding was determined using a rotarod (Columbus Instruments, Columbus, OH) and was rated on a scale of 5 to 0: (5) normal use and (0) complete lack of use. To monitor the general health of the animal and to ensure consistency in food consumption, weights were recorded at the beginning and end of the experiment.

Bone Destruction and Osteoclastogenesis.
Radiographs (Faxitron X-ray Corporation, Wheeling, IL) of femora were obtained at 14 days to assess bone destruction. The extent of bone destruction was assessed at x4 magnification as described previously (6 , 7) using a 0 to 5 scale: (0) normal bone with no signs of destruction and (5) full thickness bicortical bone loss and displaced skeletal fracture. Right (internal control) and left (tumor-bearing) femora were fixed in 4% zinc-buffered formalin at 4°C overnight, decalcified in 10% EDTA (pH 7.4) for 2 weeks, and embedded in paraffin. Femoral sections 7-µm thick were cut in the frontal plane and stained with tartrate-resistant acid phosphatase and H&E to visualize activated osteoclasts. Osteoclasts were counted as described previously (6) .

Tumor Burden.
Three different manners to assess tumor burden were used in the present study. Sarcoma cancer cells were genetically manipulated to express enhanced GFP to visualize tumor cells as described previously (26) . Dissected femora containing GFP-expressing sarcoma cells were illuminated using a light source and a blue 470/40 nm excitation filter (Chroma Technology, Brattleboro, VT). Fluorescence area and intensity were measured using Image Pro Plus v.3.0 software (Media Cybernetics, Silver Spring, MD) and were expressed as IODs. Femoral sections 7-µm thick were stained with either conventional H&E to visualize normal marrow elements and sarcoma cells under bright field microscopy or with an antibody raised against GFP to visualize sarcoma cells using fluorescence microscopy. Tumor mass and intramedullary space were then selected and measured using image analysis, and results are expressed as percentage of intramedullary space occupied by tumor.

The tumor characteristics of sarcoma cells transfected with GFP such as growth rates, rate of bone resorption, and the ability to induce bone cancer-related pain behaviors, were temporally, behaviorally, and physically identical to nontransfected sarcoma cells.

Immunohistochemistry and Quantification.
Mice were sacrificed and processed for immunohistochemical analysis of bone marrow aspirates and spinal cord as described previously (6 , 7) . Animals received a normally non-noxious mechanical stimulation of the injected knee 1.5 h and 5 min before euthanasia for visualization of c-Fos and SPR expression, respectively (27) . Spinal cord segments L1-L5 were removed, postfixed, and cryoprotected in 30% sucrose. Serial frozen spinal cord sections, 60-µm thick, were cut on a sliding microtome, collected in phosphate buffered saline, and processed as free-floating sections. Bone marrow aspirates of sham and tumor-bearing femora were smeared onto glass slides to air dry. COX-2 expression was determined using an antibody raised against mouse COX-2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Spinal cord markers included c-Fos protein (1:20,000; Oncogene Research, San Diego, CA), SPR (1:5,000; raised in our laboratory), dynorphin (1:1,000; Neuromics, Minneapolis, MN), and GFAP (1:800; Dako, Carpinteria, CA). To confirm the specificity of the primary antibody, controls included were preabsorption with the corresponding synthetic peptide or omission of the primary antibody.

Fluorescent images were obtained using either an MRC 1024 confocal imaging system (Bio-Rad, Philadelphia, PA) or a SPOT II digital camera on an Olympus BX-60 fluorescence microscope with Image Pro Plus software. Fluorescence intensities of GFAP staining were expressed as percentage of increase in fluorescence relative to sham levels. The number of c-Fos-IR and dynorphin-IR neurons were expressed as mean number of neurons per section. The number of SPR internalized neurons within the superficial laminae (laminae I-II) of the spinal cord was expressed as percentage of internalization within SPR+ neurons. An internalized neuron was defined as having >10 endosomes within the soma of SPR+ neurons. All of the results were obtained from 10 randomly selected L3 coronal sections per animal.

