Oral mucosal tumor inhibition by Ibuprofen

SCIENTIFIC ARTICLES J Oral Maxillofac Surg 41:795-800. 1983

Oral Mucosal Tumor Inhibition by Ibuprofen HOWARD CORNWALL, BA, ONATOLU ODUKOYA, BDS, M MED Sc, AND GERALD SHKLAR, DDS, MS Eighty young adult male and female golden hamsters (Mesocricetus auratus) were divided into four equal groups. The left buccal pouches of the animals in group 1 were painted three times weekly for 24 weeks with 0.1% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in heavy mineral oil. The left buccal pouches of the animals in group 2 were similarly painted with DMBA for 24 weeks, but the animals also received 10 mg ibuprofen (Motrin) administered systemically by the oral route twice weekly. The animals in group 3 received only ibuprofen, and those in group 4 served as untreated controls. After 24 weeks the animals were killed in groups of 16 (two males and two females from each of the four experimental groups) per week and their left buccal pouches studied for the presence of tumors. Sizes of tumors were also recorded. Ibuprofen was found to prevent tumor formation at 25-27 weeks. Some tumors developed in the animals receiving DMBA and ibuprofen by the 28-29 week, but they were fewer in number and smaller than those of the animals receiving only DMBA and not ibuprofen.

A delay in the formation of carcinomas of the buccal pouch of hamsters by the administration of aspirin and indomethacin was recently demonstrated by Perkins and Shklar.! This finding was consistent with previous studies that have used the growth of tumors transplanted into mice and rats as an index of the tumor-inhibiting effect of these nonsteroidal anti-inflammatory drugs.I-' The mechanism of tumor inhibition by aspirin and indomethacin was thought to be related to their known function as inhibitors of prostaglandin synthesis.t-' This concept was supported by Lupulescu, who found that prostaglandins could enhance both the induction of squamous cell carcinoma in mice, and the transplantability and cellular atypicality of chemically induced squamous cell carcinoma transplanted to syngeneic mice.f-? We felt that it would be of interest to study the

effect of other prostaglandin inhibitors on carcinogenesis in an oral tumor system using a lower concentration of carcinogen than the standard thrice-weekly application of a 0.5% solution of 7, 12-dimethylbenz[a]anthracene (DMBA).8,9 This carcinogen is so potent in 0.5% solution that, in a wide variety of studies of systemically administered tumor-inhibiting agents such as retinoids.J? vitamin E,l1 and immunoenhancing agents such as bacillus Calmctte-Guerin12 and levarnisole;'! tumor formation was delayed but was not prevented in any of the experimental animals. Ibuprofen (Motrin) was selected as the prostaglandin inhibitor and DMBA, in a 0.1% solution applied thrice weekly, as the carcinogen to be applied to hamster buccal pouch. Ibuprofen (2-4-isobutylphenyl propionic acid) is widely used in the treatment of rheumatoid arthritis and osteoarthritis. 14-18 Its pharmacology has been adequately studied. 19-2o It is promptly absorbed following oral administration and exists in circulation as a free fraction in equilibrium with a fraction bound to plasma proteins. The mechanism of action of ibuprofen as a pros-

Received from the Department of Oral Medicine and Oral Pathology, Harvard School of Dental Medicine, Boston, Massachusetts. Address correspondence and reprint requests to Dr. Shklar: Department of Oral Medicine and Oral Pathology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston MA 02115.

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Table 1. Average Numbers and Sizes of Tumors

Week Week Week Week Week

25 26 27 28 29

Group I (DMBA, n = 20)

Group 2 (DMBAfIbuprofen,

I tumor; 1.0 mm 2 tumors; 1.5 mm 3 tumors; 2.5 mm 3 tumors; 4.0 mm 5 tumors; 4.0 mm

Normal mucosa, no tumors Erythema, no tumors Leukoplakia, no tumors I tumor; 2.0 mm 1.5 tumors; 2.0 mm

taglandin inhibitor is thought to be due to the inhibition of the cyclo-oxygenase enzyme responsible for prostaglandin biosynthesis.P In this respect it differs from the action of steroidal anti-inflammatory agents, whose inhibitory effect on prostaglandin is due to the biosynthesis of a phospholipase A2-inhibitor that prevents prostaglandin generation.P

Il

=

20)

Group 3 (Ibuprofen, Il = 20) Normal Normal Normal Normal Normal

mucosa, mucosa, mucosa, mucosa, mucosa,

no tumors no tumors no tumors no tumors no tumors

Group 4 (Untreated, n = 20) Normal Normal Normal Normal Normal

mucosa, mucosa, mucosa, mucosa, mucosa,

no no no no no

tumors tumors tumors tumors tumors

pouches of each animal were photographed at that time. Tumors, if present, were counted and measured. Specimens obtained from the buccal pouches as well as from the heart, liver, kidney, adrenal, lung, and pancreas were fixed in 10% formalin, sectioned in paraffin, and stained with hematoxylin and eosin for histologic study. Results

