PET/CT com 18F-FDG como ferramenta de avaliação do desenvolvimento de carcinoma hepatocelular secundário a doença hepática gordurosa não alcoólica em modelo experimental / 18f-fdg pet/ct as an assessment tool of hepatocellular carcinoma secondary to non-alcoholic fatty liver disease development in experimental model

ABSTRACT BACKGROUND: Hepatocellular carcinoma (HCC) can be the last step of non-alcoholic fatty liver disease (NAFLD) evolution. Experimental models are crucial to elucidate the pathogenesis of HCC secondary to NAFLD. The 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) plays an important role in evaluating HCC development and progression. OBJECTIVE: To standardize the imaging method of PET/CT with 18F-FDG as an evaluation tool of the experimental model of HCC secondary to NAFLD. METHODS: Ten male Sprague-Dawley rats were fed with choline-deficient high-fat diet and diethylnitrosamine (DEN) in the drinking water for 16 weeks and then received 1 mL of saline solution (0.9%) daily by gavage for three weeks. At the 16th and 19th weeks, abdominal ultrasonography (USG) was performed. 18F-FDG PET/CT images were obtained before the beginning of experiment (week 0) and at the end (week 19). Histological and immunohistochemically analysis were also performed. RESULTS: The USG results showed a homogeneous group at the 16th week with an average of 4.6±2.74 nodules per animal. At the 19th week, PET/CT findings demonstrated an average of 8.5±3.7 nodules per animal. The mean values of SUVmed and SUVmax were 2.186±0.1698 and 3.8±1.74, respectively. The average number of nodules per animal in the histological analysis was 5.5±1.5. From all nodules, 4.6% were classified as well-differentiated HCC and 81.8% were classified as poorly-differentiated HCC. CONCLUSION: 18F-FDG PET/CT was able to evaluate the development of HCC in an experimental model of NAFLD non-invasively. From the standardization of PET/CT in this model, it is possible to use this tool in future studies to monitor, in vivo and non-invasively, the progression of HCC.

RESUMO BACKGROUND: O carcinoma hepatocelular (CHC) pode ser a última fase da doença hepática gordurosa não alcoólica (DHGNA). Modelos experimentais são cruciais para elucidação da patogênese do CHC secundário a DHGNA. A tomografia por emissão de pósitrons/tomografia computadorizada (PET/TC) com 2-desoxi-2-(18F)fluoro-D-glicose (18F-FDG) desempenha um importante papel na avaliação do desenvolvimento e progressão do CHC. OBJETIVO: Padronizar a metodologia de imagem por PET/TC com 18F-FDG como uma ferramenta de avaliação do modelo experimental de CHC secundário a DHGNA. MÉTODOS: Dez ratos Sprague-Dawley machos foram alimentados com dieta hiperlipídica deficiente em colina associada a dietilnitrosamina (DEN) na água de beber por 16 semanas e depois receberam 1 mL de solução salina (0,9%) por gavagem diariamente por três semanas. Nas 16ª e 19ª semanas, foi realizada a ultrassonografia abdominal. As imagens do PET/TC com 18F-FDG foram obtidas antes do início do experimento (semana 0) e no final (semana 19). Análises histológica e imunohistoquímica também foram realizadas. RESULTADOS: Os resultados da ultrassonografia demonstraram um grupo homogêneo na 16ª semana com uma média de 4,6±2,74 nódulos por animal. Na 19ª semana, os achados do PET/CT demonstraram uma média de 8,5±3,7 nódulos por animal. Os valores médios de SUVmed e SUVmáx foram 2,186±0,1698 e 3,8±1,74, respectivamente. A média do número de nódulos na análise histológica foi de 5,5±1,5. De todos os nódulos, 4,6% foram classificados como bem diferenciados e 81,8% foram classificados como CHC pouco diferenciado. CONCLUSÃO: O PET/TC com 18F-FDG foi capaz de avaliar o desenvolvimento do CHC secundário a DHGNA de forma não invasiva. A partir da padronização do PET/CT neste modelo, faz-se possível a utilização desta ferramenta em futuros estudos para monitorar, in vivo e de forma não invasiva, a progressão do CHC.

