Etoricoxib in the Prevention of Rat Mammary Carcinogenesis

Orendáš P., I. Ahlers, P. Kubatka, E. Ahlersová, B. Bojková, M. Kassayová, L. Friedmanová, J. Kisková, I. Ďatelinka, M. Starostová: Etoricoxib in the Prevention of Rat Mammary Carcinogenesis. Acta Vet. Brno 2007, 76: 613-618. Several experimental studies suggest that non-steroidal antiinflammatory drugs have chemopreventive effects in mammary carcinogenesis. In this study, tumour suppressive effects of a selective inhibitor of cyclooxygenase-2 (COX-2) etoricoxib in the prevention of N-methyl-Nnitrosourea (NMU)-induced mammary carcinogenesis in Sprague-Dawley rats were evaluated. Etoricoxib was administered in the diet, at two concentrations: 1) 0.01 mg/g (ETO 0.001%) and 2) 0.025 mg/g (ETO 0.0025%). Although the chemopreventive effects were not statistically significant, remarkable tumour suppressive effects with the concentration of ETO 0.0025% were recorded. The incidence decreased by 4.31% and tumour frequency per group decreased by 6.67% when compared to the control group. Latency (the period from carcinogen administration to the first tumour appearance) increased by 7.28% in dose-dependent manner. The results of our experiments point to dose-dependent tumour suppressive effects of a higher concentration of etoricoxib (ETO 0.0025%) when compared to the control group. They suggest that higher etoricoxib concentrations may enhance its tumour suppressive effects. Mammary carcinogenesis, prevention, rats, coxibs, NSAIDs Mammary gland cancer presents a serious health issue worldwide. Chemoprevention is assumed to be an effective way to combat carcinogenesis. Non-steroidal anti-inflammatory drugs (NSAIDs) are important drugs suppressing manifestations of inflammation. They are frequently administered in the treatment of rheumatoid arthritis to alleviate pain and fever. Recently, their preventive effects in carcinogenesis were reported. Clinical studies aimed at long-term application of various NSAIDs reported a pronounced decline in the risk of colon (Thun et al. 1991), mammary gland (Harris et al. 1999), prostatic (Nelson and Harris 2000), and lung cancer (Harris et al. 2002). The experimental studies with laboratory animals proved chemopreventive effects of NSAIDs in various types of neoplasia, including mammary gland cancer. Similarly, our group proved the chemopreventive effects of indomethacin administered in drinking water (Môciková-Kalická et al. 2001), nimesulide applied subcutaneously (Kubatka et al. 2002) and those of rofecoxib administered with the diet in mammary carcinogenesis in female rats (Kubatka et al. 2003). The primary effect of NSAIDs is the suppression of prostanoid synthesis by inhibition of cyclooxygenase (COX) activity. Two isoforms of COX were identified: constitutive COX1 and inducible COX-2 isoform found predominantly under pathological conditions, e.g. inflammation and neoplasia. Expression of COX-2 may influence the process of carcinogenesis via several mechanisms: peroxidase activity of COX-2 supports the conversion of procarcinogens to carcinogens (Fosslien 2000), inhibitory effect of prostaglandins in immune reactions was ACTA VET. BRNO 2007, 76: 613-618; doi:10.2754/avb200776040613 Address for correspondence: Prof. MUDr. Ivan Ahlers, DrSc. Department of Animal Physiology Institute of Biology and Ecology Faculty of Science, P.J. Šafárik University Moyzesova 11, 041 67 Košice, Slovak Republic Phone: +421 905 133 775 Fax: +421 556 222 124 E-mail:iahlers@kosice.upjs.sk http://www.vfu.cz/acta-vet/actavet.htm found (Rita and Young 1994), as well as angiogenetic (Tsujii 1998) and antiapoptotic effects (Fosslien 2000). On the other hand, enhanced invasion of tumour cells with increased expression of COX-2 was observed. Brueggemeier et al. (1999) pointed to linear dependence between aromatase gene (CYP 19) expression and expression of COX-2 in mammary carcinoma. The above mentioned facts predetermine the use of NSAIDs in mammary carcinogenesis prevention. From the pharmaco-dynamic point of view, inhibitors COX-1 (acetylsalicylic acid, indomethacin, ibuprofen) display serious side effects on the gastrointestinal tract associated with their long-term application. These undesirable effects are markedly reduced in administration of COX-2 inhibitors (nimesulide, meloxicam, coxibs), supporting their preference in long-term use. Etoricoxib represents new NSAID generation selectively inhibiting COX-2 activity in human cells in vitro with minimal COX-1 activity inhibition. The present study was aimed at evaluation of chemopreventive effects of etoricoxib in mammary carcinogenesis in female Sprague-Dawley rats. Materials and Methods Female Sprague-Dawley rats obtained from Charles River Laboratories (Budapest, Hungary) aged 33 37 days, weighing 101 125 g were used in the experiment. The animals were adapted to standard vivarium conditions with the temperature of 23 ± 2 °C, relative humidity 60 70%, artificial regimen light : dark = 12 : 12 h, with lights on from 7:00 h (light intensity 150 lux per cage, light source: fluorescent lamps Tesla 40 W). During the experiment, animals had access to tap water ad libitum. Etoricoxib (Merck Research Laboratories, Merck Frosst, Montreal, Canada) was administered in a chow ST-1 Bergman (Jeseničky u Prahy, Czech Republic). Etoricoxib was administered in the chow at two concentrations: 0.01 mg/1 g (ETO 0.001% or ETO10) and 0.025 mg/1 g (ETO 0.0025% or ETO25). Mammary carcinogenesis was induced by N-methyl-N-nitrosourea (NMU) (Sigma, Deisenhofen, Germany), administered at two intraperitoneal doses (50 mg/kg) in a 6-day interval between postnatal days 42 48. NMU was freshly prepared by dissolving in isotonic saline solution (0.5 ml per animal). Chemoprevention with etoricoxib began 7 days before carcinogen administration and lasted until the end of the experiment (20 weeks after NMU administration). Animals were divided into three experimental groups: 1) control group without chemoprevention; 2) chemoprevention with etoricoxib at a concentration of 0.001% (ETO 0.001%); 3) chemoprevention with etoricoxib at a concentration of 0.0025% (ETO 0.0025%). Each group consisted of 20 animals. The animals were weekly weighed and palpated in order to record the incidence, number, location and size of tumours. In the 21st week of the experiment (dated from the first NMU application), the animals were sacrificed by quick decapitation. Mammary tumours were excised, weighed and tumour size was recorded. The following basic variables of mammary carcinogenesis were assessed in each group: 1) tumour incidence as the percentage representation of tumour-bearing animals 2) tumour frequency per group as the average number of tumours per group 3) tumour frequency per animal as average number of tumours per tumour-bearing animals 4) average tumour volume 5) latency period determined by the period from carcinogen administration to the appearance of the first tumour in an animal. The effect of a chemopreventive agent on the body weight gain (evaluated from the initiation until the end of the experiment), food and water intake was observed. Food and water intake during 24 h was observed in the 6th, 11th and 20th week after carcinogen administration in 6 measurements (twice in a given week). Tumour incidence was evaluated by Mann-Whitney U-test, other variables by one-way analysis of variance or Kruskal-Wallis test (P < 0.05 level). Tumour volumes were calculated according to formula V (mm3) =  x S1 x S2 /12; where (S1  S2), S1 is tumour width and S2 tumour length, S1 and S2 were measured perpendicularly. The experiment was carried out from October 2005 to March 2006.