Statistical Analysis.
A one-way ANOVA was used to compare parameters between the experimental groups. For multiple comparisons, the Fisher’s protected least significant difference post hoc test was used. An unpaired Student’s t test was used for tumor burden analyses. Results were considered statistically significant at P < 0.05. In all of the cases, the investigator was blind to the experimental status of each animal.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selective COX-2 Inhibition Significantly Reduces Bone Cancer Pain-related Behaviors.
Fourteen days after tumor implantation, sarcoma-bearing mice that received regular diet (sarcoma + vehicle) exhibited an increased number of spontaneous flinches as compared with sham controls (P < 0.0001), a reflection of ongoing cancer pain (Fig. 1A)Citation . These mice also exhibited pain-induced impairment of their limbs as evidenced by a reduction in limb use during normal ambulation (P < 0.001 versus sham; Fig. 1BCitation ) and enhanced guarding of the sarcoma-bearing limb during forced ambulation (P < 0.002 versus sham; Fig. 1CCitation ), both a reflection of ambulatory pain. Sarcoma + vehicle mice also exhibited a greater number of palpation-evoked flinches as compared with shams (P < 0.0001), a measure of palpation-evoked pain (Fig. 1D)Citation . Acute administration of the selective COX-2 inhibitor NS-398 reduced the number of spontaneous and palpation-evoked flinches (P < 0.0001 versus sarcoma + vehicle) and improved limb use score (P < 0.05 versus sarcoma + vehicle). However, guarding during forced ambulation did not improve. Chronic treatment of sarcoma-bearing mice with the selective COX-2 inhibitor MF tricyclic (sarcoma + chronic COX-2 inhibitor) significantly reduced all of the bone cancer pain behaviors examined (P < 0.005 versus sarcoma + vehicle; Fig. 1, A–DCitation ).



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  		Fig. 1. Selective COX-2 inhibition attenuates ongoing and movement-evoked bone cancer pain behaviors. The number of spontaneous flinches of the cancerous limb over a 2-min observation period was used as a measure of ongoing pain (A). Parameters of movement-evoked pain include assessment of the sarcoma-bearing limb during normal ambulation in an open field (B) and the extent of guarding of tumor-bearing limb during forced ambulation on a rotarod (C; both are measures of ambulatory pain). Quantification of the number of flinches evoked by normally non-noxious palpation of the sarcoma-bearing limb over a 2-min observation period following palpation (D) was used as a measure of palpation-evoked pain. Whereas acute inhibition of COX-2 reduced some pain behaviors 14 days after sarcoma injection, note that all pain behaviors were significantly reduced with chronic COX-2 inhibition; bars, ± SE. *P < 0.05 versus sham; #P < 0.05 versus sarcoma + vehicle group (one-way ANOVA and Fisher’s PLSD).

 
COX-2 Inhibition Reduces Peripheral Sensitization of Sensory Neurons.
Nociceptive or painful conditions have been associated with increases in the number of c-Fos-IR neurons in the spinal cord (21 , 27 , 28) . The expression of c-Fos in the superficial dorsal horn (laminae I-II) has been used as a marker of sensitization of primary afferent terminals in naïve (27, 28, 29, 30) , inflammatory (21 , 31) , and sarcoma-induced bone cancer pain (5, 6, 7 , 21) states. Normal, non-noxious palpation of uninjured tissues resulted in minimal expression of c-Fos in superficial laminae (Fig. 2ACitation ; Table 1Citation ). In the bone cancer state, sarcoma + vehicle mice exhibited an increased number of c-Fos-IR neurons (P < 0.05 versus sham; Fig. 2CCitation ), and chronic treatment with a COX-2 inhibitor significantly reduced this expression to sham levels (Fig. 2ECitation ; Table 1Citation ).



