Chemopreventive effect of montelukast in n-nitroso n-methyl urea induced mammary carcinogenesis in female Sprague-Dawley rats.

OBJECTIVE: The objective of the study was to evaluate the chemopreventive effect of montelukast sodium; selective reversible cysteinyl leukotriene D4-receptor antagonist in N-nitroso N-methyl urea (NMU) induced mammary carcinogenesis in virgin female Sprague-Dawley rats.
MATERIALS AND METHODS: Thirty rats were divided into five groups (normal control, disease control, montelukast1 mg/kg, montelukast10 mg/kg, tamoxifen10 mg/kg) of six animals each. The drug was administered in two doses,1 mg/kg,and 10 mg/kg orally and compared with the standard drug tamoxifen (10 mg/kg)p.o.
RESULTS: Montelukast sodium 1 mg/kg,10 mg/kg, and tamoxifen10 mg/kg decreased the tumor incidences by 50%,66.67%, and 83.33% and the total number of tumors in group by 41.67%, 58.33% and 91.67% respectively, when compared to the disease control. Montelukast sodium 1 mg/kg,10 mg/kg,and tamoxifen10 mg/kg decreased the average tumor burden by 86.41%,94.8% and 95.97%and average tumor volume by 89.52%, 95.84%, and 95.4%respectively, when compared to disease control group.
CONCLUSION: The results revealed that montelukast sodium prevent the mammary carcinogenesis and confirms the role of cysteinyl leukotriene D4-receptor in mammary gland neoplasia.

Introduction

Mammary gland cancer is the leading cause of morbidity and mortality in females all-over the world, and is the second most common type of cancer after lung cancer and the fifth most common cause of cancer death.[12] Chemoprevention is assumed to be an effective way to combat mammary gland neoplasia. Arachidonic acid and linoleic acid, as well as their metabolites (eicosanoids) derived from the arachidonic acid pathway have been implicated in the pathogenesis of cancer, and are now believed to play important roles in tumor promotion, progression, and metastatic disease.[3]5-lipoxygenase pathway may influence the mammary carcinogenesis. Inhibitory effect of 5-lipoxygenase inhibitors nor dihydroguaiaretic acid (NDGA) and esculetin in mammary carcinogenesis was reported. Montelukast inhibited the peripheral tumor metastasis and increased the survival rates of mice in Lewis lung carcinoma metastasis model.[4] In the present study, we have evaluated the chemopreventive efficacy of montelukast sodium in chemically induced mammary carcinogenesis in virgin female Sprague-Dawley rats.

Materials and Methods

Experimental design

A total of 30 virgin female Sprague-Dawley rats of 55 days old were randomly assigned to one of the five experimental groups, each group containing 6 animals. Group 1 animals received vehicle (0.9% sodium chloride (NaCl) intraperitoneally and 1% W/V carboxy methyl cellulose CMC orally) and served as normal controls. Rats in groups 2, 3, 4,and 5 were induced mammary cancer by two intraperitoneal doses of N-methyl urea (NMU) (50 mg/kg/body weight each)in 0.9% NaCl (maintained at 4°C)[5] at 55 days of age and 4 weeks after the prior administration. Group 2 rats received 1% W/V CMC orally and served as disease controls. Treatment groups 3, 4,and 5 rats were orally administered with montelukast sodium 1 mg/kg,[6]10 mg/kg,[4] and tamoxifen10 mg/kg[7] suspended in 1%W/V CMC respectively. Treatment with montelukast sodium, tamoxifen, and 1%W/V CMC started 1 week before and continued upto 8 weeks after the first NMU administration.

The first administration of NMU was considered as day1. Animals were weighed and palpated twice in a week for tumors for a period of 122 days. The time of appearance of the first tumor (latency period) was recorded for individual animals in each group. After 122 days, all the animals were sacrificed by decapitation and observed for the presence of mammary tumors. Relative organ weights of liver, uterus, ovary, and adrenal gland were noted. Tumor incidence, tumor multiplicity (mean ± SEM) and the total number of tumors in each group were recorded. Wet weight and diameter of individual excised tumor was measured. Tumor volume was calculated using the formula 4/3 πr3, where ‘r’is half the average diameter. The tumor tissues and normal mammary gland tissues were dissected fixed in 10% buffered formalin and were further processed for histopathological examination.