Mammary gland cancer presents a serious health issue worldwide.Chemoprevention is assumed to be an effective way to combat carcinogenesis.Non-steroidal anti-inflammatory drugs (NSAIDs) are important drugs suppressing manifestations of inflammation.They are frequently administered in the treatment of rheumatoid arthritis to alleviate pain and fever.Recently, their preventive effects in carcinogenesis were reported.Clinical studies aimed at long-term application of various NSAIDs reported a pronounced decline in the risk of colon (Thun et al. 1991), mammary gland (Harris et al. 1999), prostatic (Nelson and Harris 2000), and lung cancer (Harris et al. 2002).The experimental studies with laboratory animals proved chemopreventive effects of NSAIDs in various types of neoplasia, including mammary gland cancer.
Similarly, our group proved the chemopreventive effects of indomethacin administered in drinking water (Môciková-Kalická et al. 2001), nimesulide applied subcutaneously (Kubatka et al. 2002) and those of rofecoxib administered with the diet in mammary carcinogenesis in female rats (Kubatka et al. 2003).
The primary effect of NSAIDs is the suppression of prostanoid synthesis by inhibition of cyclooxygenase (COX) activity.Two isoforms of COX were identified: constitutive COX-1 and inducible COX-2 isoform found predominantly under pathological conditions, e.g.inflammation and neoplasia.
Expression of COX-2 may influence the process of carcinogenesis via several mechanisms: peroxidase activity of COX-2 supports the conversion of procarcinogens to carcinogens (Fosslien 2000), inhibitory effect of prostaglandins in immune reactions was found (Rita and Young 1994), as well as angiogenetic (Tsujii 1998) and antiapoptotic effects (Fosslien 2000).
On the other hand, enhanced invasion of tumour cells with increased expression of COX-2 was observed.Brueggemeier et al. (1999) pointed to linear dependence between aromatase gene (CYP 19) expression and expression of COX-2 in mammary carcinoma.The above mentioned facts predetermine the use of NSAIDs in mammary carcinogenesis prevention.
From the pharmaco-dynamic point of view, inhibitors COX-1 (acetylsalicylic acid, indomethacin, ibuprofen) display serious side effects on the gastrointestinal tract associated with their long-term application.These undesirable effects are markedly reduced in administration of COX-2 inhibitors (nimesulide, meloxicam, coxibs), supporting their preference in long-term use.Etoricoxib represents new NSAID generation selectively inhibiting COX-2 activity in human cells in vitro with minimal COX-1 activity inhibition.
The present study was aimed at evaluation of chemopreventive effects of etoricoxib in mammary carcinogenesis in female Sprague-Dawley rats.
Chemoprevention with etoricoxib began 7 days before carcinogen administration and lasted until the end of the experiment (20 weeks after NMU administration).Animals were divided into three experimental groups: 1) control group without chemoprevention; 2) chemoprevention with etoricoxib at a concentration of 0.001% (ETO 0.001%); 3) chemoprevention with etoricoxib at a concentration of 0.0025% (ETO 0.0025%).Each group consisted of 20 animals.The animals were weekly weighed and palpated in order to record the incidence, number, location and size of tumours.In the 21 st week of the experiment (dated from the first NMU application), the animals were sacrificed by quick decapitation.Mammary tumours were excised, weighed and tumour size was recorded.The following basic variables of mammary carcinogenesis were assessed in each group: 1) tumour incidence as the percentage representation of tumour-bearing animals 2) tumour frequency per group as the average number of tumours per group 3) tumour frequency per animal as average number of tumours per tumour-bearing animals 4) average tumour volume 5) latency period determined by the period from carcinogen administration to the appearance of the first tumour in an animal.
The effect of a chemopreventive agent on the body weight gain (evaluated from the initiation until the end of the experiment), food and water intake was observed.Food and water intake during 24 h was observed in the 6 th , 11 th and 20 th week after carcinogen administration in 6 measurements (twice in a given week).
Tumour incidence was evaluated by Mann-Whitney U-test, other variables by one-way analysis of variance or Kruskal-Wallis test (P < 0.05 level).Tumour volumes were calculated according to formula V (mm 3 ) =  x S 1 2 x S 2 /12; where (S 1  S 2 ), S 1 is tumour width and S 2 tumour length, S 1 and S 2 were measured perpendicularly.The experiment was carried out from October 2005 to March 2006.