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  		Fig. 2. Neurochemical changes associated with peripheral and central sensitization are attenuated by chronic inhibition of COX-2. Representative camera lucida tracings of neurons expressing c-Fos protein (A, C, and E) and confocal images of neurons expressing dynorphin (B, D, and F) in representative 60-µm sections of the L3 segment of the spinal cord in sham-injected mice (A and B), sarcoma-bearing mice maintained on regular rodent diet (sarcoma + vehicle; C and D), or diet containing the COX-2 inhibitor MF-tricyclic (sarcoma + MF; E and F). After normally non-noxious palpation of tumor-bearing limbs, sarcoma + vehicle mice showed increased expression of c-Fos protein in neurons within the superficial and deep laminae (C, dashed lines separate laminae) and expression of the prohyperalgesic peptide dynorphin in neurons within the deep dorsal horn of the spinal cord (D, arrows). In sarcoma animals that received chronic administration of a COX-2 inhibitor, there was a marked reduction of the expression of c-Fos protein and dynorphin (E and F). Spinal cord confocal images were constructed from a collection of six scans obtained from 60-µm thick tissue sections acquired with a x40 lens. Scale bar = 100 µm.

 

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  		Table 1 Markers of peripheral and central sensitization are attenuated by chronic COX-2 inhibition

Summary of changes associated with the sensitization of primary afferent fibers, and the sensitization of the spinal cord involved in the conduction of pain information in sham, sarcoma + vehicle, and sarcoma + chronic COX-2 inhibitor-treated mice 14 days after sham or sarcoma injections. For peripheral sensitization, the results are expressed as the mean number of c-Fos-expressing neurons in laminae I–II and the percentage of SPR-expressing neurons in lamina I that show internalization in 60-µm sections of spinal cord at the L3 spinal segment after normally non-noxious mechanical stimulation (palpation) of the tumor-bearing femur. To assess central sensitization, we quantified the mean number of c-Fos-expressing neurons in laminae III–VI of the spinal cord, the number of dynorphin-IR neurons in laminae III–VI of the spinal cord, and the immunofluorescence levels of the astrocyte marker GFAP in spinal laminae I–X in 60-µm sections of spinal cord at the L3 spinal segment. Sham animals received injection of tissue culture medium alone, sarcoma + vehicle animals received injection of sarcoma cells and were maintained on regular rodent diet (vehicle), and sarcoma + chronic COX-2 inhibitor received rodent diet containing the COX-2 inhibitor MF tricyclic. Values represent mean ± SE from 10 60-µm thick sections within the L3 segment of the spinal cords from each animal, at least 4 animals per treatment group. Chronic MF tricyclic treatment reduced the expression of all spinal markers except for GFAP which remained unchanged.

 
Similarly, the internalization of the SPR has been used as a marker of activation and sensitization of substance P-containing primary afferent fibers (5 , 32, 33, 34) . Normally non-noxious palpation of sham-operated femora resulted in SPR internalization within few SPR-expressing (SPR+) neurons in lamina I of the spinal cord. Fourteen days after tumor implantation, non-noxious palpation of the tumor-bearing limb resulted in an increase in SPR internalization (P < 0.05 versus sham), which was reduced with chronic COX-2 inhibition (Table 1)Citation .

COX-2 Inhibitor Reduces Bone Cancer-induced Central Sensitization in the Spinal Cord.
Spinal markers of central sensitization used in the current experiment include deep spinal expression (laminae III-VI) of c-Fos and dynorphin, and astrocyte expression of GFAP (5, 6, 7 , 21) . Expression of c-Fos has been noted previously in animals with bone cancer pain (5) . In sham-operated and naïve mice, relatively few c-Fos-IR neurons were observed in laminae III-VI (Fig. 2A)Citation , whereas the number of c-Fos-IR neurons increased significantly in sarcoma + vehicle mice 14 days after tumor implantation (P < 0.0001 versus sham; Fig. 2CCitation ). Chronic COX-2 inhibition significantly reduced the number of c-Fos-expressing neurons (Fig. 2ECitation ; Table 1Citation ).

Spinal expression of dynorphin, a member of the opioid family, which has been shown to induce pain (35, 36, 37) , has been observed in various persistent pain states (5 , 38 , 39) . In sham-operated mice, few spinal neurons expressed dynorphin in deep spinal laminae (Fig. 2B)Citation . Increased expression of dynorphin in laminae III-VI was observed in sarcoma + vehicle mice (P < 0.005 versus sham; Fig. 2DCitation ), and this expression was reduced with chronic COX-2 inhibition (Fig. 2FCitation ; Table 1Citation ).