Polydatin inhibits cell proliferation, invasion and migration, and induces cell apoptosis in hepatocellular carcinoma

Polydatin, a small molecule from Polygonum cuspidatum, has many biological functions, particularly anti-cancer effects. However, the anti-cancer effects of polydatin in hepatocellular carcinoma (HCC) have not been examined yet. In the present study, MTT assay, BrdU assay, transwell invasion assay, and wound healing assay were performed to determine cell proliferation, invasion and migration. Flow cytometry and TUNEL assay were used to measure cell apoptosis. Quantitative real-time PCR and western blotting assays were used to determine mRNA and protein expression levels. Xenograft experiment was performed to determine the in vivo anti-tumor effect of polydatin. Immunostaining was performed to analyze the expression of caspase-3 and Ki-67. Our results showed that polydatin inhibited cell proliferation in a concentration-dependent and time-dependent manner in the HCC cell lines. Polydatin also induced cell apoptosis in a concentration-dependent manner possibly via increasing the caspase-3 activity, and up-regulating the protein expression of caspase-3, caspase-9, Bax, and down-regulating the protein expression of Bcl-2. In addition, polydatin treatment had an inhibitory effect on cell proliferation, invasion and migration in HCC cell lines. Polydatin treatment also suppressed the Wnt/beta-catenin signaling activities in HCC cells. Polydatin treatment significantly reduced tumor growth in nude mice inoculated with HepG2 cells, suppressed the expression of Ki-67, and increased caspase-3 expression and TUNEL activity. Our data indicated the important role of polydatin for the suppression of HCC progression.

In vivo effects of adenosine 5´-triphosphate on rat preneoplastic liver / Efecto in vivo de adenosina 5´-trifosfato sobre el hígado preneoplásico de la rata

The utilization of adenosine 5´-triphosphate (ATP ) infusions to inhibit the growth of some human and animals tumors was based on the anticancer activity observed in in vitro and in vivo experiments, but contradictory results make the use of ATP in clinical practice rather controversial. Moreover, there is no literature regarding the use of ATP infusions to treat hepatocarcinomas. The purpose of this study was to investigate whether ATP prevents in vivo oncogenesis in very-early-stage cancer cells in a well characterized two-stage model of hepatocarcinogenesis in the rat. As we could not preclude the possible effect due to the intrinsic properties of adenosine, a known tumorigenic product of ATP hydrolysis, the effect of the administration of adenosine was also studied. Animals were divided in groups: rats submitted to the two stage preneoplasia initiation/promotion model of hepatocarcinogenesis, rats treated with intraperitoneal ATP or adenosine during the two phases of the model and appropriate control groups. The number and volume of preneoplastic foci per liver identified by the expression of glutathione S-transferase placental type and the number of proliferating nuclear antigen positive cells significantly increased in ATP and adenosine treated groups. Taken together, these results indicate that in this preneoplastic liver model, ATP as well as adenosine disturb the balance between apoptosis and proliferation contributing to malignant transformation.

La utilización de adenosina 5´-trifosfato (ATP ) para inhibir el crecimiento de algunos tumores en humanos y en animales se basa en la actividad anticancerígena observada en experimentos in vitro e in vivo. El uso del ATP en la práctica clínica es discutido debido a resultados contradictorios. Por otra parte, no existen antecedentes del uso de ATP en el tratamiento de hepatocarcinomas. El objetivo del presente estudio fue determinar si el ATP previene la oncogénesis in vivo en un modelo de preneoplasia hepática murina de dos etapas. Para determinar la probable contribución de la adenosina, producto de la hidrólisis de ATP y descrita como tumorigénica, se estudió también el efecto del nucleósido exógeno sobre los focos preneoplásicos. Los animales se dividieron en grupos: ratas sometidas al modelo de preneoplasia de iniciación/promoción, ratas tratadas con ATP o adenosina intraperitonealmente durante las dos fases del modelo y los correspondientes grupos controles. El número y el volumen de focos preneoplásicos por hígado, identificados por la expresión de la forma placentaria de la glutation S- transferasa de rata y el número de células positivas para el antígeno nuclear proliferante, aumentaron significativamente en los grupos tratados con ATP y adenosina. Los resultados en su conjunto indican que en este modelo preneoplásico, el ATP y la adenosina alteran el balance entre apoptosis y proliferación, contribuyendo a la transformación maligna.

