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Effect of Ketoprofen in Topical Formulation on Vascular Endothelial Growth Factor Expression and Tumor Growth in Nude Mice With Osteosarcoma

Posted on: Saturday, 6 November 2004, 03:00 CST

Abstract

OST cells, a low metastatic cell line established from human osteosarcoma, were inoculated under the periosteum of the ossa cranii of nude mice. Four weeks later, tumors were percutaneously treated for an additional 4 weeks with a patch containing either placebo or ketoprofen (KP). In the placebo group, OST cells formed osteoid and invaded the cranial bone. Tumor mass weighed 3.54 g. Approximately 85% of cells within the tumor expressed proliferating cell nuclear antigen (PCNA), indicating that they were proliferating with a high mitotic activity. Many feeder vessels were located within the tumor. The majority of tumor cells expressed intensely vascular endothelial growth factor (VEGF). In the KP group, invasion of OST cells into the cranial bone was suppressed and the tumor mass was 47% of that of the placebo group. Approximately 65% of cells within the tumor were PCNA-negative, indicating that their growth was arrested. There were considerably fewer feeder vessels within the tumor in the KP group than in the placebo group. Only a small number of cells expressed VEGF. Based on these findings, we concluded that topical administration of KP to nude mice with osteosarcoma inhibited VEGF expression, reduced the development of feeder vessels for supply of nutrients and oxygen, and suppressed tumor growth.

2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: Osteosarcoma; Ketoprofen; Topical formulation; Tumor growth; Vascular endothelial growth factor

Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known as not only anti-inflammatory agents but also anti-tumor agents. For example, in humans, the regular use of aspirin was reported to reduce the risk of digestive tumors including those in the stomach [34] and colon [35]. Sulindac also causes regression of intestinal polyps in patients with familial adenomatous polyposis [11]. Ketoprofen (KP) has been used as an analgesic to relieve the acuity of post-operative pain [32] and chronic cancer pain [31]. In rodents, dietary exposure to KP reduced the incidence of colon tumor induced by chemical carcinogens [21]. Other NSAIDs including aspirin [20] and indomethacin [19] also inhibited the growth of colon tumors induced by chemical carcinogens.

Osteosarcoma is a malignant bone tumor occurring mainly in the metaphyseal region of the long bones of young people. The most common sites affected are the distal femur, the proximal tibia and the proximal humerus. Osteosarcoma expands the cortex of the bone and later erupts through the cortex into the soft tissues. It often metastasizes hematogenously to the lungs during the early stage of tumor development. The main treatment of patients with osteosarcoma is to remove the tumor by wide excision. These patients also receive pre- and post-operative treatment with anti-mitotic drugs such as methotrexate, adriamycin, and ifosfamide [8]. To date, however, treatment of osteosarcoma patients with NSAIDs has not been reported.

In humans, KP has been used in topical formulation in a variety of musculoskeletal conditions [3,23,24]. The kinetics of KP after topical plaster application is a slow and continuous release form [24]. Using nude mouse skin as the barrier, Sheu et al. [30] showed that KP can penetrate percutaneously from poultices in nude mice. The aim of the present study is to examine whether percutaneous treatment of tumor-bearing mice with KP is a useful means for cancer therapy. For this, osteosarcoma takase (OST) cells, a low metastatic cell line established from human osteosarcoma [14,18], were inoculated under the periosteum of the ossa cranii of nude mice and then treated percutaneously with KP, and the effects of KP on tumor growth, distribution of blood vessels and expression of vascular endothelial growth factor (VEGF) within the tumor were examined.

Materials and methods

Materials

Ketoprofen-containing patches (286 g of KP/cm^sup 2^), placebo- containing patches and ketoprofen powder were generously donated by Hisamitsu Pharmaceutical Co., Ltd. (Tokyo, Japan). Female BALB/cA Jclnu/nu strain nude mice (3 weeks old) were from Clea Japan, Inc. (Tokyo, Japan) and allowed free access to standard laboratory chow and tap water. A mouse monoclonal antibody to proliferating cell nuclear antigen (PC 10), an immunostaining kit (LSAB kit) and peroxidase-conjugated ENVISION+ were from DAKO Japan (Tokyo, Japan). A mouse monoclonal antibody to vascular endolhelial growth factor (sc-7269) was from Santa Cruz Biotechnology (Santa Cruz, CA). A kit for alkaline phosphatase (Liquiteck ALP) was from Nippon Roche Co., Ltd. (Tokyo, Japan).