Materials and Methods GROSS OBSERVATIONS

Eighty noninbred adult male and female golden hamsters tMesocricetus auratus)" were divided into four equal groups of ten males and ten females. A 0.1% solution of DMBA (Sigma Chemical Co.) in heavy mineral oil (USP) was painted on the left buccal pouches of group-I and group-2 animals three times weekly for 24 weeks with a number four sable brush. Group-2 and group-3 animals received 10 mg of ibuprofen (Motrin", Upjohn Company) twice weekly, administered orally by pipette in an aqueous suspension. Animals in group 4 served as untreated controls. The animals were maintained in a controlled environment with an alternating 12hour light-dark cycle and with water and laboratory chow ad libitum. Two males and two females were randomly selected from each of the four experimental groups and were killed weekly from week 25 to week 29 using carbon dioxide. The right and left buccal

* Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHEW Publication No. (NIH) 78-23, revised 1978).

At the twenty-fifth week, small tumors were developing in the left buccal pouches of several of the animals in group 1 (DMBA) (Fig. I). The group-2 animals (DMBA and ibuprofen) showed some erythema of the buccal pouches but no gross evidence of tumor formation (Fig. 2). At 26 weeks, tumors in the group-I animals had increased both in number and in size (Table I) and all the animals had developed tumors. No tumors were observed in the group-2 animals at this time. At 27 weeks, tumors in the group-I animals had again increased both in number and size (Fig. 3). No tumors were observed in the group-2 animals, but some areas of leukoplakia were observed in several animals (Fig. 4). At 28 weeks, the size of the tumors continued to increase in the group-I animals, and some tumors were observed in several animals in group 2, but the lesions were small. At 29 weeks, the buccal pouches in the group-I animals all had multiple large tumors (Fig. 5). Some of the animals in group 2 had only one or two small tumors at this time (Fig. 6). No lesions were observed in the buccal pouches of the group-3 (ibuprofen) and group-4 (control) an-

FIGURE I. Top left, left buccal pouch of a group-I hamster at 25 weeks, showing a moderately sized tumor and several areas of erythema and tissue proliferation. FIGURE 2. FIGURE 3. FIG URE 4. can be seen. FIGURE 5. FIGURE 6. leukoplakia.

Top right, left buccal pouch of a group-2 hamster at 25 weeks showing regular surface with no tumors. Middle left, left buccal pouch of a group-I animal at 27 weeks showing two proliferating tumor masses. Middle right, left buccal pouch of a group-2 animal at 27 weeks showing erythema but no tumors. Some area of leukoplakia Bottom left, left buccal pouch of a group-I animal at 29 weeks showing multiple tumors, some of which are now large. Bottom right, left buccal pouch of a group-2 animal at 29 weeks showing one small tumor adjacent to an area of

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FIGURE 7. Top, low-power microscopic view of papillary epidermoid carcinoma in a group-I animal at 29 weeks . The tumor is well differentiated and keratin pearls can be seen . Hematoxylin and eosin; x 80. FIGURE 8. Bottom, high-power microscopic view showing cellular atypism of invading cords of epithelium. Surface necrosis is visible. Hematoxylin and eosin; x 250

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CORNWALL ET AL

FIGURE 9. High-power microscopic view of early epidermoid carcinoma in a group-2 animal at 29 weeks. The carcinoma is developing in an area of leukoplakia characterized by hyperkeratosis and dysplasia. Hematoxylin and eosin; x 250

imals. No gross pathologic alterations were observed in the major organs of any of the animals at autopsy. MICROSCOPIC OBSERVATIONS

The tumors in both group-l and group-Z animals were epidermoid carcinomas. The lesions were papillary in form and were well differentiated, with keratin formation clearly defined (Figs. 7,8). Bizarre mitoses and cell forms were rarely observed. The tumors in some group-2 animals were seen to develop in areas of leukoplakia (Fig. 9). No microscopic evidence of dysplasia or neoplasia was observed in the buccal pouches of animals from groups 3 and 4. No microscopic evidence of pathology was observed in the heart, lung, liver, kidney, adrenal, pancreas, or spleen in any of the animals from groups 1-4. None of the animals receiving ibuprofen died during the experiment, and there were no pathologic changes observed in the major organs at autopsy. This is in accord with clinical observations that ibuprofen is a safe drug.U STATISTICAL ANALYSIS

Means and standard deviations of the number of tumors were calculated for groups 1 and 2 and Stu-

dent's t test for paired data was used to determine if there was a statistically significant difference. The mean number of tumors in group 1 was 2.84 :t 1.53 (SD) and that in group 2 was 0.45 ± 0.68. This difference was statistically significant (P ,,:;; 0.01). The results were even more significant, however, in that no tumors developed in the group-2 animals until the twenty-eighth week (Fig. 10). No differences in tumor development were found between male and female animals.