Morphine does no promote esophageal carcinogenesis in rats exposed to diethylnitrosamine

BACKGROUND: The high incidence of esophageal cancer in the north of Iran has been associated to the consumption of opium and exposure to nitrosamines. Diethylnitrosamine has an established potential of producing experimental cancer in the esophagus and liver. AIM: To evaluate by histopathology the effect of oral administration of morphine and diethylnitrosamine during 23 weeks on the hepatic and esophageal carcinogenesis on 176 rats. METHODS: We divided the rats into the following groups: Morph: morphine; Den: diethylnitrosamine; Den+morph: Den and morphine in the same solution; Den/morph: Den and morphine in different solutions and days. RESULTS: Morphine did not promote neoplasias. The highest neoplastic incidents were found: a) in the esophagus, Den in relation to Den/morph and Den+morph (71.1 percent, 55.8 percent, and 50.0 percent); b) in the liver, Den and Den/morph in relation to Den+morph (73.8 percent, 81.4 percent, and 40.9 percent); c) higher incident of hepatic neoplasia than esophageal in Den/morph (81.4 percent and 55.8 percent). Different doses of diethylnitrosamine were ingested among the groups Den, Den/morph, and Den+morph, respectively 2.9, 2.8, and 2.3 mg/kg/day. CONCLUSIONS: These results show that the morphine did not promote esophageal carcinogenesis and may have stimulated the hepatic metabolism of the first pass of the carcinogen.

RACIONAL: A alta incidência de câncer esofagiano no norte do Irã foi associada ao consumo de ópio e exposição às nitrosaminas. A dietilnitrosamina possui potencial estabelecido de produzir câncer experimental em esôfago e fígado. OBJETIVO: Avaliar por histopatologia o efeito da administração oral de morfina e de dietilnitrosamina na carcinogênese esofágica e hepática em ratos. MÉTODOS: Durante 23 semanas, 176 ratos ingeriram diferentes soluções, sendo divididos em grupos: Morf: morfina; Den: dietilnitrosamina; Den+morf: dietilnitrosamina e morfina numa mesma solução; Den/morf: dietilnitrosamina e morfina em diferentes soluções e dias. RESULTADOS: Morf não promoveu neoplasias. Encontraram-se maiores incidências neoplásicas: a) no esôfago, Den em relação à Den/morf e Den+morf (71,1 por cento, 55,8 por cento e 50,0 por cento); b) no fígado, Den e Den/morf em relação à Den+morf (73,8 por cento, 81,4 por cento e 40,9 por cento); c) maior incidência de neoplasia hepática do que esofágica em Den/morf (81,4 por cento e 55,8 por cento). Diferentes doses de dietilnitrosamina foram ingeridas entre os grupos Den, Den/morf e Den+morf, respectivamente 2,9, 2,8 e 2,3 mg/kg/dia. CONCLUSÕES: A morfina não promoveu a carcinogênese esofágica e pode ter estimulado o metabolismo hepático de primeira passagem do carcinógeno.