Inoculation of OST cells into nude mice and percutaneous treatment with KP

OST cells were generously donated by Dr. Katsuro Tomita (Department of Orthopaedic Surgery, Kanazawa University School of Medicine, Japan). In nude mice, OST cells injected into the abdominal cavity proliferated and invaded the peritoneum and abdominal wall where bone tissue was induced in the fascia [18]. In this study, OST cells were inoculated under the periosteum of the ossa cranii of nude mice. Briefly, OST cells were cultured in RPMI- 1640 medium containing 10% fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin and 0.25 ng/ml amphotericin B in 60-mm plates. When cells had grown to approximately 80% confluence, they were harvested by trypsinization, centrifuged for 3 min at 1500 rpm and resuspended in culture medium. Under ether anesthesia, 0.4 ml of cell suspension (2.5 10^sup 6^ cells/ml) was inoculated under the periosteum of the ossa cranii of nude mice.

After 4 weeks of inoculation, tumor-bearing mice were divided into two groups (10 mice/group), the KP group and the placebo group. In the KP group, the tumor was treated percutaneously for 4 weeks with a KP-containing patch measuring 10 mm 15 mm. A patch contained approximately 429 of KP. In the placebo group, the tumor was treated percutaneously for 4 weeks with a placebo-containing patch of the same size. In both groups, the patches were changed daily. After 4 weeks of treatment, animals were sacrificed under nembutal anesthesia. The tumor was excised and weighed. Then, body weight was measured. The tumor was fixed in 10% formalin and embedded in paraffin. After deparaffinization, elastica-Goldner staining of the sections was performed to examine the effect of KP on the distribution of feeder vessels within the tumor.

All animal experiments were approved by the local animal ethics committee at Ehime University School of Medicine.

Immunohistochemistry

Immunohistochemistry for proliferating cell nuclear antigen (PCNA) in tumor sections was performed using a mouse monoclonal antibody to PCNA and an LSAB kit as described previously [26]. Cells were considered to contain PCNA when the nucleus was stained brown. The PCNA-labeling index was evaluated by determining the percentage of positive nuclei present in approximately 400 cells. Values for 5- 6 determinations per mouse were averaged to obtain the value for each mouse. The results were expressed as mean SD for four mice.

Immunohistochemistry for VEGF in tumor sections was performed by the polymer immunocomplex method. A mouse monoclonal antibody to VEGF was incubated for l h with peroxidase-conjugated ENVISION+. Then this mixture was treated for l h with normal mouse serum to block remaining binding sites of ENVISION+. The formalin-fixed, paraffin-embedded tissue sections were deparaflinized followed by antigen retrieval with autoclaving (121 C, 15 min) and endogenous peroxidase blocking by H^sub 2^O^sub 2^ (37 C, 10 min), and then incubated for l h with an antibody-ENVISION+ complex. VEGF-positive cells were visualized by adding diaminobenthidine to the sections. Nucleus was counterstained with Hematoxylin. Four different microscopic fields per tumor were photographed and VEGF-positive cells present in approximately 1000 cells per photograph were counted. Since the staining intensity varied significantly, the VEGF expression was estimated "O" if negative, "1+" if week intensity, and "2+" for intermediate or strong intensity. The VEGF-labeling score was evaluated as follows:

VEGF-labeling score = [(1 number of "1+" cells + 2 number of "2+" cells)/number of total cells] 100 The number of total cells is the sum of numbers of "0", "1+" and "2+" cells. The results were expressed as mean SD for four determinations.

Measurements of ALP activity and DNA

Serum ALP activity was measured using a kit for ALP. DNA in the homogenates was measured fluorometrically using calf thymus DNA as the standard [13].

Statistical analysis

Statistical analysis of group means was conducted by ANOVA followed by post hoc comparisons using Fisher's protected least significant difference test. Statistical analysis of differences in tumor weight (Table 1), PCNE-labeling index, and VEGF-labeling score was performed using Student's t-test. All data were expressed as means SD.

