In vitro and in vivo studies of urinary bladder cancer
Ficheiros
Data
2013
Autores
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
O carcinoma da bexiga constitui uma doença frequente com elevado impacto sócioeconómico
em todo o mundo, pelo que o uso de modelos pré-clínicos é fundamental na
pesquisa de novos tratamentos mais eficazes.
Através da utilização de três linhas celulares comerciais humanas (T24, 5637, HT1376) de
carcinoma da bexiga e do modelo animal de indução química de lesões uroteliais com
recurso à exposição de murganhos ICR à N-butil-N-(4-hidroxibutil) nitrosamina (BBN), foram
estabelecidos como objetivos para esta tese: estudar os efeitos in vitro de várias
concentrações de cisplatina, malato de sunitinibe e meloxicam; analisar in vitro os efeitos da
associação de várias concentrações de malato de sunitinibe com a cisplatina e com o
meloxicam; estudar in vivo os efeitos do meloxicam e da infusão do chá verde nas lesões
uroteliais do murganho induzidos pela administração da BBN; caracterizar
citogeneticamente um carcinoma papilar urotelial de baixo grau de murganho induzido pela
BBN.
As linhas celulares T24, 5637 e HT1376 foram expostas à cisplatina, ao malato de sunitinibe
e ao meloxicam, como drogas isoladas e em diferentes combinações. A proliferação celular,
o ciclo celular, a apoptose, os danos no DNA e a autofagia foram avaliados pelos seguintes
métodos: 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio, citometria de fluxo, anticorpo M30
CytoDEATH, ensaio do cometa, e colorações monodansilcadaverina e laranja de acridina.
Para o estudo da associação de dois fármacos foi calculado o índice de combinação com
recurso ao método de Chou e Talalay. No estudo in vivo para a avaliação do efeito do
meloxicam, murganhos machos foram expostos à BBN na água de bebida durante 12
semanas, sendo depois administrado durante 6 semanas por via intraperitoneal este
fármaco. As lesões uroteliais foram avaliadas pela coloração convencional de hematoxilina e
eosina (H&E). Foi estudado o efeito do meloxicam sobre a função hepática e renal. No
estudo in vivo do efeito da exposição contínua ao chá verde durante 20 semanas, a
administração da BBN foi feita por entubação gástrica durante 10 semanas, sendo
caracterizadas, após sacrifício dos animais, as lesões uroteliais por H&E. A constituição
cromossómica do carcinoma papilar urotelial de baixo grau do murganho, foi analisada por
citogenética convencional e por hibridização in situ por fluorescência.
Isoladamente, a exposição aos três fármacos diminuiu significativamente a proliferação
celular das linhas celulares de carcinoma da bexiga (p<0.05). A cisplatina induziu a
acumulação de células na fase S do ciclo celular, em contrapartida o malato de sunitinibe e
o meloxicam induziram a acumulação de células na fase G0/G1. A percentagem de células
na fracção sub-G0/G1 aumentou após a exposição das mesmas aos três fármacos. O
meloxicam provocou um ligeiro, mas significativo, aumento da apoptose nas linhas celulares
T24 (p=0.006) e 5637 (p<0.001). O malato de sunitinibe e o meloxicam induziram danos no
DNA quando aplicados sobre as linhas celulares em separado. Os três fármacos
isoladamente induziram autofagia. Após a exposição conjunta à cisplatina e ao malato de
sunitinibe, assim como do malato de sunitinibe e ao meloxicam, observamos um índice de
combinação inferior a 1 nas células T24, 5637 e HT1376, o que se traduz num efeito
sinérgico; identificamos também uma redução na proliferação celular, um aumento da
apoptose, danos no DNA e autofagia.
In vivo, a incidência das lesões pré-neoplásicas induzidas pela BBN não foi afetada pelo
tratamento com o meloxicam. No entanto, apesar de não ser estatisticamente significativo, o
desenvolvimento de lesões uroteliais de características malignas foi menor nos animais
tratados com o meloxicam, sem alterações significativas dos parâmetros renais e hepáticos.