Results
Preventive effects of etoricoxib in female Sprague-Dawley rat mammary carcinogenesis are summarized in Table 1.
Etoricoxib administered at a lower dose in the experimental group ETO 10 did not significantly influence the observed indicators of mammary carcinogenesis (incidence, frequency, tumour volume, latency).In the experimental group ETO 25 the incidence slightly decreased by 4.31%, tumour frequency per group by 6.67%, frequency per animal by 2.44%.The latency period was prolonged by 7 days.No macroscopic changes were observed in the selected organs (liver, kidneys, small intestine, stomach and lungs).
Daily food intake per rat in ETO 10 ranged between 19.95 g and 21.53 g and daily drug doses ranged between 0.629 -0.885 μg etoricoxib/g b.w.In group ETO 25 the daily food intake per rat was between 19.25 and 20.43 g and daily drug dose was between 1.66 and 2.19 μg etoricoxib/g/b.w.The food intake revealed a significant difference in the 11 th week between groups ETO 10 and ETO 25 (10.47%decrease in food intake in group ETO 25 as opposed to ETO 10) and in the 20 th week -between CONT and ETO 25 (10.55%decrease in food intake in group ETO 25 in comparison to CONT) (Table 2).
Daily water intake per rat in the 6 th week ranged between 34.75 ml and 37.5 ml, in the 11 th week between 35.25 ml and 42.29 ml, in the 20 th week between 41.5 ml and 47.09 ml.Significant difference in water intake was recorded in the 11 th week between groups CONT and ETO 25 (13.5% decrease in water intake in group ETO 25 as opposed to CONT) and between ETO 10 and ETO 25 (7.94% decrease in water intake in group ETO 25 as opposed to ETO 10) (Table 3).

