Glioblastoma (GBM) is the most aggressive form of primary brain tumour, with dismal patient outcome. Treatment failure is associated with intrinsic or acquired apoptosis resistance and the presence ...of a highly tumourigenic subpopulation of cancer cells called GBM stem cells. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising novel therapy for some treatment-resistant tumours but unfortunately GBM can be completely resistant to TRAIL monotherapy. In this study, we identified Mcl-1, an anti-apoptotic Bcl-2 family member, as a critical player involved in determining the sensitivity of GBM to TRAIL-induced apoptosis. Effective targeting of Mcl-1 in TRAIL resistant GBM cells, either by gene silencing technology or by treatment with R-roscovitine, a cyclin-dependent kinase inhibitor that targets Mcl-1, was demonstrated to augment sensitivity to TRAIL, both within GBM cells grown as monolayers and in a 3D tumour model. Finally, we highlight that two separate pathways are activated during the apoptotic death of GBM cells treated with a combination of TRAIL and R-roscovitine, one which leads to caspase-8 and caspase-3 activation and a second pathway, involving a Mcl-1:Noxa axis. In conclusion, our study demonstrates that R-roscovitine in combination with TRAIL presents a promising novel strategy to trigger cell death pathways in glioblastoma.
The proteasome has emerged as a novel target for antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous system. To identify cell death ...pathways activated in response to inhibition of the proteasome system in cancer cells, we treated human SH-SY5Y neuroblastoma cells with the selective proteasome inhibitor (PI) epoxomicin (Epoxo). Prolonged exposure to Epoxo was associated with increased levels of poly-ubiquitinylated proteins and p53, release of cytochrome c from the mitochondria, and activation of caspases. Analysis of global gene expression using high-density oligonucleotide microarrays revealed that Epoxo triggered transcriptional activation of the two Bcl-2-homology domain-3-only (BH3-only) genes p53 upregulated modulator of apoptosis (PUMA) and Bim. Subsequent studies in PUMA- and Bim-deficient cells indicated that Epoxo-induced caspase activation and apoptosis was predominantly PUMA-dependent. Further characterization of the transcriptional response to Epoxo in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; with deficiency in either p53 or PUMA significantly protected HCT116 cells against Epoxo-induced apoptosis. Our data suggest that p53 activation and the transcriptional induction of its target gene PUMA play an important role in the sensitivity of cancer cells to apoptosis induced by proteasome inhibition, and imply that antineoplastic therapies with PIs might be especially useful in cancers with functional p53.
In patients suffering from cerebrovascular diseases and traumatic brain damage, increases in serum levels of protein S100B are positively correlated with the severity of the insult. Since high ...concentrations of S100B have been shown to exert neurotoxic effects, the objective of this study was to characterize the regulatory mechanisms underlying control of S100B release from astrocytes. To that end, we analyzed the kinetics and amount of S100B release in correlation with regulation of S100B gene expression in an in vitro ischemia model. Astrocyte cultures were treated with combined oxygen, serum and glucose deprivation, serum and glucose deprivation or hypoxia alone for 6, 12 and 24 h, respectively. While oxygen, serum and glucose deprivation triggered the most rapid release of S100B, serum and glucose deprivation provoked comparable levels of released S100B at the later time points. In contrast to oxygen, serum and glucose deprivation and serum and glucose deprivation, hypoxia alone elicited only marginal increases in secreted S100B. Parallel analysis of extracellular lactate dehydrogenase and the number of viable cells revealed only moderate cell death in the cultures, indicating that S100B was actively secreted during in vitro ischemia. Interestingly, S100B mRNA expression was potently downregulated after 12 and 24 h of oxygen, serum and glucose deprivation, and prolonged oxygen, serum and glucose deprivation for 48 h was associated with a significant reduction of S100B release at later time intervals, whereas lactate dehydrogenase levels remained constant. Our data suggest that secretion of S100B during the glial response to metabolic injury is an early and active process.
There are no blood-based molecular biomarkers of temporal lobe epilepsy (TLE) to support clinical diagnosis. MicroRNAs are short noncoding RNAs with strong biomarker potential due to their ...cell-specific expression, mechanistic links to brain excitability, and stable detection in biofluids. Altered levels of circulating microRNAs have been reported in human epilepsy, but most studies collected samples from one clinical site, used a single profiling platform or conducted minimal validation.
Using a case-control design, we collected plasma samples from video-electroencephalogram-monitored adult TLE patients at epilepsy specialist centers in two countries, performed genome-wide PCR-based and RNA sequencing during the discovery phase and validated findings in a large (>250) cohort of samples that included patients with psychogenic non-epileptic seizures (PNES).