In previous studies, an ipsilateral hypertrophy of astrocytes labeled with GFAP was observed after implantation of sarcoma cells into mouse femora (5, 6, 7 , 21) . Similarly, gliosis was observed 14 days after tumor injection in the present study. Unlike other markers of peripheral and central sensitization, GFAP expression was not altered by chronic treatment of sarcoma-bearing mice with MF tricyclic (Table 1)Citation .

Selective COX-2 Inhibition Reduces Tumor-induced Osteoclast Proliferation and Hypertrophy.
Sham-injected mice did not demonstrate significant bone destruction (mean bone score of 0.5 ± 0.3; Fig. 3ACitation ). In sarcoma + vehicle mice, extensive bone destruction characterized by multifocal radiolucencies was observed (mean score of 2.8 ± 0.3; P < 0.0007 versus sham + vehicle mice) 14 days after tumor implantation (black defects highlighted by arrow; Fig. 3BCitation ). Chronic treatment of tumor-bearing mice with a COX-2 inhibitor markedly reduced the excessive resorption of bone (mean score of 1.3 ± 0.4; P < 0.005 versus sarcoma + vehicle; Fig. 3CCitation ).



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  		Fig. 3. COX-2 promotes tumor-induced bone destruction and osteoclastogenesis. High-resolution anterior-posterior radiographs of sham-injected (A) and sarcoma-injected mice that received regular rodent diet (B) or diet containing a COX-2 inhibitor (C). Fourteen days after sarcoma implantation, extensive bone resorption shown as black defects was observed in the femora of sarcoma-bearing mice (B, arrow), whereas sarcoma-bearing mice treated with a COX-2 inhibitor showed reduced bone destruction as demonstrated by the persistence of mineralized bone (C, white lattice structure). Dental grade amalgam (A–C, dense white plug at the ends of bone) was used to confine tumor cells within bone. Digital photomicrographs of distal femoral sections 7-µm thick that were stained with tartrate-resistant acid phosphatase (dark staining in B1 and C1) and H&E (B2 and C2) to visualize number and size of osteoclasts, respectively. Bones of sarcoma-bearing mice demonstrated an increased proliferation (B1, arrows) and hypertrophy (B2, arrows) of osteoclasts. Note that chronic treatment of sarcoma-bearing mice with a COX-2 inhibitor markedly reduced the number (C1 versus B1) and size (C2 versus B2) of osteoclasts. Scale bar = 1.5 mm in A–C, 150 µm in B1 and C1, and 40 µm in B2 and C2.

 
The increased resorption in the tumor-bearing bone was in large part because of sarcoma-induced osteoclast proliferation and hypertrophy (40) . Fourteen days after tumor injection, there was a significant increase in the number of activated osteoclasts (100 ± 8 osteoclasts/mm2; Fig. 3B1Citation ) and size of osteoclasts (expressed as the number of osteoclasts that exhibit >3 nuclei; Ref. 41 ; 38 ± 6.4 osteoclasts/mm2 had >3 nuclei; Fig. 3B2Citation ) as compared with sham animals (34 ± 10 osteoclasts/mm2; P < 0.05; and 9.6 ± 3.6 osteoclasts/mm2 had >3 nuclei; P < 0.005). Chronic COX-2 inhibition significantly reduced sarcoma-induced osteoclast proliferation (34 ± 6 osteoclasts/mm2; P < 0.005; Fig. 3C1Citation ) and hypertrophy (7.5 ± 0.7 osteoclasts/mm2 had >3 nuclei; P < 0.001; Fig. 3C2Citation ). Chronic treatment did not alter the number or size of osteoclasts in sham-injected mice as compared with naïve mice and, therefore, did not appear to affect normal bone homeostasis over the time period examined.