Results

Effect of KP on body weight, tumor weight and serum ALP activity

The tumor was treated percutaneously with either placebo or KP for 4 weeks. The body weight, tumor weight and serum ALP activity were measured (Table 1). In the placebo group, OST cells formed o\steoid and invaded the cranial bone (Fig. 1 (center)). The tumor mass weighed 3.54 g. Progressive weight loss occurred in this group. Body weight was 62% of that of age-matched normal nude mice, termed the normal group. The level of ALP activity in serum of this group was 5.6-times higher than that of the normal group. In the KP group, invasion of the tumor into the cranial bone was suppressed (Fig. 1 (right)). The tumor mass decreased to 47% of that of the placebo group. Body weight recovered to 88% of that of the normal group. Serum ALP activity of the KP group was decreased to a level similar to that of the normal group.

Table 1

Effect of KP on body weight, tumor weight and serum ALP activity

Fig. 1. X-ray film of the head of nude mice with osteosarcoma. OST cells were inoculated under the periosteum of the ossa cranii of nude mice and grew for 4 weeks. Then, the tumor was treated topically with either placebo or KP for an additional 4 weeks. (Arrow) invasion of tumor; (left) normal group; (center) placebo group; (right) KP group.

Effect of KP on tumor growth in vivo and in vitro

Since the results of tumor weight and serum ALP measurements suggested that topical treatment with KP might suppress tumor growth, PCNA, which appears in the nuclei of proliferating cells [5,22,33], was detected in tumor sections by immunohistochemistry for PCNA. In the placebo group, the majority of cells in the tumor were immunostained with a high intensity of staining (Fig. 2A). In the KP group, there were fewer PCNA-positive cells than the placebo group and their intensity of staining was low (Fig. 2B). The PCNA- labeling index was considerably lower in the KP group than in the placebo group (Fig. 3A). These results indicate that KP had the ability to suppress proliferation of OST cells in vivo.

Next, to examine whether KP can directly suppress cell proliferation, OST cells were cultured in the absence or presence of KP for 10 days. The DNA contents of the cultures were measured to monitor cell proliferation. The DNA contents of cultures not treated with KP increased linearly over the 10-day experimental period. The DNA contents of cultures treated with KP at either 1 or 10 g/ml also increased linearly and were identical to those of the untreated cultures on the corresponding day. Fig. 4 shows the result of KP at 10 g/ml. However, in cultures treated with KP at 100 g/ml for more than 2 days, cells began to detach from the bottom of the plates. Thus, KP at 100 g/ml was toxic to OST cells.

Effect of KP on feeder vessel distribution and expression of VEGF in the tumor

Since angiogenesis plays a pivotal role in growth of many solid tumors [10], the distribution of feeder vessels in the tumor was analyzed by elastica-Goldner staining of tumor sections. The placebo group contained many feeder vessels within the tumor (Fig. 2C), while there were considerably fewer feeder vessels in the KP group than in the placebo group (Fig. 2D). The KP group, but not the placebo group, contained much necrotic tissues in the central region of the tumor (Fig. 2D, arrowhead).

Fig. 2. Immunohistochemical staining and elastica-Goldner staining. (A, B) show immunohistochemistry for PCNA within the tumor, bar = 25 m. (C, D) show elastica-Goldner staining of tumor sections. Arrow, feeder vessels. Arrowhead, necrotic tissue, bar= 100 m. (E, F) show immunohistochemistry for VEGF within the tumor, bar = 25 m. (A), (C) and (E), placebo group; (B), (D) and (F), KP group.

Fig. 3. PCNA-labeling index and VEGF-labeling score. (A) PCNA- labeling index is shown. Values given are means + SD for 4 mice. * P < 0.0001, vs the placebo group. (B) VEGF-labeling score is shown. Values given are means SD for 4 determinations. ** P < 0.001, vs the placebo group.

Fig. 4. Effect of KP on proliferation of OST cells in cultures. OST cells were cultured in the absence ([white circle]) or presence ([black circle]) of 10 g/ml KP in a 60-mm plate. At indicated intervals, cells were harvested and sonicated briefly at 0 C, and DNA in the homogenate was measured. Values given are means SD for 4 plates.

Next, expression of an angiogenic factor, VEGF, in tumor cells was analyzed by immunohistochemistry for VEGF. Positive immunostaining with VEGF was predominantly observed in the cytoplasm of tumor cells (Fig. 2E and F). In the placebo group, the majority of cells in the tumor were intensely immunostained (Fig. 2E), indicating that they expressed a high concentration of VEGF. In the KP group, there were a small number of cells that were slightly immunostained (Fig. 2F). The VEGF-labeling score also was considerably lower in the KP group than in the placebo group (Fig. 3B). These results indicate that KP inhibited VEGF expression in tumor cells in vivo.