Discussion Ibuprofen, in the dosage used in this experiment, significantly inhibited the development of chemically-induced tumors of the hamster" buccal pouch, probably due to its pharmacologic action as an inhibitor of prostaglandin synthesis. Similar results have been demonstrated with aspirin and indomethacin using the standard buccal pouch tumor system consisting of thrice-weekly topical applications by brush of a 0.5% solution of DMBA in mineral oil. I Tumors were found to develop at 23-26 weeks rather than 8-10 weeks when a 0.1% solution of DMBA was used. The ibuprofen prevented tumors from developing until the twenty-eighth week, and even at this time only a few small tumors developed. This experiment suggests that ibuprofen

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might not only delay tumor formation but actu ally might prevent it if the ca rcinogen used were less active than DMBA.

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References I. Perkins TM, Shklar G: Delay in hams ter buccal pouch carcinogene sis by aspirin and indomethacin. Oral Surg 53:170,1982 2. Hial V, Horako va Z, Shaff RE , et al: Alteration of tumor grow th by aspiri n a nd indome thacin: studies with two transplantable tumors in mouse . Eur J Pharmacol37: 367, 1976 . 3. Powles T, Clark S, Easty D, et al: Th e inhibition by aspirin and indome thacin of ost eolytic tumor deposits and hypercalcemia in rats with Walker tumor, a nd its possible app lication to human cancer. Br J Can cer 28:316, 1973 4. Vane JR: Inhibit ion of pro stagland in synthes is as a mechanism of action for aspirin-like drugs. Natu re New BioI 231:232, 1971 5. Flower RJ: Drugs which inhibit prostaglandin biosynthesis. Pharmacol Rev 26:33, 1974 6. Lupulescu A: Enh ancement of carci noge nesi s by pro stagland ins. Nature 272:634, 1978 7. Lup ulescu A: Effects of prostaglandins on tumor transplantat ion. Oncology 37:418, 1980 8. Salley 11: Experimental ca rcinogenesis in the cheek pouch of the Syrian hamster. J Dent Res 33:253, 1954 9. Morris AC: Factors influencin g expe rimen tal carcinogenesis in hamster cheek pouch. J Dent Res 40:3, 1961 10. Shklar G, Schwartz J, Grau 0, et al: Inhibition of hamster

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buccal pouch carcinogenesis by 13-cis-retino ic acid. Oral Surg 50:45, 1980 Shklar G: Inhibition of oral mucosal carcinogenesis by Vitamin E. J Nat! Can cer Inst 68:791, 1982 Giunt a JL, Reif AE, Shklar G: Bacillus Calmette-Guerin and antilymphocyte serum in carcinogenesis: effects on hamster buccal pouch. Arch PathoI 98:237, 1974 Eisenberg E, Shklar G: Levam isole and hamster pouch carcinogenesis. Oral Surg 1977;43:562,571. Michell OM, MacDonald G: Controll ed trial of ibuprofen in rheum atoid arthritis. J Rheumatol I:(suppl 1)59, 1974 Boardman PL , Nuki G, Hart FD : Ibuprofen in the treatment of rheumatoid art hritis and osteoarth ritis. Ann Rheum Dis 26:560, 1967 Hu skisson EC, Hart FD , Shenfield GS: Ibuprofen: a review. Practitioner. 1971 ;207:639,648. Chalm ers TM : Clinical ex perience with ibuprofen in the treatment ofrheumatoid arthritis. Am Rheum Dis 28:513, 1969 Boardm an PL , Nu ki G , Hart FD: Ibuprofen in the treat ment of rheumatoid art hritis and osteo arthritis. Am Rheum Dis 26:560, 1968 Adams SS , Bough RG, Cliffe W, et al: Some aspect s of the pharmacology, met abolism, and toxicology of ibuprofen . Rheumatol Phys Med Suppl 9, 1970 Broo ks CD , Schlagel CA, Sekhar NC, et al: Tolerance and pharmacology of ibup rofen . Curr ent Ther Res 15:180, 1973 Ferreira SH, Vane JR : New aspects of the mode of act ion of nonsteroidal anti-inflammatory drugs. Am Rev Pharo macol 14:57, 1974 Flower RJ, Blackwell GJ: Anti-inflammatory steroids induce biosynthesis of a phospholip ase A 2 inhibitor which prevents prostaglandin generation. Nature. 278:456, 1979