Inhibition of bladder tumour growth by sirolimus in an experimental carcinogenesis model.
OBJECTIVE
To investigate the anticarcinogenic effects of sirolimus 2 mg/kg/day on a rat model of urinary bladder carcinogenesis induced with N-butyl-N(4-hydroxybutyl)nitrosamine (BBN).


METHODS
Thirty-six male Wistar rats were divided into four groups: 1, a control group (eight), given tap water only; 2, a sirolimus control group (eight), given 2 mg/kg/day; 3, a carcinogen (BBN) group (12) exposed to 0.05% BBN; 4, a treatment group (sirolimus/BBN; eight) given 2 mg/kg/day + 0.05% BBN. In the tumour-induction phase, from week 1 to week 8, rats from groups 3 and 4 received BBN ad libitum in drinking water. In the treatment phase, from week 8 to week 20, rats from groups 2 and 4 received sirolimus 2 mg/kg/day by an oesophageal cannula. At week 20 the rats were killed humanely, and the number and size of tumours recorded. The bladders were collected for histological, immunohistochemical and gene expression evaluation. Blood was collected for the determination of several serum proliferative and inflammatory markers. Lipid peroxidation, through serum malondialdehyde (MDA) content, and total antioxidant status (TAS) were also evaluated.


RESULTS
Sirolimus caused a marked inhibition of bladder tumour growth. When compared with group 3, group 4 had a reduced proportion of rats with tumour (three of eight vs eight of 12), and significantly fewer tumours per rat, with a mean (sd) of 1.00 (0.0) vs 1.88 (0.35), and tumour volume per tumour, of 0.30 (0.11) vs 66.1 (48.9) mm³, with less aggressive histological changes, i.e. a marked reduction in hyperplasia (four of eight vs 12/12), high-grade dysplasia (four of eight vs 11/12) and urothelial tumour. Rats in group 4 had no infiltrative bladder cancers and had a lower incidence of high-grade tumours than rats in group 3. The rats in group 4 had decreased serum levels of transforming growth factor-β1, higher levels of tumour necrosis factor-α, and higher levels of serum TAS and a better serum MDA/TAS ratio, a marker of more favourable redox status. Furthermore, the down-regulation of bladder caspase 3 gene expression and the increased Ki67 immunostaining in group 3 were significantly attenuated in group 4.


CONCLUSIONS
Sirolimus given as an oral agent, 2 mg/kg/day, significantly inhibited rat bladder carcinogenesis. Sirolimus reduced the number and volume of tumours and induced a less aggressive histological behaviour. This might be due to antiproliferative and antioxidant properties, as well as to the restoration of apoptotic pathways.
Parada
Belmiro
B
Institute of Pharmacology & Experimental Therapeutics, IBILI, Medicine Faculty, Coimbra University, Portugal. parada.belmiro@gmail.com
Reis
Flávio
F
Figueiredo
Arnaldo
A
Nunes
Pedro
P
Teixeira-Lemos
Edite
E
Garrido
Patrícia
P
Sereno
José
J
Pinto
Rui
R
Cunha
Maria F X
MF
Neto
Paula
P
Santos
Paulo
P
Velada
Isabel
I
Mota
Alfredo
A
Teixeira
Frederico
F
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000627:therapeutic use*; D002085:Butylhydroxybutylnitrosamine; D002273:Carcinogens; D053148:Caspase 3 / Q000378:metabolism; D015536:Down-Regulation; D019394:Ki-67 Antigen / Q000378:metabolism; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D020123:Sirolimus / Q000627:therapeutic use*; D001743:Urinary Bladder / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced / Q000188:drug therapy* / Q000473:pathology
D000903:Antibiotics, Antineoplastic; D002273:Carcinogens; D019394:Ki-67 Antigen; D002085:Butylhydroxybutylnitrosamine; D053148:Caspase 3; D020123:Sirolimus