Discussion

KP has been used in oral and topical formulations [3,21,23,24,31,32]. In the present study, we evaluated the anti- tumoral activity of topical patch application of KP in nude mice with osteosarcoma. In the placebo group, OST cells inoculated under the periosteum of the ossa cranii invaded the cranial bone and the tumor mass reached 21.5% of body weight 8 weeks after the inoculation. This result and a high level of serum ALP activity of this group suggested that OST cells were growing aggressively in nude mice. To confirm this, we immunohistochemically examined PCNA expression in tumor sections, because expression of PCNA in cells is closely linked to the cell cycle [5,33]. The level of PCNA in the nucleus begins to increase during the late Gl phase immediately before the onset of DNA synthesis, peaks during the S phase, then decreases again during the G2 and M phases. Robbins et al. [22] reported that PCNA positivity in human solid tumors correlated with mitotic activity and the grade of tumors. Since approximately 85% of cells within the tumor of the placebo group were PCNA-positive showing a high staining intensity, this indicated that the great majority of OST cells were proliferating with a high mitotic activity. In the KP group, invasion of OST cells into the cranial bone was suppressed and the tumor mass was decreased to 7.1% of body weight. Only 35% of cells within the tumor were PCNA-positive and their intensity of staining was faint, indicating that they proliferated with a low mitotic activity but the remaining 65% were arrested in the GO/ Gl phase. Based on these findings, we concluded that KP in topical formulation had the ability to suppress the growth of osteosarcoma in vivo.

The finding that OST cells in cultures treated with KP at either 1 or 10 g/ml replicated at a normal proliferative rate during the 10- day treatment period indicates that KP at <10 g/ml was nontoxic to OST cells. In the present study, we used a patch containing 429 g of KP for topical treatment of the tumor. Since we did not measure the amount of KP actually absorbed through the skin from a patch, whether the amount of KP delivered to the tumor was less than 10 g is unknown. In general, the patch/plaster contains a large excess of KP as compared to the absorption capacities of the skin. Rolf et al. [23] reported that when the patients with knee disorders requiring arthroscopy were treated with the plaster containing 30 mg of KP in multiple applications for 5 consecutive days, the median maximum concentration of KP at removal of the last of five plasters was 57 ng/g in the synovial tissue and 569 ng/g in the cartilage. They also reported that KP disappeared with short half-lives from the tissues. Therefore, it is likely that KP in topical formulation suppressed growth of OST cells inoculated under the periosteum of the ossa cranii by an indirect action in vivo but not by direct cytotoxicity to tumor cells.

Tumor growth is preceded by increased vascular supply of nutrients and oxygen to the tumor. Therefore, the sprouting of new blood vessels, termed angiogenesis, within tumors plays a pivotal role in the maintenance, growth and metastasis of many solid tumors [1O]. To visualize tumor vessels, immunohistochemistry for constitutive marker CD31 on endothelial cells often has been performed. There are some reports that NSAIDs reduced the density of CD31-positive vessels within the tumor in vivo [27,28]. In the present study, we used elastica-Goldner staining to visualize feeder vessels within the tumor, because it was hard to distinguish feeder vessels from other vessels by CD31 immunohistochemical examination. The results of elastica-Goldner staining showed that the density of feeder vessels within the tumor was considerably lower in the KP group than in the placebo group. Taken together, our finding indicated that KP-induced inhibition of angiogenesis caused a reduction in the development of feeder vessels for supply of nutrients and oxygen, suppressing osteosarcoma growth and inducing the formation of extensive necrotic tissue within the central region.

