In vivo effects of ACTH and N-POMC peptide in the expression of proteins and genes related to cell cycle control in rat adrenals.

A proliferação das células do córtex adrenal é um fenômeno que envolve genes e proteínas relacionados com o controle do ciclo celular. Um dos principais fatores envolvidos na proliferação do córtex adrenal é o ACTH (hormônio corticotrófico), mas outros fatores, como o peptídeo N-terminal da POMC, parece estar envolvido. Para contribuir com o entendimento dos mecanismos envolvidos na resposta proliferativa do córtex adrenal analisamos os efeitos desencadeados por ambos os peptídeos em ratos cujo eixo HPA foi inibido pela dexametasona. Foram utilizados os métodos de incorporação de BrdU; de análise da expressão proteica das ciclinas D, E e p27 através de imunistoquímica e immunoblotting e da expressão de genes relacionados ao controle do ciclo celular utilizando placas de qRT-PCR. Nossos resultados sugerem que ambos os peptídeos induzem efeito proliferativo mas zona-específico no córtex adrenal, e que esse efeito pode ser mediado pelo controle da expressão das ciclinas D2, D3 e E, e pelo inibidor de ciclo celular, a proteína p27kip1.The proliferation of adrenal cortical cells involves genes and proteins related with the control of the cell cycle. The main factor involved in the proliferation of the adrenal cortex is the ACTH (corticotrophin), but other factor, such as the peptide N-terminal of POMC, seems to be involved. To contribute to the understanding of the mechanisms involved in the proliferative response of the adrenal cortex, we analyzed the effects triggered by both peptides in rats with the HPA axis inhibited by dexamethasone. The methods used were the incorporation of BrdU; analysis of protein cyclins D, E and p27 expression through immunohistochemistry and immunoblotting and expression of genes involved in cell cycle regulation by using qRT-PCR arrays. The results suggest that both peptides induced proliferative effect in adrenal cortex in a zone-specific manner. This effect may be mediated by the control of cyclins D2, D3 and E expression, and also by the cell cycle inhibitor, protein p27KIP1

Insights from systems biology in physiological studies: learning from context

Systems biology presents an integrated view of biological systems, focusing on the relations between elements, whether functional or evolutionary, and providing a rich framework for the comprehension of life. At the same time, many low-throughput experimental studies are performed without influence from this integrated view, whilst high-throughput experiments use low-throughput results in their validation and interpretation. We propose an inversion in this logic, and ask which benefits could be obtained from a holistic view coming from high-throughput studies-and systems biology in particular-in interpreting and designing low-throughput experiments. By exploring some key examples from the renal and adrenal physiology, we try to show that network and modularity theory, along with observed patterns of association between elements in a biological system, can have profound effects on our ability to draw meaningful conclusions from experiments

The tyrosine kinase inhibitor nilotinib is more efficient than mitotane in decreasing cell viability in spheroids prepared from adrenocortical carcinoma cells

Abstract Background New drugs for adrenocortical carcinoma (ACC) are needed because most patients undergo rapid disease progression despite surgery and adjuvant therapy with mitotane. In this study, we aimed to investigate the in vitro effects of different chemotherapy drugs, alone or combined with mitotane, on the viability of adrenocortical carcinoma cells. Methods Everolimus, sunitinib, zoledronic acid, imatinib and nilotinib cytotoxicity, alone or combined with mitotane were tested on ACC H295R cells in monolayer or spheroid cultures using MTS assays and confocal microscopy. Moreover, the nilotinib effects were investigated in spheroids cultured from patient tumor-derived ACC-T36 cells. Results Morphological characterization of H295R cell spheroids using histochemistry was performed and showed that dense, homogenously sized, multicellular spheroids were obtained. We observed that sunitinib and nilotinib alone were equally effective in a monolayer preparation, whereas mitotane was the most effective even at a low dose. A combination of sunitinib and mitotane was the most effective treatment, with only 23.8% of cells in the monolayer remaining viable. Spheroid preparations showed resistance to different drugs, although the poor effect produced by mitotane alone was surprising, with a cell viability of 84.6% in comparison with 13.1% in monolayer cells. The most ineffective drugs in spheroid preparations were everolimus, zoledronic acid and imatinib. In both cell types, nilotinib, either alone or in combination with mitotane induced more significant cell viability inhibition in monolayer and spheroid preparations. In addition, the mechanism of nilotinib activity involves the ERK1/2 pathway. Conclusion Taken together, our data identified nilotinib as a cytotoxic drug that combined with ERK inhibitors deserves to be tested as a novel therapy for adrenocortical carcinoma

Anticancer Activities of the Quinone-Methide Triterpenes Maytenin and 22-β-hydroxymaytenin Obtained from Cultivated Maytenus ilicifolia Roots Associated with Down-Regulation of miRNA-27a and miR-20a/miR-17-5p

Natural triterpenes exhibit a wide range of biological activities. Since this group of secondary metabolites is structurally diverse, effects may vary due to distinct biochemical interactions within biological systems. In this work, we investigated the anticancer-related activities of the quinone-methide triterpene maytenin and its derivative compound 22-β-hydroxymaytenin, obtained from Maytenus ilicifolia roots cultivated in vitro. Their antiproliferative and pro-apoptotic activities were evaluated in monolayer and three-dimensional cultures of immortalized cell lines. Additionally, we investigated the toxicity of maytenin in SCID mice harboring tumors derived from a squamous cell carcinoma cell line. Both isolated molecules presented pronounced pro-apoptotic activities in four cell lines derived from head and neck squamous cell carcinomas, including a metastasis-derived cell line. The molecules also induced reactive oxygen species (ROS) and down-regulated microRNA-27a and microRNA-20a/miR-17-5p, corroborating with the literature data for triterpenoids. Intraperitoneal administration of maytenin to tumor-bearing mice did not lead to pronounced histopathological changes in kidney tissue, suggesting low nephrotoxicity. The wide-ranging activity of maytenin and 22-β-hydroxymaytenin in head and neck cancer cells indicates that these molecules should be further explored in plant biochemistry and biotechnology for therapeutic applications