Cancer Letters, 43(1988)15-19 Elsevier Scientific Publishers Ireland Ltd. Modulation of methylcholanthrene-induced carcinogenesis in

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2 Cancer Letters, 43(1988)15-19 Elsevier Scientific Publishers Ireland Ltd. Modulation of methylcholanthrene-induced carcinogenesis in the uterine cervix of mouse by indomethacin A. Ramesh Rao and S.P. Hussain Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi (India) Received 24 July 1988 Accepted 2 August 1988 Keywords: Cervical carcinogenesis in mice; 3-methyl- cholanthrene; indomethacin. 164

3 SlJM'1.6.RY The present paper reports the chemopreventive action of indomethacin on methylcholanthrene (MCA)-induced carcinogenesis in the uterine cervix of virgin young adult Swiss albino mouse. Placement of a sterile cotton thread impregnated with beeswax containing approx. 600 ug of MCA produces cervical tumors in 91% mice in 16 weeks. When such carcinogen-containing threads are inserted into the uterine cervix of mice fed diets containing indomethacin at the dose levels of 10 mg/kg, 20 mg/kg and 40 mg/kg diet for two preinsertion weeks plus 16 post-insertion weeks, the cervical tumor incidences were 83%, 65% and 26% (P<'O.01),. respectively. It is concluded that indomethacin, when given in the diet at sufficiently high concentration, significantly inhibits MeA-induced cervical carcinogenesis. INTROUDCTION The modulation of chemical carcinogenesis has been demonstrated to be feasible with the administration of several chemically diverse species of naturally occuring or synthetic compounds [29,30]. Indomethacin, a non-steroidal anti-inflanmatory drug, has been reported to inhibit the induction of tumors in the rat colon [20,21], mouse oesophagus [22], rat manmary.gland [16,17] and mouse skin 165

4 [27]. The present investigation is designed to evaluate the possible chemopreventive action of indomethacin on MCAinduced carcinogenesis in the uterine cervix. of virgin young adult Swiss albino mouse. MATERIALS AND METHODS Animals Random-bred, weeks old, virgin Swiss albino mice procured from Small, Disease-Free Animal House, Haryana Agricultural University, Hissar, India, were maintained in an air-conditioned animal facility providing (unless otherwise stated) standard food pellets '(Hindustan Lever Ltd., India) and tap-water ad libitum. Chemicals MCA and indomethacin were purchased from Sigma (USA). Yellow variety of beeswax was obtained from Mysore (India) and filtered in molten state (60 0 C) 4 times to remove dust particles. Indomethacin was added, along with sucrose carrier (10 mg/kg diet), to the pulvarized diet at concentrations of 10, 20 and 40 mg/kg diet. The control diet contained sucrose carrier alone. 166

5 Tumor Induction Murphy's string method [18,19] as described by Manoharan and Rao [14] was followed for the induction of tumors in the mouse uterine cervix. Briefly, sterile, double cotton thread impregnated with beeswax containing approx. 600 ug of MCA(3:1, w/w) was inserted into the cana 1 of uterine cervix by means of laparotomy under mild ether anesthesia. EXPERIMENTAL DESIGN The animals were sorted into control and experimental groups (Table 1) and indomethacin feeding was commenced 2 weeks before the laparotomy for the insertion of the cotton threads into the uterine cervix. Animals of group I were inserted intracervically with MCA-plus-wax Impregnated threads, and were on control diet. Animals of groups II, III and IV were also inserted intracervically with MCA-plus-wax impregnated threads, and were put on diets containing 10, 20 and 40 mg/kg of indomethacin, respectively. Animals of groups V, VI and VII were inserted intracervically with waxonly impregnated threads, and in addition, were put on qiets containing 10, 20 and 40 mg/kg of indomethacin, respectively. Animals of group VIII were inserted intracervically with wax-only impregnated threads and put on control diet. 167

6 Animals were weighed initially, at weekly intervals and at autopsy. Mice in a moribund state were killed and their uterine cer~iees were fixed in Bouin's fluid. Sixteen weeks after the insertion of cotton threads, the surviving animals were killed and their uterine cervices were fixed in Bouin's fluid. The tissues were processed for histopathological assessment of precancerous and cancerous lesions in the cervical epithelium. Standard statistical test (chi-square test) was employed to evaluate the effect of indomethacin on MCAinduced cervical tumor incidences by analysing the significance level of differences between control and experimental values. RESULTS The animals in the present study did not seem to suffer from any toxic effect of indomethacin at the given dietary dose levels. Only a small number of animals died in each group, and there was no significant fall in the body weight gain in experimental animals. The incidence of tumors in different groups are depicted in Table 1. Animals which were inserted 168