One mechanism by which tumors induce angiogenesis has been reported to be expression of angiogenic factors including VEGF and fibroblast growth factors [9,10, 17,27]. A variety of tumor cells expresses VEGF mRNA and protein and secretes VEGF [12,16,25,29]. The intensity of VEGF expression in soft tissue sarcomas correlated with their tumor grade [6]. Kaya et al. [16] reported that 63% of biopsy tumor specimens of 27 patients with osteosarcoma were VEGF-positive while the remaining 27% were VEGF-negative, and that local microvessel density in osteosarcoma was higher in the VEGF-positive tumors than in the VEGF-negative tumors. Using immunohistochemistry for VEGF, we found that not only the placebo group but also the KP group expressed VEGF within osteosarcoma. However, VEGF expression within osteosarcoma was considerably lower in the KP group than in the placebo group. Such results suggest that reduced development of feeder vessels in the KP group may result from inhibition of osteosarcoma-induced VEGF expression. This interpretation is supportedby findings of Kim et al. [17] that blocking the action of tumor-produced VEGF by treating tumor-bearing nude mice with a monoclonal antibody to VEGF reduced vascular density within the tumor. However, we should note the involvement of other angiogenic/ proangiogenic factors, because it has been reported that there is no apparent correlation between VEGF expression and microvessel density in synovial sarcoma [15].

Cyclooxygenase (COX) also regulates angiogenesis [27,28]. There are at least two isoforms of COX, COX-1 and COX-2. COX-1 is expressed constitutively in many tissues, while COX-2 is induced in inflammatory cells and cancer cells [38]. Using an endothelial cell/ colon carcinoma coculture model system, Tsujii et al. [36] showed that COX regulated colon carcinoma-induced angiogenesis by two mechanisms: COX-1 regulated angiogenesis in endothelial cells, while COX-2 modulated production of angiogenic factors by colon carcinoma. Sawaoka et al. [27] reported that oral administration of COX inhibitors to nude mice with gastrointestinal tumors suppressed angiogenesis and tumor growth by suppressing expression of angiogenic factors by inhibiting COX-2, and by suppressing vascular endothelial cell growth and angiogenesis by inhibiting COX-1. In the present study, immunohistochemistry for COX-2 did not show any evidence of COX-2 expression in the tumors of both placebo and KP groups (data not shown). Therefore, whether KP, a nonselective COX inhibitor [37], inhibited both COX-1 and COX-2 and consequently reduced VEGF expression and development of feeder vessels remains unclear.

Osteosarcoma often has already formed micrometastases in the distant organs, especially the lung, during the early stage of tumor development. In humans, VEGF expression in primary osteosarcoma correlated with the development of pulmonary metastasis and poor prognosis [16]. In rodents, higher expression of VEGF mRNA in an osteosarcoma cell line (LM8) was reported to facilitate neovascularization at the site of metastasis, resulting in extremely high metastatic potency after intravenous injection [2]. In our case, despite a high VEGF expression within the tumor, radiographs of the whole bodies of the placebo group did not exhibit any evidence of bone tissue formation in other organs including the lung (data not shown). This is consistent with the findings of others that OST cells are osteosarcoma cells with a low metastatic ability [14,18].

Oral use of NSAIDs causes serious side effects such as gastrointestinal damage and bleeding for patients. For example, indomethacin administered orally developed ulceration and necrosis in the stomach and jejunum [4]. The advantage of the topical use of NSAIDs is a marked reduction in the incidence of side effects [7]. Although KP has side effects for skin, these have been reported to be benign [I]. The present finding that topical administration of KP to nude mice with osteosarcoma suppressed tumor growth may offer a new strategy against human osteosarcoma using topical application of KP.

Acknowledgements

We thank Professor Katsuro Tomita (Department of Orthopaedic Surgery, Kanazawa University, School of Medicine, Japan) for donating OST cells. We also thank Hisamitsu Pharmaceutical Co., Ltd. for donating KPcontaining patches, placebo-containing patches and KP powder.

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Kenshi Sakayama(a,*), Teruki Kidani(a), Tatsuhiko Miyazaki(b), Haruo Shirakata(a), Yoshiyuki Kimura(c), Junji Kamogawa(a), Hiroshi Masuno(d), Hamyasu Yamamoto(a)

a Department of Orthopaedic Surgery, School of Medicine, Ehime University, \Shigenobu, Onsen-gun, Ehime 791-0295, Japan

b Second Department of Pathology, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-0295, Japan

c Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-0295, Japan

d Department of Medical Laboratory Technology, Ehime College of Health Science, Takooda, Tobe-cho, lyo-gun, Ehime 791-2101, Japan

Received 22 December 2003; accepted 12 March 2004

* Corresponding author. Tel.: +81-89-960-5343; fax: +81-89-960- 5346.

E-mail address: kenshi@rn.ehime-u.ac.jp (K. Sakayama).

Copyright Journal of Bone and Joint Surgery, Inc. Nov 2004

Source: Journal of Orthopaedic Research

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