After profiling and validation, we identified miR-27a-3p, miR-328-3p and miR-654-3p with biomarker potential. Plasma levels of these microRNAs were also changed in a mouse model of TLE but were not different to healthy controls in PNES patients. We determined copy number of the three microRNAs in plasma and demonstrate their rapid detection using an electrochemical RNA microfluidic disk as a prototype point-of-care device. Analysis of the microRNAs within the exosome-enriched fraction provided high diagnostic accuracy while Argonaute-bound miR-328-3p selectively increased in patient samples after seizures. In situ hybridization localized miR-27a-3p and miR-328-3p within neurons in human brain and bioinformatics predicted targets linked to growth factor signaling and apoptosis.
This study demonstrates the biomarker potential of circulating microRNAs for epilepsy diagnosis and mechanistic links to underlying pathomechanisms.
The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 ...mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.
•Pro-survival Bcl-2 proteins are key regulators of apoptosis and autophagy.•Autophagy can exert pro-survival and pro-death functions depending on the context.•The Bcl-2 antagonist AT-101 triggers apoptosis and a cytoprotective autophagy response in MCF7 breast carcinoma cells.•AT-101-induced cytoprotective autophagy is independent of the expression levels and subcelluar localisation of Bcl-xL and Bcl-2.
MicroRNAs function as negative regulators of posttranscriptional gene expression, having major roles in cellular differentiation. Several neuroblastoma cell lines can be induced to undergo ...differentiation by all-trans-retinoic acid (ATRA) and are used for modeling signaling pathways involved in this process. To identify miRNAs contributing to differentiation, we profiled 364 loci following ATRA treatment of neuroblastoma cell lines and found miR-10a and miR-10b to be highly overexpressed in SK-N-BE, LAN5 and SHSY-5Y. Ectopic overexpression of these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by ATRA in each of these cell lines. One of the predicted downregulated miR-10a/b targets was nuclear receptor corepressor 2 (NCOR2), a corepressor of gene transcription, which is known to suppress neurite outgrowth. NCOR2 was experimentally validated as a direct target of miR-10a/b, and siRNA-mediated inhibition of this mRNA alone resulted in neural cell differentiation. Moreover, induction of differentiation could be blocked by ectopic upregulation of NCOR2 using an expression construct lacking the miR-10a/b 3' untranslated region target site. We conclude that miR-10a/b has major roles in the process of neural cell differentiation through direct targeting of NCOR2, which in turn induces a cascade of primary and secondary transcriptional alterations, including the downregulation of MYCN.
Apoptotic desensitization is a hallmark of cancer cells, but present knowledge of molecular systems controlling apoptosis has yet to provide significant prognostic insights. Here, we report findings ...from a systems study of the intrinsic pathway of apoptosis by BCL2 family proteins and clinical translation of its findings into a model with applications in colorectal cancer (CRC). By determining absolute protein quantifications in CRC cells and patient tumor samples, we found that BAK and BAX were expressed more highly than their antiapoptotic inhibitors. This counterintuitive finding suggested that sole inhibition of effector BAX and BAK could not be sufficient for systems stability in nonstressed cells. Assuming a model of direct effector activation by BH3-only proteins, we calculated that the amount of stress-induced BH3-only proteins required to activate mitochondrial apoptosis could predict individual death responses of CRC cells to 5-fluorouracil/oxaliplatin. Applying this model predictor to protein profiles in tumor and matched normal tissue samples from 26 patients with CRCs, we found that differences in protein quantities were sufficient to model the increased tumor sensitivity to chemotherapy compared with normal tissue. In addition, these differences were sufficient to differentiate clinical responders from nonresponders with high confidence. Applications of our model, termed DR_MOMP, were used to assess the impact of apoptosis-sensitizing drugs in lowering the necessary dose of state-of-the-art chemotherapy in individual patients. Together, our findings offer a ready clinical tool with the potential to tailor chemotherapy to individual patients.
Purpose
Resistance to bevacizumab (BEV) in glioblastoma is believed to occur via activation of molecular networks including the mTOR/PI3K pathway. Using an MR/PET molecular imaging biomarker ...approach, we investigated the response to combining BEV with the mTOR/PI3K inhibitor BEZ235.
Methods
Tumours were established by orthotopically implanting U87MG-luc2 cells in mice. Animals were treated with BEZ235 and/or BEV, and imaged using diffusion-weighted-MRI, T2-weighted and T2*-weighted before and after administration of superparamagnetic iron oxide contrast agent. Maps for changes in relaxation rates (Δ
R
2, Δ
R
2* and apparent diffusion coefficient) were calculated. Vessel size index and microvessel density index were derived. 3′-Deoxy-3′-
18
Ffluorothymidine (
18
FFLT) PET and
O
-(2-
18
Ffluoroethyl)-
l
-tyrosine (
18
FFET) PET were further performed and tumour endothelium/proliferation markers assessed by immunohistochemistry.
