RESUMO
Induced pluripotent stem cells (iPSC) have a great potential, but their clinical application depends on finding strategies to abolish their tumorigenic potential. The use of Oct4, Sox2, Klf4, c-Myc and Nanog to generate iPSC demonstrated the already known importance of these genes to maintain stemness. Therefore, the presence of these genes is responsible for iPSC-derived teratomas. Similar to iPSC, P19 teratocarcinoma cell line also has characteristics of embryonic carcinoma cells and the ability to differentiate into many cell types. We separately silenced the transcription factors Oct4, Sox2, Klf4, c-Myc and Nanog in P19 cells and measured the impact of this silencing in vivo. All silenced cells generated tumors when injected in immunosuppressed mice, but silencing of Oct4, Sox2 and Klf4 generated mainly teratomas with mesoderm tissue. Our results suggest that downregulation of these transcription factors is not enough to avoid the formation of teratomas, but their silencing affect their differentiation potential.
Assuntos
Inativação Gênica , Teratoma/genética , Fatores de Transcrição/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular , Proliferação de Células , Feminino , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Teratoma/patologiaRESUMO
Glioblastoma (GBM) is an aggressive brain tumor with temozolomide (TMZ)-based chemotherapy as the main therapeutic strategy. Doxorubicin (DOX) is not used in gliomas due to its low bioavailability in the brain; however, new delivery strategies and low doses may be effective in the long term, especially as part of a drug cocktail. Our aim was to evaluate the chronic effects of low doses of DOX and TMZ in GBM. Human U87-ATCC cells and a primary GBM culture were chronically treated with TMZ (5 µM) and DOX (1 and 10 nM) alone or combined. DOX resulted in a reduction in the number of cells over a period of 35 days and delayed the cell regrowth. In addition, DOX induced cell senescence and reduced tumor sphere formation and the proportion of NANOG- and OCT4-positive cells after 7 days. Low doses of TMZ potentiated the effects of DOX on senescence and sphere formation. This combined response using low doses of DOX may pave the way for its use in glioma therapy, with new technologies to overcome its low blood-brain barrier permeability.
Assuntos
Neoplasias Encefálicas/patologia , Doxorrubicina/farmacologia , Glioblastoma/patologia , Temozolomida/farmacologia , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
Glioblastoma is the most aggressive tumor in the CNS and is characterized by having a cancer stem cell (CSC) subpopulation essential for tumor survival. The purinergic system plays an important role in glioma growth, since adenosine triphosphate (ATP) can induce proliferation of glioma cells, and alteration in extracellular ATP degradation by the use of exogenous nucleotidases dramatically alters the size of gliomas in rats. The aim of this work was to characterize the effect of the purinergic system on glioma CSCs. Human U87 glioma cultures presented tumor spheres that express the markers of glioma cancer stem cells CD133, Oct-4, and Nanog. Messenger RNA of several purinergic receptors were differently expressed in spheres when compared to a cell monolayer not containing spheres. Treatment of human gliomas U87 or U343 as well as rat C6 gliomas with 100 µM of ATP reduced the number of tumor spheres when grown in neural stem cell medium supplemented with epidermal growth factor and basic fibroblast growth factor. Moreover, ATP caused a decline in the number of spheres observed in culture in a dose-dependent manner. ATP also reduces the expression of Nanog, as determined by flow cytometry, as well as CD133 and Oct-4, as analyzed by flow cytometry and RT-PCR in U87 cells. The differential expression of purinergic receptor in tumor spheres when compared to adherent cells and the effect of ATP in reducing tumor spheres suggest that the purinergic system affects CSC biology and that ATP may be a potential agonist for differentiation therapy.
RESUMO
Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.