COX-2 Is Expressed by Tumor Cells, and COX-2 Inhibition Reduces Tumor Burden.
The expression of COX-2 has been demonstrated in multiple tumor cells (17 , 42) . In the present experiment, the expression of COX-2 was observed in sarcoma cells stably transfected with GFP obtained from bone marrow aspirates (Fig. 4, A–C)Citation or when cultured in vitro (Fig. 4, D–F)Citation . COX-2 was also expressed in a few cells that were presumably inflammatory cells from bone marrow aspirates of sarcoma-bearing femora (data not shown).



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  		Fig. 4. Expression of COX-2 by osteolytic 2472 sarcoma cells in vitro and in vivo. A and B are confocal images obtained from bone marrow aspirates in mice 14 days after sarcoma implantation, whereas C and D are confocal images of sarcoma cells grown in culture. A and C demonstrate COX-2 staining within sarcoma cells, and B and D show GFP fluorescence in sarcoma cells. Note that the great majority of COX-2 expressing cells also appear to correspond to GFP-expressing sarcoma cells (arrows). Confocal images of sarcoma cells were projected from four optical sections acquired at 0.8-µm intervals with a x60 lens. Scale bar = 25 µm in A–D.

 
The degree of tumor burden within femora was quantified by measuring the area and intensity of GFP fluorescence of whole bones (Fig. 5, B and C)Citation , and expressing results as IOD (IOD = area x intensity of fluorescence) and quantifying the area occupied by tumor within the intramedullary space using an antibody raised against GFP (Fig. 5, B1 and C1)Citation or standard H&E stain (Fig. 5, B2 and C2)Citation . Fourteen days after tumor implantation, sarcoma cells proliferated and completely infiltrated the intramedullary space of the mouse femur when whole bones were examined (IOD of 79.8 ± 11.4) and when femoral sections were analyzed (97.1 ± 2.1% of marrow space was occupied by tumor). Tumor burden was reduced by ~50% with chronic COX-2 inhibitor treatment at the whole bone (IOD of 49 ± 6.7; P < 0.05 versus sarcoma + vehicle; Fig. 5CCitation ) and sectioned bone levels (50.0 ± 6.9% of marrow space was occupied by tumor; P < 0.0003 versus sarcoma + vehicle; Fig. 5, C1 and C2Citation ) as demonstrated by the incomplete replacement of normal marrow (arrowheads) by tumor cells (*). Similar reductions in tumor burden after chronic COX-2 administration were obtained in femoral sections immunostained with a GFP antibody (data not shown).



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  		Fig. 5. Chronic inhibition of COX-2 reduces tumor burden in mice with bone cancer. Photomicrographs of the anterior aspect of a sarcoma-bearing femur 14 days after sarcoma injections (A). When the same bone in A was illuminated with a light source and band pass filters to visualize sarcoma cells transfected with GFP (B), GFP-expressing tumor cells had completely filled the intramedullary space. Femora in C were identical to B, except these animals received chronic treatment of a COX-2 inhibitor from day 6 to day 14 (C). Note the reduction in GFP fluorescence after chronic COX-2 inhibition (B versus C). Fluorescent photomicrographs of 7-µm thick tissue sections of the distal femur from sarcoma-bearing mice that received regular diet (B1) or diet containing a COX-2 inhibitor (C1) were stained with an antibody raised against GFP to label sarcoma cells. Serial 7-µm thick sections were stained with H&E to additionally define and confirm the presence of tumor cells within the intramedullary space (B2, C2, *). Note that animals that received chronic administration of a COX-2 inhibitor demonstrated a reduction in tumor burden (C, C1, and C2) as compared with vehicle-treated mice (B, B1, and B2). The presence of normal marrow cells in animals treated with the COX-2 inhibitor are delineated by arrowheads. Scale bar = 1.5 mm in A–C and 750 µm in B1, B2, C1, and C2.

