RESUMO
The placental stem cells have called the focus of attention for their therapeutic potential to treat different diseases, including cancer. There is plenty evidence about the antiproliferative, antiangiogenic and proapoptotic properties of the amniotic membrane. Liver cancer is the fifth cause of cancer in the world, with a poor prognosis and survival. Alternative treatments to radio- or chemotherapy have been searched. In this work we aimed to study the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma cells. In addition, we have analyzed the regulation of pro and antiOncomiRs expression involved in hepatocarcinoma physiology. We have determined by 3H-thymidine incorporation assay that AM-CM inhibits DNA synthesis in HepG2 cells after 72 h of treatment. AM-CM pure or diluted at 50% and 25% also diminished HepG2 and HuH-7 cells viability and cell number. Furthermore, AM-CM induced cell cycle arrest in G2/M. When proliferation mechanisms were analyzed we found that AM-CM reduced the expression of both Cyclin D1 mRNA and protein. Nuclear expression of Ki-67 was also reduced. We observed that this CM was able to promote the expression of p53 and p21 mRNA and proteins, leading to cell growth arrest. Moreover, AM-CM induced an increase in nuclear p21 localization, observed by immunofluorescence. As p53 levels were increased, Mdm-2 expression was downregulated. Interestingly, HepG2 and HuH-7 cells treatment with AM-CM during 24 and 72 h produced an upregulation of antiOncomiRs 15a and 210, and a downregulation of proOncomiRs 206 and 145. We provide new evidence about the promising novel applications of human amniotic membrane in liver cancer.
Assuntos
Âmnio/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Meios de Cultivo Condicionados/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Âmnio/crescimento & desenvolvimento , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Ciclina D1/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Placenta/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-mdm2/genética , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.
Assuntos
Âmnio/citologia , Proliferação de Células , Sobrevivência Celular , Fígado/citologia , Biomarcadores/metabolismo , Células Cultivadas , Células Epiteliais/citologia , Feminino , Humanos , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Fosforilação , Gravidez , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)(2)cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.