7 intracervically with MCA-plus-wax impregnated threads and fed on control diet (group I) displayed 91% tumor inciderice in their uterine cervices. Animals that were inserted intracervically with wax-only impregnated threads did not develop any tumors in their uterine cervices no matter whether they were on cont rol diet (group VI I I) or on indomethacin diets (group V, VI and VII). When the animals th3t were on diets containing indomethacin at the dose levels of 10 mg/kg (group II). 20 rng/kg (group III) and 40 mg/kg (group IV), received the MCA-plus-wax impregnated threads into their uterine cervices, the tumor incidences at the site were 83%, 65% and 26% (P < 0.01), respectively. The tumors were squamous cell carcinomas of either carcinoma situ type or infiltrating carcinoma type. The incidences in of hyperplasia and dysplasia did not have any correlations with different treatments. DISCUSSION The present study demonstrates that indomethacin, when given at appropriate dose levels, inhibits MCA-induced carcinogenesis in the uterine cervix of mice. This inhibitory action of indomethacin was not significant at 10 mg/kg and,20 mg/kg dietary dose levels whereas i twas significant (P <0.01) at 40 mg/kg dietary dose level. 169

8 Indomethacin has displayed its chemopreventive action on chemically-induced carcinogenesis in such diverse sites as colon [20,21], oesophagus [22], mammary gland [16,17] and skin [27]. Epidemiological study suggests a significant decline in breast cancer incidences among women who use indomethacin [6]. How this drug achieves its inhibitory action on carcinogenesis is unclear at present. Indomethacin is a pathway potent of inhibitor of the prostaglandin synthatase arachidonic acid metabolism [4,5,11]. It can modulate carcinogen metabolism by inhibiting prostaglandin synthatase [15,32]. At several extrahepatic sites, cooxidation of xenobiotic chemicals including polycyclic aromatic hydrocarbons is known to depend upon prostaglandin synthatase [2,25,26]. If there is any "involvement of prostaglandin synthatase co-oxidation of polycyclic aromatic hydrocarbons in activation process in the epithelial cells of uterine cervix, the suppression of this pathway by indomethacin would conceivably inhibit carcinogenesis. Further, indomethacin is known to inhibit the proliferation of many mammalian normal as well as neoplastic cells [1,7,10,12,23] and this property of the drug could also contribute to its tumor inhibitory action. Indomethacin inhibits the induction of ornithine decarboxylase activity by tumor promoters [~7] and a recent study indicates that decline in polyamine synthesis may adversely affect the 170

9 process of carcinogenesis [28]. Another property of indomethacin is to inhibit angiogenesis whereas prostaglandin may augment it [12,46]. This anti-angiogenesis action promotes tumor suppression. It has also been suggested that the immunomodulatory effects [3,8,13,24] of indomethacin may contribute to the inhibition of carcinogenesis. Whether its inhibitory action on tumorigenesis in the uterine cervix is mediated through the modulation of hormonal secretion or action has yet to be known. 171

10 ... Teble 1. IIodbletory influence of ind.etheeln on MCA-induced earelnogenesls in the uteride cervix of.ouse Group Treatments No. of mice Body weight (g) No. of mice with cervical lesions Tumor mean + S.E.M incidence Initial Effective Hyper- Dys- Squamous (%) Initial Finar piasia plash cell carcinoma I. MCA + wax thread /23 Normal diet (25) (23) (91) II. KeA + Wax thread /24 Indomethacin diet (25' (24) - (83) (10 mg/kg) lit. KCA + wax thread /23 Indomethacin diet (25) (23) (65) (20 mg/kg) -..J t-,) tv. MeA + wax thread /23* Indomethacin diet (25) (23) (26) (40 mg/kg) V. Wax thread /14 Indomethacin diet (15) (14) (0) (10 mg/kg) VI Wax thread /13 Indomethacin diet (15) (13) (0) (20 mg/kg) VII Wax thread /14 Indomethacin diet (15) (14) (0) (40 mg/kg) VIII Wax thread /13 Normal diet (15) (13) (0) *P < 0.01 (group I vs. group IV).