RESUMO
The tumor microenvironment has a dynamic and usually cancer-promoting function during all tumorigenic steps. Glioblastoma (GBM) is a fatal tumor of the central nervous system, in which a substantial number of non-tumoral infiltrated cells can be found. Astrocytes neighboring these tumor cells have a particular reactive phenotype and can enhance GBM malignancy by inducing aberrant cell proliferation and invasion. The tumor suppressor p53 has a potential non-cell autonomous function by modulating the expression of secreted proteins that influence neighbor cells. In this work, we investigated the role of p53 on the crosstalk between GBM cells and astrocytes. We show that extracellular matrix (ECM) from p53(+/-) astrocytes is richer in laminin and fibronectin, compared with ECM from p53(+/+) astrocytes. In addition, ECM from p53(+/-) astrocytes increases the survival and the expression of mesenchymal markers in GBM cells, which suggests haploinsufficient phenotype of the p53(+/-) microenvironment. Importantly, conditioned medium from GBM cells blocks the expression of p53 in p53(+/+) astrocytes, even when DNA was damaged. These results suggest that GBM cells create a dysfunctional microenvironment based on the impairment of p53 expression that in turns exacerbates tumor endurance.
RESUMO
Glioblastomas (GBMs) are devastating tumors of the central nervous system, with a poor prognosis of 1-year survival. This results from a high resistance of GBM tumor cells to current therapeutic options, including etoposide (VP-16). Understanding resistance mechanisms may thus open new therapeutic avenues. VP-16 is a topoisomerase inhibitor that causes replication fork stalling and, ultimately, the formation of DNA double-strand breaks and apoptotic cell death. Autophagy has been identified as a VP-16 treatment resistance mechanism in tumor cells. Retinoblastoma protein (RB) is a classical tumor suppressor owing to its role in G1/S cell cycle checkpoint, but recent data have shown RB participation in many other cellular functions, including, counterintuitively, negative regulation of apoptosis. As GBMs usually display an amplification of the EGFR signaling involving the RB protein pathway, we questioned whether RB might be involved in mechanisms of resistance of GBM cells to VP-16. We observed that RB silencing increased VP-16-induced DNA double-strand breaks and p53 activation. Moreover, RB knockdown increased VP-16-induced apoptosis in GBM cell lines and cancer stem cells, the latter being now recognized essential to resistance to treatments and recurrence. We also showed that VP-16 treatment induced autophagy, and that RB silencing impaired this process by inhibiting the fusion of autophagosomes with lysosomes. Taken together, our data suggest that RB silencing causes a blockage on the VP-16-induced autophagic flux, which is followed by apoptosis in GBM cell lines and in cancer stem cells. Therefore, we show here, for the first time, that RB represents a molecular link between autophagy and apoptosis, and a resistance marker in GBM, a discovery with potential importance for anticancer treatment.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Glioblastoma/patologia , Proteína do Retinoblastoma/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/ultraestrutura , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/ultraestrutura , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Interferência de RNA/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Gamma radiation induces apoptosis in the proliferative zone (neuroblastic layer) of the developing rat retina. We asked whether sensitivity to apoptosis might be related to distinct phases of the cell cycle. Explants of newborn rat retina or newborn pups were gamma-irradiated and apoptosis was detected by chromatin condensation, DNA fragmentation in situ and DNA electrophoresis. After 6 hours, early appearing apoptotic bodies were located mainly towards the outer tier of the neuroblastic layer. In contrast, after 24 hours, late-appearing apoptotic cells were located towards the inner margin of the neuroblastic layer, a region associated with the S phase of the cell cycle. Labeling of a cohort of cells with the nucleotide analog bromo-deoxyuridine (BrdU) at the time of irradiation, showed that these cells die in the late wave of apoptosis. BrdU given 3 hours before fixation labeled a large number of late apoptotic cells, but no early apoptotic cells. After labeling of all cycling cells with BrdU, 40% of the early apoptotic profiles were unlabeled, and thus post-mitotic. The same schedules of cell death were identified after gamma irradiation in vivo. The results show that irradiation leads to two waves of apoptosis in distinct cell populations. An early wave comprises both post-mitotic cells and proliferating cells out of the S phase. The late wave comprises cells in S phase, which pass through this phase again to die. The antioxidant pyrrolidinedithiocarbamate prevented the early but not the late wave of apoptosis following irradiation, and blocked lipid peroxidation at 6 hours after the insult, suggesting that the two waves of apoptosis are indeed mediated by distinct mechanisms.
Assuntos
Apoptose/efeitos da radiação , Raios gama , Retina/efeitos da radiação , Animais , Animais Recém-Nascidos , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Cromatina/fisiologia , Cromatina/efeitos da radiação , Cromatina/ultraestrutura , Fragmentação do DNA , Cinética , Técnicas de Cultura de Órgãos , Ratos , Retina/citologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos da radiaçãoRESUMO
Studies of programmed cell death in the developing retina in vitro are currently reviewed. The results of inhibiting protein synthesis in retinal explants indicate two mechanisms of apoptosis. One mechanism depends on the synthesis of positive modulators ('killer proteins'), while a distinct, latent mechanism appears to be continuously blocked by negative modulators. Extracellular modulators of apoptosis include the neurotrophic factors NT-4 and BDNF, while glutamate may have either a positive or a negative modulatory action on apoptosis. Several protein kinases selectively modulate apoptosis in distinct retinal layers. Calcium and nitric oxide were also shown to affect apoptosis in the developing retinal tissue. The protein c-Jun was found associated with apoptosis in various circumstances, while p53 seems to be selectively expressed in some instances of apoptosis. The results indicate that the sensitivity of each retinal cell to apoptosis is controlled by multiple, interactive, cell type- and context-specific mechanisms. Apoptosis in the retina depends on a critical interplay of extracellular signals delivered through neurotrophic factors, neurotransmitters and neuromodulators, several signal transduction pathways, and the expression of a variety of genes.
Assuntos
Apoptose/fisiologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Técnicas de Cultura , Degeneração Retiniana/metabolismo , Transdução de Sinais/fisiologia , Animais , Camundongos , RatosRESUMO
Studies of programmed cell death in the developing retina in vitro are currently reviewed. The results of inhibiting protein synthesis in retinal explants indicate two mechanisms of apoptosis. One mechanism depends on the synthesis of positive modulators ('killer proteins'), while a distinct, latent mechanism appears to be continuously blocked by negative modulators. Extracellular modulators of apoptosis include the neurotrophic factors NT-4 and BDNF, while glutamate may have either a positive or a negative modulatory action on apoptosis. Several protein kinases selectively modulate apoptosis in distinct retinal layers. Calcium and nitric oxide were also shown to affect apoptosis in the developing retianl tissue. The protein c-Jun was found associated with apoptosis in various circumstances, while p53 seems to be selectively expressed in some instances of apoptosis. The results indicate that the sensitivity of each retinal cell to apoptosis is controlled by multiple, interactive, cell type- and context-specific mechanisms. Apoptosis in the retina depends on a critical interplay of extracellular signals delivered through neurotrophic factors, neurotransmitters and neuromodulators, several signal transduction pathways, and the expression of a variety of genes.