cytochrome-c-t and Astrocytoma

The aim of the present study was to investigate whether silencing of Hsp27 or Hsp72 expression in glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cells increases their sensitivity to programmed cell death induction upon temozolomide and/or quercetin treatment. Transfection with specific siRNA was performed for the Hsp gene silencing. As revealed by microscopic observation and flow cytometry, the inhibition of Hsp expression was correlated with severe apoptosis induction upon the drug treatment studied. No signs of autophagy were detected. This was correlated with a decreased mitochondrial membrane potential, increased level of cytochrome c in the cytoplasm, and activation of caspase 3 and caspase 9. All these results suggest that the apoptotic signal was mediated by an internal pathway. Additionally, in a large percentage of cells treated with temozolomide, with or without quercetin, granules within the ER system were found, which was accompanied by an increased level of caspase 12 expression. This might be correlated with ER stress. Quercetin and temozolomide also changed the shape of nuclei from circular to "croissant like" in both transfected cell lines. Our results indicate that blocking of Hsp27 and Hsp72 expression makes T98G cells and MOGGCCM cells extremely vulnerable to apoptosis induction upon temozolomide and quercetin treatment and that programmed cell death is initiated by an internal signal.

Topics: Apoptosis; Astrocytoma; Autophagy; Caspase 12; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Dacarbazine; Endoplasmic Reticulum Stress; Gene Silencing; Glioblastoma; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Membrane Potential, Mitochondrial; Molecular Chaperones; Quercetin; Temozolomide

Acrylamide (ACR) can be produced during food processing and has neurotoxic effects in humans. This study aims to determine ACR induced apoptotic responses in human astrocytoma U-1240 MG cells to realize the incurred toxic mechanisms. Under 1 and 2mM ACR exposure, cell viability decreased as time increased. The increments in sub-G(1) phase were 87.5-fold, and pro-caspase 3 and PARP protein expressions decreased 35% and 54.5% respectively relative to the control after 2mM ACR treatment. Molecular evidence of Bax/bcl-2 ratio and cytochrome c expression increased 8.86-fold and 6.81-fold as well as pro-caspase 9 decreased 67.8% relative to the control respectively under 2mM ACR exposure. Trolox, an ROS scavenging agent, attenuated cell death and induced ROS production by 2mM ACR. The ultrastructure alterations of mitochondria showed marked vesicular matrix compartmentalization and cytoplasmic vacuole formation after 2mM ACR was treated for 48h, whereas those treated for 72h showed chromatin condensation, pyknosis, and swelling. These results indicate long-term exposure to ACR induced mitochondria collapse and finally led to apoptosis. Although 2mM ACR is higher than average daily intake dosage, workers in chemical industries may be exposed to sufficient doses to entail health risks.

Topics: Acrylamide; Apoptosis; Astrocytoma; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Chromans; Cytochromes c; Dose-Response Relationship, Drug; G1 Phase; Humans; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Toxicity Tests

Adenosine deaminase, which catalyzes the deamination of adenosine and deoxyadenosine, plays a central role in purine metabolism. Indeed, its deficiency is associated with severe immunodeficiency and abnormalities in the functioning of many organs, including nervous system. We have mimicked an adenosine deaminase-deficient situation by incubating a human astrocytoma cell line in the presence of deoxycoformycin, a strong adenosine deaminase inhibitor, and deoxyadenosine, which accumulates in vivo when the enzyme is deficient, and have monitored the effect of the combination on cell viability, mitochondrial functions, and other metabolic features. Astrocytomas are the most common neoplastic transformations occurring in glial cell types, often characterized by a poor prognosis. Our experimental approach may provide evidence both for the response to a treatment affecting purine metabolism of a tumor reported to be particularly resistant to chemotherapeutic approaches and for the understanding of the molecular basis of neurological manifestations related to errors in purine metabolism. Cells incubated in the presence of the combination, but not those incubated with deoxyadenosine or deoxycoformycin alone, underwent apoptotic death, which appears to proceed through a mitochondrial pathway, since release of cytochrome c has been observed. The inhibition of adenosine deaminase increases both mitochondrial reactive oxygen species level and mitochondrial mass. A surprising effect of the combination is the significant reduction in lactate production, suggestive of a reduced glycolytic capacity, not ascribable to alterations in NAD⁺/NADH ratio, nor to a consumption of inorganic phosphate. This is a hitherto unknown effect presenting early during the incubation with deoxyadenosine and deoxycoformycin, which precedes their effect on cell viability.

Topics: Adenosine Deaminase Inhibitors; Astrocytoma; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Mitochondria; NAD; Reactive Oxygen Species; Spectrometry, Fluorescence

To evaluate the association of signals of apoptosis namely, TGF-beta1, TNF-alpha and cytochrome c release in cytoplasm with survival rate to determine the potential use of such parameters as predictive markers for patients with astrocytomas.. We measured TGF-beta1, TNF-alpha and cytoplasmic cytochrome c in 30 astrocytic tumors Grade II, III and IV.. We found that TNF-alpha and cytochrome c release in Grade IV tends to be significantly lower than those in Grade II, whereas TGF-beta1 did not significantly change in the different grades. Patients with astrocytic tumors having elevated cytochrome c showed a better survival rate compared to those with less release. There is neither a correlation shown between TNF-alpha and cytochrome c release nor between TNF-alpha and patient survival. TGF-beta1 was positively correlated with cytochrome c release. Patients showing such correlation had increased survival rate over 18 months follow up period.. These data suggest that TGF-beta1 and cytochrome c may be useful prognostic markers that help patients' stratification and in adjusting the disciplines of therapy.

Topics: Adolescent; Adult; Aged; Apoptosis; Astrocytoma; Biomarkers, Tumor; Brain Neoplasms; Child; Child, Preschool; Cytochromes c; Female; Humans; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Astrocytoma; Brain; Brain Neoplasms; Caspases; Cytochromes c; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Medulloblastoma; Neurons; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Transcription, Genetic