 
No differences in terminal weights were observed between sham + vehicle (26 ± 0.8 g), sham + chronic COX-2 inhibitor (24 ± 0.8 g), sarcoma + vehicle (26 ± 0.5 g), or sarcoma + chronic COX-2 inhibitor (25 ± 0.7 g) animals. Mean plasma concentrations were obtained 14 days after tumor implantation in mice that received chronic COX-2 inhibitor (1.8 ± 0.05 µg/ml) and appeared consistent within treatment group (range, 1.79–1.89 µg/ml). The plasma levels in the present study were in the range where COX-2 inhibitors exert antitumorigenic effects (22 , 24) . At these drug and plasma concentrations, no observable side effects such as enhanced bleeding, ataxia, decreased food intake, anorexia, cachexia, or bone malformations were observed in the present study, which is consistent with previous studies (22 , 23 , 25 , 42) .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, chronic inhibition of COX-2 attenuated ongoing and movement-evoked cancer pain-related behaviors, and a major reason for the analgesic efficacy of COX-2 inhibition may be because of its simultaneous inhibition of PG synthesis at multiple sites in both the tumor-bearing bone and within the nervous system. Previous results have shown that tumor cells up-regulate COX-2 and COX-2 mediated synthesis of PGs (17 , 43 , 44) , and release of PGs by tumor cells would be expected to directly sensitize primary afferent neurons (45, 46, 47) innervating the tumor-bearing bone (48) . Additionally, expression of COX-2 by neurons in the spinal cord plays an important role in generating and maintaining hyperalgesia, as blockade of spinal COX-2 activity is effective in attenuating several chronic pain states (46 , 49, 50, 51) .

In animals with bone cancer pain, a stereotypic set of neurochemical changes occurs in the spinal cord that appears to reflect the peripheral and central sensitization of pathways involved in the generation and maintenance of bone cancer pain (5, 6, 7 , 21 , 52 , 53) . Thus, in the areas of the spinal cord that receive sensory input from the tumor-bearing bone, there is an up-regulation of the neuronal activation marker cFos, release of substance P from primary afferent neurons in response to normally non-noxious stimulation of the bone, an up-regulation of the prohyperalgesic peptide dynorphin, and hypertrophy of astrocytes. Chronic inhibition of COX-2 attenuated many of these neurochemical changes suggesting that COX-2 inhibition reduces both peripheral and central sensitization of sensory and spinal cord neurons that appear to be intimately involved in the generation and maintenance of bone cancer pain.

Two weeks after injection of the sarcoma cells into the intramedullary space of the femur, extensive tumor-induced osteolysis is observed throughout the femur. Chronic inhibition of COX-2 resulted in a significant reduction in the proliferation and hypertrophy of osteoclasts and a reduction in tumor-induced bone resorption. Osteoclasts resorb bone by maintaining an acidic extracellular microenvironment (pH 4.0–5.0) at the osteoclast-mineralized bone interface (54) . As osteolytic bone cancers induce an increase in the number and size of activated osteoclasts (40) , this osteoclast-induced increase in acidity may activate acid-sensing ion channels/receptors, such as the vanilloid receptor 1, that are expressed by sensory neurons that innervate the bone (11 , 52) . As bone resorption occurs, growth factors that are embedded in mineralized bone are released (55) , and many of the growth factors from bone may directly activate pain fibers, which innervate the bone (48) . Finally, the loss of mechanical stability of bone because of excessive tumor-induced bone destruction will result in mechanical deformation of the periosteum, the richly innervated fibrous sheath, which encompasses bony structures (48 , 56 , 57) . Deformation of the periosteum generates an intense, sharp, and stabbing pain, and, as COX-2 inhibition reduces both bone destruction and mechanical weakening of bone, this would be expected to significantly reduce movement-evoked pain.

A mechanism by which chronic inhibition of COX-2 reduces tumor-induced bone resorption is suggested by previous reports that demonstrate that PGs (particularly PGE2) may modulate osteoclast function (12 , 14 , 58 , 59) . Several PG receptors (including EP2 and EP4) are expressed by osteoblasts in vivo, and knockout of these receptors results in reduced osteoclast activity (13) . PGs also have been shown to induce the expression of OPGL by osteoblasts (13 , 60, 61, 62, 63) , and increased expression of OPGL by osteoblasts results in the proliferation and activation of nearby osteoclasts (64 , 65) . Both NSAID treatment (60) and deletion of receptors for PGE2 (EP receptors; Refs. 59 , 66 ) have been shown to reduce OPGL expression. In light of the previous and present findings, determining the efficacy of combined multimodal antiresorptive therapies (such as a combination of bisphosphonates or osteoprotegerin with a selective COX-2 inhibitor) to treat osteolytic bone diseases would be of significant interest (67) .