Effects of acetylsalicylic acid and acetic acid solutions on VX2 liver carcinoma in rabbits: in vivo analysis

PURPOSE: To analyze, in vitro, the effects of acetylsalicylic acid (aspirin) and acetic acid solutions on VX2 carcinoma cells in the liver of rabbits with VX2 hepatic tumors; to determine the histolytic and anatomopathological characteristics of the solutions; and to evaluate the eventual biochemical and hepatic changes. METHODS: A total of 48 rabbits were evaluated. The animals were randomized into two groups, protocol 3 (study group) and protocol 4 (controls), and each group was then subdivided into 3 subgroups. Four days after implantation of the tumor in the liver, median laparotomy was performed with a 0.4-ml injection of a solution of either aspirin (5.0 percent), acetic acid (5.0 percent) or saline. The animals were sacrificed after 24 hours (protocol 3) or after 11 days (protocol 4). Body weight, clinical evolution and biochemical levels, as well as the abdominal and thoracic cavities, were evaluated, and liver microscopy was performed. RESULTS: No changes in clinical evolution, body weight or biochemical levels were reported. However, an increase in alkaline phosphatase was observed in protocol 4 (controls). The tumor was eliminated in both protocols. CONCLUSION: Acetylsalicylic acid and acetic acid solutions cause the destruction of experimental hepatic tumors.

OBJETIVO: Analisar os efeitos das soluções de aspirina e de ácido acético, in vivo, em fígado de coelhos portadores de tumor hepático VX2, verificando o efeito histolítico e anatomo-patológico das soluções e eventuais alterações bioquímicas hepáticas. MÉTODOS: Utilizou-se 48 coelhos, divididos em 2 protocolos experimentais(3 e 4), subdivididos em 3 grupos cada. Após 4 dias da implantação do tumor no fígado, procedeu-se a laparotomia mediana, com injeção de 0,4 ml da solução de aspirina (5,0 por cento), de ácido acético (5,0 por cento) e solução salina; o sacrifício ocorreu apos 24 horas (protocolo 3) e 11 dias (protocolo 4); avaliou-se o peso, evolução clinica, dosagens bioquímicas, cavidade abdominal e torácica e microscopia do fígado. RESULTADOS: Não foram observadas alterações na evolução clinica, peso e nas dosagens bioquímicas, apenas elevação da fosfatase alcalina no grupo controle do protocolo 4. Observamos desaparecimento do tumor em ambos os protocolos. CONCLUSÃO: As soluções de ácido acético e ácido acetilsalicílico acarretam destruição do tumor hepático experimental.

A relação do uso crônico de fenobarbital com áreas potencialmente pré-neoplásicas em fígado de ratos / Potentially pre-neoplasics areas in rat's liver associated to chronic use of phenobarbital

RACIONAL: O fenobarbital é utilizado em modelos experimentais não só por ser um importante agente promotor da carcinogênese em fígado de ratos, como também por ser não-genotóxico, órgão-específico e dose-dependente. OBJETIVOS: Avaliar o efeito da administração diária de fenobarbital em ratos, desde o nascimento até os 24 meses de idade, na ausência concomitante de administração de agentes químicos iniciadores da carcinogênese. MATERIAL E MÉTODOS: Um grupo controle de ratos machos Wistar recebeu dieta básica e a esta, do outro grupo, foi adicionado diariamente, fenobarbital a 0,05 por cento, durante 24 meses. Cortes dos lobos médio e direito do fígado foram submetidos ao processamento histológico e corados pela hematoxilina-eosina e coloração imunoistoquímica para a glutationa S-transferase forma placentária. RESULTADOS: Detectaram-se áreas glutationa S-transferase forma placentária positivas em ambos os grupos e as imagens foram analisadas quanto ao número e à extensão da superfície, mediante análise de imagem por histomorfometria. CONCLUSÃO: O uso crônico de fenobarbital não alterou o número de áreas glutationa S-transferase forma placentária positivas, havendo, no entanto, aumento no tamanho médio de áreas glutationa S-transferase forma placentária positivas, com conseqüente aumento da superfície glutationa S-transferase forma placentária positiva, sendo este aumento provavelmente relacionado a maior capacidade evolutiva dessas lesões e possível irreversibilidade das mesmas.