Experimental groups
CONT -control group of animals with administered carcinogen without chemoprevention ETO 10 (0.001%) -animal group with administered carcinogen + chemoprevention by etoricoxib received in chow at a concentration of 0.01 mg/g ETO 25 (0.0025%) -group with administered carcinogen + chemoprevention by etoricoxib received in chow at a concentration of 0.025 mg/g 615  et al. 2000;Alshafie et al. 2000;Abou-Issa et al. 2001).We observed a clear dose-dependent rofecoxib action resulting in decreased mammary tumour incidence, frequency and volume in female Sprague-Dawley rats (Kubatka et al. 2003).
Our new findings are the first report on possible preventive effects of etoricoxib in experimental mammary carcinogenesis.The results indicate some influence on carcinogenesis associated with a higher daily dose of coxib.It may be assumed that the dose was insufficient and lowered by decreased food (and water) intake in these experimental animals between the 11 th and 20 th weeks.The issue of dosage may play a vital role in further concerns about possible prevention of tumorigenesis with long-term administration of NSAIDs.Alternative modes of construction and use of new NSAIDs (e.g.NO-containing drugs) will be searched in the future, for ways to prevent imbalance in blocked synthesis of prostaglandin, thromboxan, prostacycline and other substances production.The effort to attain highly selective COX-systems is likely to be supported by reduction of undesirable side effects mainly in cardiovascular events.

Table 1 .
Chemopreventive effects of etoricoxib in NMU-induced mammary carcinogenesis in female Sprague-Dawley rats in the final 21 st week of the experiment *Data are expressed as means ± SEM **Data are expressed as means ± SEM Significantly different, s P < 0.05 vs. CONT

Table 2 .
Effects of etoricoxib on food intakeData on food intake (g) are expressed as means ± SEM.Values in brackets are calculated as percentage deviation ETO 25 vs. CONT (100%).Significantly different, a P < 0.05 vs. CONT.b Values in brackets are calculated as percentage deviation ETO 25 vs. ETO 10 (100%).Significantly different, b P < 0.05 vs. ETO 10.

Table 3 .
Effects of etoricoxib on water intake Data on water intake (ml) are expressed as means ± SEM.