Expressão das conexinas 26, 32, 43 e panexina 1 em diferentes etapas da hepatocarcinogênese química associada à fibrose em camundongos C3H/HeJ.
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Data
2022-05-27
Autores
Sensulini, Luis Artur
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Universidade Estadual Paulista (Unesp)
Resumo
O câncer de fígado é a quarta maior causa de mortes relacionadas ao câncer com crescente aumento de incidência e mortalidade no mundo nas últimas décadas, tendo como principal representante o carcinoma hepatocelular (CHC). Um dos mecanismos de controle da homeostasia celular é a comunicação intercelular realizada por canais específicos que inclui as famílias de proteínas das conexinas (Cxs) e panexinas (Panxs). No fígado, a Cx32 e Cx26 são as principais formadoras de junções GAP hepáticas, além das células endoteliais sinusoidais que expressam, preferencialmente, a Cx43. Estudos demonstraram que a Cx26 e Cx32 tem expressão elevada no fígado normal e até mesmo em lesões pré-neoplásicas, mas baixa expressão no CHC. Outros estudos demonstraram correlação positiva entre a hiperexpressão da Panx1 com pior prognóstico e risco maior de ocorrência de metástase nos pacientes. Assim, no presente trabalho avaliou-se o comportamento da expressão das Cxs 26, 32 e Panx1 ao longo do processo de hepatocarcinogênese associada à fibrose em camundongos C3H/HEJ, em lesões pré-neoplásicas e neoplasias pela técnica de imunofluorescência. Dessa maneira, camundongos C3H/HEJ, com 14 dias de idade, receberam injeção intraperitoneal (i.p) de 25 mg/kg peso corpóreo (p.c.) de dietilnitrosamina (DEN), ou seu veículo (NaCl 0,9%) no caso do grupo controle. A partir da 8ª semana de vida, por um período de 8 a 29 (grupo interrupção) ou 8 a 46 semanas (grupo contínuo), os camundongos receberam 3 doses i.p semanais de tetracloreto de carbono (CCl4, solução a 10% em doses escalonadas de 0,25 a 1,50 µL/g peso corpóreo, sendo esta última mantida até o final das aplicações). Nas semanas 17, 30 e 47, os animais foram eutanasiados. Amostras de soro e de fígado (com ou sem tumores) foram coletadas para análise sérica de alanina aminotransferase (ALT) ou para histologia/immunoistoquímica e imunofluorescência. A incidência e multiplicidade de lesões hepáticas pré-neoplásicas e neoplásicas foram comparadas nos diferentes pontos de eutanásia. Em geral, os resultados demonstraram que a continuidade de aplicações de CCl4 da 30-46ª semana não influenciaram no tamanho e incidência das lesões macroscópicas ou de adenomas e CHC quando comparado com o grupo interrupção, embora com níveis séricos de ALT maiores no grupo contínuo. Os níveis de proliferação celular Ki-67+, nos adenomas e CHC, foram similares nos grupos de interrupção e contínuo de aplicações de CCl4. A deposição de colágeno nos grupos tratados com CCl4 foi mais intensa no grupo interrompido. Além disso, a expressão da Cx26 e Cx32 foi menor nas lesões hepáticas pre-neoplásicas e neoplásicas em comparação com tecido hepático normal (grupo controle) e no tecido adjacente (grupo DEN/CCl4), com correlação negativa de ambas Cxs com os níveis de Ki-67+. Não houve diferença significativa de expressão de Panx1 6 entre os grupos tratados ou controle, nos diferentes períodos de eutanásia ou evolução da hepatocarcinogênese. Em conclusão, os resultados indicam o modelo animal utilizado pode ser reproduzido com menor número de aplicações de CCl4 e que a expressão das Cxs 26 e 32 são marcadores precoces do processo de carcinogênese associado a fibrose. As próximas etapas, consistem na realização e análise da expressão gênicas das Cxs e Panx1, em amostras de RNA extraídas a partir de microdissecção a laser já realizada de áreas de FHA, adenomas e CHC e tecido hepático normal, além da quantificação de imunofluorescência da Cx43.