BACKGROUND: Phenobarbital has been used in experimental models because it is an important agent of carcinogenesis promotion in the liver of rats, and it is also non-genotoxic, organ-specific and dose-dependent. AIM: To evaluate the effects of the daily administration of phenobarbital in old rats treated with phenobarbital since their birth up to 24 months of age, in the absence of concomitant administration of chemical agents, which initiate carcinogenesis. PATIENTS AND METHODS: A control group of male Wistar rats was fed with a basic diet and a second group was fed with the same basic diet added of 0.05 percent of phenobarbital, for a period of 24 months. Medium and right liver fragments were submitted to the histological processing and they were stained by hematoxiciline and eosin and were immunohystochemically colored to glutathione S-transferase placentary form. RESULTS: Glutathione S-transferase placentary positive zones were detected in both groups and the images were analyzed concerning their number and surface extension through the technique of histometry analyses. CONCLUSION: Chronic use of phenobarbital did not modify the number of glutathione S-transferase placentary form positive areas. Although, data indicates that glutathione S-transferase placentary form positive areas media size are increased, probably because there are an increase in their evolution capacity and irreversibility.

Effects of cessation of alcohol exposure on rat hepatocarcinogenesis.

Cessation of long-term alcohol exposure is reported to enhance rat hepatocarcinogenesis. The purpose of the present study was to assess this possibility using glutathione-S transferase placental form (GST-P) positive foci as end point lesions. All rats were treated with a single i.p. injection of diethylnitrosamine (DEN) (200 mg/kg body weight) and then given a MF pellet diet for 2 weeks. Thereafter, the animals were maintained on: alcohol liquid diet in which 36% of total calories were provided by alcohol (5% Al diet) for 6 weeks (group 1); control liquid diet (C diet) for 6 weeks (group 2); 5% Al diet for 6 weeks and subsequently C diet for 4 weeks (group 3); 5% Al diet for 10 weeks (group 4); or C diet for 10 weeks (group 5). All rats were subjected to two thirds partial hepatectomy at 3 weeks after DEN injection. The number and area of GST-P positive foci per cm2 of liver tissue were slightly increased in group 1 compared to the group 2 and significantly elevated in the group 4 compared to group 5. However, numbers in group 3 were significantly lower in group 4 and similar to the group 5 values. PCNA positive cells in the GST-P positive foci in the group 1 and group 4 were significantly increased as compared with respective controls (groups 2 and 5, respectively), while indices in the group 3 were again similar to values for group 5. Cessation of short-term alcohol administration thus had no promoting effects on development of GST-P foci, suggesting that the duration of alcohol treatment may be important. The results also imply the existence of a cumulative exposure time or dose threshold for alcohol if promoting effects of cessation are to be seen on rat hepatocarcinogenesis.

Tratamento percutâneo sob orientaçäo ultra-sonográfica, dos tumores hepáticos: experimento em modelo animal / Percutaneous treatment, under ultrasound guidance, of hepatic tumors: an experiment in animal model