No estudo do chá verde, os animais expostos à BBN e ao chá verde não desenvolveram
lesões neoplásicas.
A análise citogenética convencional e molecular do carcinoma papilar urotelial de baixo grau
do murganho revelou a presença de células normais e de células tetraplóides. Em 42% das
células tetraplóides, foi detetada uma translocação recíproca envolvendo os cromossomas 4
e 14 (onde estão localizados os genes humanos CDKN2A e RB1, respetivamente). No
Homem, estes genes estão localizados nos cromossomas 9 e 13, os quais estão associados
ao carcinoma da bexiga.
Urinary bladder carcinoma is a public health issue around the world. The use of preclinical models of urinary bladder carcinoma is an important tool in the search for new and more efficient treatments. Using three human urinary bladder-cancer cell lines and the mouse model of urinary bladder tumours chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), this thesis had as purposes: to evaluate the in vitro effects of cisplatin, sunitinib malate and meloxicam, as single drugs; to evaluate the ability of sunitinib malate to potentiate cisplatin effect and to evaluate the ability of meloxicam to enhance the sunitinib malate activity; to determine the in vivo effects of meloxicam and green tea infusion, as well as to characterize cytogenetically a mouse low-grade papillary urothelial carcinoma induced by BBN. T24, 5637 and HT1376 cell lines were exposed to cisplatin, sunitinib malate and meloxicam as single drugs and in different combinations. Cell proliferation, cell cycle, apoptosis, DNA damage and autophagy were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, flow cytometry, M30 CytoDEATH antibody, comet assay, monodansylcadaverine and acridine orange staining. In the combined approaches, the combination index was calculated based on the Chou and Talalay method. In in vivo studies and in order to evaluate the effects of meloxicam, male mice were exposed to BBN in drinking water, over the course of 12 weeks. Meloxicam was administrated by intraperitoneal route and for 6 consecutively weeks. Tumour development was assessed by haematoxylin and eosin staining (H&E). Renal and hepatic functions were also analyzed. To determine the effects of green tea on male and female mice urinary bladder lesions, BBN was administrated by oral gavage during 10 weeks and green tea was provided ad libitum, over the course of 20 weeks. Urinary bladder lesions were analyzed by H&E staining. The chromosomal constitution of the mouse low-grade papillary urothelial carcinoma was analyzed by conventional cytogenetics and fluorescent in situ hybridization. In isolation, the three drugs decreased the cell proliferation (p<0.05) of urinary bladdercancer cell lines. Cisplatin induced a cell cycle arrest in early S phase, while sunitinib malate and meloxicam induced a cell cycle arrest in G0/G1 phase. The percentage of cells in the sub- G0/G1 fraction increased after treatment with the three drugs. Meloxicam showed a slight, but significant, increase of apoptosis on T24 (p=0.006) and 5637 (p<0.001) cell lines. DNA damage was also induced by sunitinib malate and meloxicam used as single drugs. Autophagy was detected after treatment with the three drugs in isolation. Concerning the simultaneous exposure of cisplatin and sunitinib malate, as well as sunitinib malate and meloxicam, a synergistic effect was observed, with a combination index less than 1 in T24, 5637 and HT1376 cells; we also observed an improved decrease cell proliferation, increase apoptosis, DNA damage and autophagy. In vivo, the incidence of pre-neoplastic lesions induced by BBN was not affected by meloxicam treatment. However, although not statistically significant, the development of neoplastic lesions was inhibited by meloxicam treatment, without significant alterations of renal and hepatic parameters. In the green tea study, animals exposed to BBN and green tea did not develop urinary bladder neoplastic lesions. The conventional and molecular cytogenetic analysis of the mouse low-grade papillary urothelial carcinoma revealed the presence of normal and tetraploid cells. In 42% of the tetraploid cells, a reciprocal translocation involving chromosomes 4 and 14 (where are located the CDKN2A and the RB1 human genes, respectively) was detected. In humans, these genes map to chromosomes 9 and 13, which are associated with urinary bladder cancer.