Liver cancer is the fourth leading cause of cancer-related deaths with increasing incidence and mortality worldwide in recent decades, with hepatocellular carcinoma (HCC) as the main representative. One of the mechanisms controlling cellular homeostasis is intercellular communication performed by specific channels that include the protein families of the connexins (Cxs) and pannexins (Panxs). In the liver, Cx32 and Cx26 are the main formers of hepatic GAP junctions, in addition to sinusoidal endothelial cells that preferentially express Cx43. Studies have shown that Cx26 and Cx32 have high expression in normal liver and even in preneoplastic lesions, but low expression in HCC. Other studies have shown a positive correlation between Panx1 overexpression with worse prognosis and higher risk of metastasis occurrence in patients. Thus, in the present study we evaluated the behavior of the expression of Cxs 26, 32 and Panx1 throughout the process of hepatocarcinogenesis associated with fibrosis in C3H/HEJ mice, in preneoplastic lesions and neoplasms by immunofluorescence technique. Thus, 14-day-old C3H/HEJ mice received intraperitoneal (i.p.) injection of 25 mg/kg body weight (b.w.) of diethylnitrosamine (DEN), or its vehicle (NaCl 0.9%) in the control group. From the 8th week of life, for a period of 8 to 29 (interruption group) or 8 to 46 weeks (continuous group), the mice received 3 weekly i.p doses of carbon tetrachloride (CCl4, 10% solution in doses ranging from 0.25 to 1.50 µL/g body weight, the latter maintained until the end of the applications). At weeks 17, 30 and 47, the animals were euthanized. Serum and liver samples (with or without tumors) were collected for serum alanine aminotransferase (ALT) analysis or for histology/immunohistochemistry and immunofluorescence. The incidence and multiplicity of preneoplastic and neoplastic liver lesions were compared at the different euthanasia points. Overall, the results showed that continued CCl4 applications from week 30- 46 did not influence the size and incidence of macroscopic lesions or of adenomas and HCC when compared to the interruption group, although with higher serum ALT levels in the continuous group. The levels of Ki-67+ cell proliferation in adenomas and HCC were similar in the interruption and continuous CCl4 application groups. Collagen deposition in the CCl4- treated groups was more intense in the interrupted group. In addition, the expression of Cx26 and Cx32 was lower in pre-neoplastic and neoplastic liver lesions compared with normal liver tissue (control group) and in adjacent tissue (DEN/CCl4 group), with negative correlation of both Cxs with Ki-67+ levels. There was no significant difference in Panx1 expression between the treated or control groups at the different periods of euthanasia or evolution of hepatocarcinogenesis. In conclusion, the results indicate that the animal model used can be reproduced with fewer applications of CCl4 and that the expression of Cxs 26 and 32 are early 8 markers of the process of carcinogenesis associated with fibrosis. The next steps, consist of performing and analyzing the gene expression of Cxs and Panx1, in RNA samples extracted from laser microdissection already performed from areas of FHA, adenomas and HCC and normal liver tissue, in addition to the immunofluorescence quantification of Cx43.
Liver cancer is the fourth leading cause of cancer-related deaths with increasing incidence and mortality worldwide in recent decades, with hepatocellular carcinoma (HCC) as the main representative. One of the mechanisms controlling cellular homeostasis is intercellular communication performed by specific channels that include the protein families of the connexins (Cxs) and pannexins (Panxs). In the liver, Cx32 and Cx26 are the main formers of hepatic GAP junctions, in addition to sinusoidal endothelial cells that preferentially express Cx43. Studies have shown that Cx26 and Cx32 have high expression in normal liver and even in preneoplastic lesions, but low expression in HCC. Other studies have shown a positive correlation between Panx1 overexpression with worse prognosis and higher risk of metastasis occurrence in patients. Thus, in the present study we evaluated the behavior of the expression of Cxs 26, 32 and Panx1 throughout the process of hepatocarcinogenesis associated with fibrosis in C3H/HEJ mice, in preneoplastic lesions and neoplasms by immunofluorescence technique. Thus, 14-day-old C3H/HEJ mice received intraperitoneal (i.p.) injection of 25 mg/kg body weight (b.w.) of diethylnitrosamine (DEN), or its vehicle (NaCl 0.9%) in the control group. From the 8th week of life, for a period of 8 to 29 (interruption group) or 8 to 46 weeks (continuous group), the mice received 3 weekly i.p doses of carbon tetrachloride (CCl4, 10% solution in doses ranging from 0.25 to 1.50 µL/g body weight, the latter maintained until the end of the applications). At weeks 17, 30 and 47, the animals were euthanized. Serum and liver samples (with or without tumors) were collected for serum alanine aminotransferase (ALT) analysis or for histology/immunohistochemistry and immunofluorescence. The incidence and multiplicity of preneoplastic and neoplastic liver lesions were compared at the different euthanasia points. Overall, the results showed that continued CCl4 applications from week 30- 46 did not influence the size and incidence of macroscopic lesions or of adenomas and HCC when compared to the interruption group, although with higher serum ALT levels in the continuous group. The levels of Ki-67+ cell proliferation in adenomas and HCC were similar in the interruption and continuous CCl4 application groups. Collagen deposition in the CCl4- treated groups was more intense in the interrupted group. In addition, the expression of Cx26 and Cx32 was lower in pre-neoplastic and neoplastic liver lesions compared with normal liver tissue (control group) and in adjacent tissue (DEN/CCl4 group), with negative correlation of both Cxs with Ki-67+ levels. There was no significant difference in Panx1 expression between the treated or control groups at the different periods of euthanasia or evolution of hepatocarcinogenesis. In conclusion, the results indicate that the animal model used can be reproduced with fewer applications of CCl4 and that the expression of Cxs 26 and 32 are early 8 markers of the process of carcinogenesis associated with fibrosis. The next steps, consist of performing and analyzing the gene expression of Cxs and Panx1, in RNA samples extracted from laser microdissection already performed from areas of FHA, adenomas and HCC and normal liver tissue, in addition to the immunofluorescence quantification of Cx43.
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Palavras-chave
Conexina 26, Conexina 32, Conexina 43, Panexina 1, Hepatocarcinogênese induzida quimicamente, Fígado - Câncer, Carcinoma hepatocelular, Homeostase, Carcinogênese