Results
Treatment with BEV resulted in a pronounced decrease in tumour volume (T2-weighted MRI). No additive effect on tumour volume was observed with the BEV/BEZ235 combination compared with BEV monotherapy. The Ki67 proliferation index and
18
FFLT uptake studies were used to support the observations. Using Δ
R
2* and Δ
R
2 values, respectively, the BEV/BEZ235 combination significantly reduced tumour microvessel volume in comparison to BEV alone. Decreased microvessel density index was further observed in animals treated with the combination, supported by von Willebrand factor (vWF) immunohistochemistry.
18
FFET uptake was decreased following treatment with BEV alone, but was not further reduced following treatment with the combination. vWF immunohistochemistry analysis showed that the mean tumour vessel size was increased in all cohorts.
Conclusion
Assessing MR imaging biomarker parameters together with
18
FFET and
18
FFLT PET provided information on mechanism of action of the drug combination and clues as to potential clinical responses. Following translation to clinical use, treatment with a BEV/BEZ235 combination could reduce peritumoral oedema obviating the requirement for steroids. The use of hypothesis-driven molecular imaging studies facilitates the preclinical evaluation of drug response. Studies of this kind may more accurately predict the clinical potential of the BEV/BEZ235 combination regimen as a novel therapeutic approach in oncology.
Aims/hypothesis
HNF1A-MODY is a monogenic form of diabetes caused by mutations in the
HNF1A
gene. Here we identify, for the first time, HNF1A-MODY-associated microRNAs (miRNAs) that can be detected ...in the serum of HNF1A-MODY carriers.
Methods
An miRNA array was carried out in rat INS-1 insulinoma cells inducibly expressing the common human
Pro291fsinsC-HNF1A
frame shift mutation. Differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of miRNAs in the serum of HNF1A-MODY carriers (
n
= 31), MODY-negative family members (
n
= 10) and individuals with type 2 diabetes mellitus (
n
= 17) was quantified by absolute real-time PCR analysis.
Results
Inducible expression of
Pro291fsinsC-HNF1A
in INS-1 cells caused a significant upregulation of three miRNAs (miR-103, miR-224, miR-292-3p). The differential expression of two miRNAs (miR-103 and miR-224) was validated in vitro. Strongly elevated levels of miR-103 and miR-224 could be detected in the serum of HNF1A-MODY carriers compared with MODY-negative family controls. Serum levels of miR-103 distinguished HNF1A-MODY carriers from HbA
1c
-matched individuals with type 2 diabetes mellitus.
Conclusions/interpretation
Our study demonstrates that the pathophysiology of HNF1A-MODY is associated with the overexpression of miR-103 and miR-224. Furthermore, our study demonstrates that these miRNAs can be readily detected in the serum of HNF1A-MODY carriers.
Abstract Proteasomal dysfunction and apoptosis are major hallmarks in the pathophysiology of Parkinson's disease (PD). PARK6 which is caused by mutations in the mitochondrial protein kinase PINK1 is ...a rare autosomal-recessively inherited disorder mimicking the clinical picture of PD. To investigate the cytoprotective physiological function of PINK1, we used primary fibroblasts from three patients homozygous for G309D-PINK1 as well as SHEP neuroblastoma cells stably overexpressing GFP-tagged wild type (wt) PINK1. Here we demonstrate that overexpression of wt PINK1 inhibits activation of Bax and release of cytochrome c, thereby diminishing caspase 9 processing and effector caspase activity after induction of proteasomal stress with the proteasome inhibitor (PI) MG132 in SHEP cells. Conversely, effector caspase activation induced by PIs, but not by the unrelated apoptotic stimulus staurosporine was potently enhanced in primary fibroblasts from homozygous PARK6 patients in comparison to those of heterozygous carriers or unaffected siblings. SHEP cells overexpressing wt PINK1 showed an elevated expression of the cytoprotective gene parkin , whereas PARK6 fibroblasts displayed significantly decreased expression of parkin in comparison to wild type control cells. Interestingly, overexpressed GFP-PINK1 was exclusively localized in the mitochondria of SHEP cells, but was redistributed to the cytoplasm under conditions of proteasomal stress. Our data indicate that PINK1 plays an important and specific physiological role in protecting cells from proteasomal stress, and suggest that PINK1 might exert its cytoprotective effects upstream of mitochondria engagement.
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