Chronic administration of a selective COX-2 inhibitor significantly reduced tumor burden in sarcoma-bearing bones. Prostaglandins have been shown to be involved in tumor growth, survival, and angiogenesis (15 , 24 , 42 , 68, 69, 70, 71) . Chronic COX-2 inhibition reduced tumor burden, which may, in turn, reduce factors released by tumor cells capable of exciting primary afferent fibers (72) . Interestingly, acute administration of a selective COX-2 inhibitor also showed a lesser but significant attenuation of both ongoing and movement-evoked cancer pain behaviors in animals with advanced bone destruction. These data strongly suggest that in addition to reducing tumor burden and preventing bone destruction (both of which are not affected by acute COX-2 inhibition), ongoing COX-2-mediated release of PGs contributes to the generation and maintenance of advanced bone cancer pain.

Chronic COX-2 inhibition may have reduced tumor burden in the present in vivo model by disrupting signaling pathways necessary for tumor growth and survival (18 , 73) , inhibiting vascular endothelial growth factor required for tumor angiogenesis (74) , or inhibiting PG-dependent phosphorylation of epidermal growth factor receptors essential for the growth and survival of tumors (75) . Whereas a critical question is the applicability of the present results to other tumors, expression of COX-2 has been described in head and neck cancers (76 , 77) , breast cancers (42 , 43) , colon carcinomas (42 , 44) , and prostate carcinomas (17 , 42) , thus suggesting the potential utility of COX-2 inhibitors in slowing the growth of other types of cancers. The reduction in tumor burden and sensitization of primary afferent fibers suggest that selective COX-2 inhibitors may provide simultaneous tumoricidal and analgesic effects.

In the present in vivo model of bone cancer pain, acute or chronic administration of a selective COX-2 inhibitor significantly attenuated both ongoing and movement-evoked pain. Whereas acute administration of a COX-2 inhibitor presumably reduces PGs capable of activating sensory or spinal cord neurons, chronic inhibition of COX-2 also appears to simultaneously reduce osteoclastogenesis, bone resorption, and tumor burden. Together, suppression of prostaglandin synthesis and release at multiple sites by selective inhibition of COX-2 may synergistically improve the survival and quality of life of patients with bone cancer pain.


   ACKNOWLEDGMENTS
 
We thank Ray Hill, Ian Rodgers, and Patricia Luk (Merck and Co., Whitehouse Station, NJ) for the gift of the selective COX-2 inhibitor MF tricyclic and quantification of drug plasma levels.


   FOOTNOTES
 
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.

1 Supported by NIH Grants from the National Institute of Neurologic Disorders and Stoke (NS23970), the National Institute for Drug Abuse (DA11986), National Institute of Dental and Craniofacial Research Dentist Scientist Award (DSA) DE00270, Training Grant DE07288, and a Merit Review from the Veterans Administration. Back

2 To whom requests for reprints should be addressed, at 18-208 Moos Towers, 515 Delaware Street S.E., Minneapolis, MN 55455. Phone: (612) 626-0180; Fax: (612) 626-2565; E-mail: manty001{at}tc.umn.edu Back

3 The abbreviations used are: COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; MF tricyclic, [3-(3,4-difluoro-phenyl)-4-(4-)methylsulfonyl]-2-(5H)-furanone; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide; GFP, green fluorescent protein; IOD, integrated optical density; GFAP, glial fibrillary acidic protein; IR, immunoreactive; SPR, substance P receptor; OPGL, osteoprotegerin ligand. Back

Received 6/ 6/02. Accepted 10/17/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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