Urinary bladder carcinoma is a public health issue around the world. The use of preclinical models of urinary bladder carcinoma is an important tool in the search for new and more efficient treatments. Using three human urinary bladder-cancer cell lines and the mouse model of urinary bladder tumours chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), this thesis had as purposes: to evaluate the in vitro effects of cisplatin, sunitinib malate and meloxicam, as single drugs; to evaluate the ability of sunitinib malate to potentiate cisplatin effect and to evaluate the ability of meloxicam to enhance the sunitinib malate activity; to determine the in vivo effects of meloxicam and green tea infusion, as well as to characterize cytogenetically a mouse low-grade papillary urothelial carcinoma induced by BBN. T24, 5637 and HT1376 cell lines were exposed to cisplatin, sunitinib malate and meloxicam as single drugs and in different combinations. Cell proliferation, cell cycle, apoptosis, DNA damage and autophagy were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, flow cytometry, M30 CytoDEATH antibody, comet assay, monodansylcadaverine and acridine orange staining. In the combined approaches, the combination index was calculated based on the Chou and Talalay method. In in vivo studies and in order to evaluate the effects of meloxicam, male mice were exposed to BBN in drinking water, over the course of 12 weeks. Meloxicam was administrated by intraperitoneal route and for 6 consecutively weeks. Tumour development was assessed by haematoxylin and eosin staining (H&E). Renal and hepatic functions were also analyzed. To determine the effects of green tea on male and female mice urinary bladder lesions, BBN was administrated by oral gavage during 10 weeks and green tea was provided ad libitum, over the course of 20 weeks. Urinary bladder lesions were analyzed by H&E staining. The chromosomal constitution of the mouse low-grade papillary urothelial carcinoma was analyzed by conventional cytogenetics and fluorescent in situ hybridization. In isolation, the three drugs decreased the cell proliferation (p<0.05) of urinary bladdercancer cell lines. Cisplatin induced a cell cycle arrest in early S phase, while sunitinib malate and meloxicam induced a cell cycle arrest in G0/G1 phase. The percentage of cells in the sub- G0/G1 fraction increased after treatment with the three drugs. Meloxicam showed a slight, but significant, increase of apoptosis on T24 (p=0.006) and 5637 (p<0.001) cell lines. DNA damage was also induced by sunitinib malate and meloxicam used as single drugs. Autophagy was detected after treatment with the three drugs in isolation. Concerning the simultaneous exposure of cisplatin and sunitinib malate, as well as sunitinib malate and meloxicam, a synergistic effect was observed, with a combination index less than 1 in T24, 5637 and HT1376 cells; we also observed an improved decrease cell proliferation, increase apoptosis, DNA damage and autophagy. In vivo, the incidence of pre-neoplastic lesions induced by BBN was not affected by meloxicam treatment. However, although not statistically significant, the development of neoplastic lesions was inhibited by meloxicam treatment, without significant alterations of renal and hepatic parameters. In the green tea study, animals exposed to BBN and green tea did not develop urinary bladder neoplastic lesions. The conventional and molecular cytogenetic analysis of the mouse low-grade papillary urothelial carcinoma revealed the presence of normal and tetraploid cells. In 42% of the tetraploid cells, a reciprocal translocation involving chromosomes 4 and 14 (where are located the CDKN2A and the RB1 human genes, respectively) was detected. In humans, these genes map to chromosomes 9 and 13, which are associated with urinary bladder cancer.
Descrição
Tese de Doutoramento em Ciências Veterinárias, Ramo Biomedicina
Palavras-chave
Cancro da bexiga , Animal de laboratório (murganho) , In vitro , Ensaios de seleção de medicamentos antitumoral , Camellia sinusis (chá verde)