The study protocol was approved by Institutional Animal Ethical Committee (IAEC), Swamy Vivekanandha College of Pharmacy (proposal No. SVCP/IAEC/M.Pharm/04/2011) and conducted in accordance with guidelines set by the (Committee for the Purpose of Control and Supervision of Experiment on Animals), India.

Statistical analysis

The values are expressed as mean ±SEM. One - way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests were carried out by using Graph-Pad In Stat software, version 3.01; P<0.05 was considered as significant.

Results

Chemopreventive effects of montelukast sodium in mammary carcinogenesis in female Sprague-Dawley rats are tabulated in Table 1. In montelukast 1 mg/kg, 10 mg/kg, and tamoxifen 10 mg/kg, the percentage of rats with tumors were decreased by 50%, 66.67%, and 83.33% respectively. The tumor latency period was increased in drug treated groups when compared to cancer control group.Total number of tumor, tumor multiplicity, increased tumor burden, and average tumor volume were decreased in treated group when compared to cancer control group.

Table 1

Chemo preventive effects of montelukast sodium in N-methyl urea induced mammarycarcinogenesis in female Sprague-Dawley rats

No macroscopic changes were observed in the selected organs (liver, kidney, stomach, intestine, and lung) due to montelukast sodium and tamoxifen administration. The body weight gain and the relative organ weights of liver, uterus, ovary, and adrenal gland didn’t showed any significant difference.

Histopathological studies of mammary glands revealed that normal control rats showed normal acini lined by single layer of cuboidal epithelium. The rats treated with NMU showed adenocarcinoma with infiltration of secretory epithelium and necrotic areas. NMU + montelukast sodium 1 mg/kg treated rat showed adenoma with almost normal architecture and few multilayered acini showing papillary projection into the lumen. Rat treated with NMU + montelukast sodium 10 mg/kg showed normal architecture of mammary gland with occasional multilayered acini. NMU + tamoxifen 10 mg/kg treated rat showing normal architecture of mammary gland with occasional multilayered acini.

Discussion

This study is the first report on chemopreventive efficacy of montelukast sodium in experimental mammary carcinogenesis in rodents. All the evaluated parameters exhibited a clear dose dependent chemo preventive effect of montelukast sodium in NMU induced mammary carcinogenesis in female Sprague-Dawley rats. The results of the present study suggested that montelukast sodium might have inhibited the tumor initiation and promotion.

In developed and developing countries, there is an increase in the consumption of high dietary fat, which has been postulated to increase the breast cancer risk. More recent studies indicate that diets containing a high proportion of arachidonic acid and linoleic acid are associated with a more advanced disease stage at the time of diagnosis of breast cancer.[891011]

The lipoxygenase inhibitors NDGA and esculetin were inhibited MDA-MB-231cell growth[12] and esculetin inhibited 7, 12-dimethyl benz(a)anthracene induced mammary carcinoma in female Sprague-Dawley rats.[13] Experiments with piroxicam, NDGA, esculetin and other inhibitors of eicosanoid biosynthesis with varying selectivity for enzymes of the prostaglandins and leukotrienes pathways, indicated that MDA-MB-231 cell growth was dependent on leukotrienes rather than prostaglandins production.[12]

Targeting upstream enzymes affects multiple downstream pathways, and may cause adverse effects by disrupting the balance between different arachidonic acid metabolizing pathways. It may be more advisable to target downstream enzymes or receptors. NDGA and esculetin, lipoxygenase inhibitors acts by inhibiting the 5-lipoxygenase enzyme and leads to enhancement of arachidonic metabolism through cyclooxygenase pathway, which is proven to be a risk-factor for the mammary carcinogenesis. In this study, inhibition of downstream receptor, cysteinyl leukotriene D4-receptor effectively inhibited the mammary carcinogenesis without altering the balance between different arachidonic acid metabolizing pathways. The cysteinyl leukotriene receptors were having a role in carcinogenesis and were recently reported to be over expressed in colon tissues derived from cancer patients.[14]

Our results revealed that montelukast sodium suppressed the development of NMU induced mammary carcinogenesis in female Sprague-Dawley rats in a dose dependent manner. Further investigations will be required to evaluate the safety, efficacy and mechanism of montelukast sodium as a chemopreventive agent for mammary cancer treatment in women.