Resumo: O objetivo deste trabalho foi avaliar e comparar a segurança e a efetividade da hipertermia por radiofrequencia e da injeçäo percutânea de álcool intramural na destruiçäo tumoral em coelhas com carcicoma VX2 implantado no fígado. Foram implantadas percutneamente, células de carcicoma VX2 no fígado de 37 coelhas New Zealand brancas. Os tumores foram tratados percutaneamente, sob orientaçäo ultra-sonográfica , com hipertermia por radiofrequencia (n=11) e com injeçäo intramural de álcool (n=14). O grupo controle recebeu injeçäo intramural de 0,5ml de soro fisiológico (n=12). O efeito das duas técnicas foi avaliado pelas medidas de crescimento tumoral, tempo de sobrevida e exame histopatológico post-mortem dos espécimes. As coelhas tratadas com radiofrequência apresentaram média de crescimento tumoral de 449,80 por cento/dia, e o tempo de sobrevida pós-tratamento foi em média 15 dias. As coelhas tratadas com álcool tiveram média de crescimento tumoral de 214,62 por cento/dia e tempo de sobrevida de 13 dias pós-tratamento. O grupo controle apresentou média de crescimento tumoral de 258,27 por cento/dia e viveu, em média, 9 dias pós-tratamento (com soro fisiológico). Metástases pulmonares causaram significativo estresse dos animais e foram a causa da morte de 31 (83,78 por cento dos pacientes) coelhas. Estudo histopatológico dos animais tratados demonstrou necrose tumoral e infarto do parênquima hepático peritumoral. Areas tumorais viáveis e metástases foram identificadas no exame post-mortem. Concluíu-se que a hiperternia por radiofrequencia e a injeçäo intratumoral de álcool säo métodos seguros para a destruiçäo tumoral no modelo animal. As coelhas tratadas apresentaram maior tempo de sobrevida que as do grupo controle, mas sem significância estatística. Näo houve diferença significativano crescimento tumoral entre os grupos. O exame anatopatológico demonstrou o efeito letal da radiofrequencia e do álcool no tumor e tecido hepático peritumoral. A incapacidade de obter-se completo controle local e sistêmico do carcicoma VX2 demonstra a agressividade desse tumor e näo necessariamentea ineficácia dos métodos testados.

Diferencias entre los ácidos ribonucleicos mensajeros de hígado normal y de hepatoma de Novikoff / Differences between the messenger ribonucleic acids of the normal liver and Novikoff's hepatoma

En la activación de varios genes en algunas células cancerosas aparentemente se requiere que se desencadene una cascada de eventos, que trae como consecuencia la transcripción de diversos genes; al mismo tiempo, otros genes son reprimidos. El propósito de este trabajo fue investigar si existen diferencias en la transcripción del ácido ribonucleico mensajero (mRNA) del hígado normal y del hepatoma de Novikoff. En todos los experimentos se utilizaron ratas macho adultas Sprague Dawley. La extracción del RNA total de bazo, hígado y células del hepatoma de Novikoff se llevó a cabo con el método de Schueltz y la separación del mRNA se realizó utilizando una columna de celulosa-ácido oligotimidílico, de acuerdo a lo señalado por Aviv y Leder. La cuantificación del mRNA mostró que las células del hepatoma de Novikoff tienen el doble de esta macromolécula comparadas con el hígado normal de rata

Synergistic promoter effect of diethylstilbesterol & phenobarbitone in diethylnitrosamine induced hepatic neoplasia in rats.

An experimental model was designed to study the role of both diethylstilbesterol (DES) and phenobarbitone (PB) singly or in combination, in diethylnitrosamine (DEN) induced hepatic neoplasia. Experimental and control rats were injected DEN (200 mg/kg) or saline, ip. Acute morphological changes were studied at days 1, 2 and 3; and at weekly intervals for 3 wk. Four weeks after DEN pretreatment the experimental and control rats were randomized into various groups and fed DES (T1), PB (T2) or a combination of both DES and PB (T3). Five rats from each experimental group were sacrificed at 10, 20 and 30 wk. Group T3 showed gross nodules with a mean nodule score of 20.5 mm at 20 wk. Nodule score in T1, T2 and T3 at 30 wk were 7, 9 and 34.5 mm respectively. The sequential morphological lesions encountered were clear cell and acidophilic foci; acidophilic, basophilic and mixed nodules. Haemorrhage within the nodules was frequent when DES was administered either alone or in combination with PB. Oval cell proliferation and cholangiocellular lesions were produced in all experimental groups. Foci and nodules generally showed loss of glucose-6 phosphatase, adenosine triphosphatase and invariable presence of gamma-glutamyl transpeptidase and glycogen. A combination of DES and PB as promoter yielded earliest and highest nodule score. This suggests that DES and PB acted synergestically as promoters or that PB caused enzyme induction thereby enhanced the promotive effect of DES.