Is there a common upstream link for autophagic and apoptotic cell death in human high-grade gliomas?

The prognosis of patients with human high-grade gliomas (HGGs) remains dismal despite major advances in their management, due mainly to the high resistance of these infiltrative tumor cells to programmed cell death (PCD). Most therapeutic strategies for HGGs are aimed to maximize PCD type I, apoptosis or type II, autophagy. These are predominantly distinctive processes, but many studies suggest a cross-talk between the two. A better understanding of the link between PCD types I and II might allow development of more effective therapies for HGGs. In this study, we examined whether there is a common upstream signaling event responsible for both apoptotic and autophagic PCD using 3 chemotherapeutic agents in human HGG cells. Our study shows that each agent caused a significant decrease in cell viability in each of the HGG cell lines tested. The increase rate of apoptosis and autophagy varied among cell lines and chemotherapeutic agents used. Increased expression of cytidine-cytidine-adenosine-adenosine-thymidine (C)/enhancer binding protein (EBP) homologous transcription factor C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 (GADD153) was documented after use of either pro-autophagic or pro-apoptotic agents. The involvement of CHOP/GADD153 in both type I and type II PCD was confirmed by overexpression and gene-silencing studies. Gene silencing by small-interfering RNA-mediated CHOP/GADD153 resulted in increased cell viability, decreased upregulation of microtubule-associated protein light-chain 3' type II (LC3II) and cleaved caspase-3, and inhibition of apoptosis and autophagy. Exogenous expression of CHOP/GADD153 triggered apoptosis and autophagy in the absence of other stimuli. The clinical significance of these findings was supported by the evidence that celecoxib, a nonsteroidal anti-inflammatory drug known to induce GADD153-mediated apoptosis, strongly increases both type I and type II PCD in HGG cells when combined with another inducer of GADD153. These data suggest that CHOP/GADD153 should be investigated as a novel targetable signaling step to improve therapies for HGGs.

In vivo gene delivery by embryonic-stem-cell-derived astrocytes for malignant gliomas.

The treatment of malignant gliomas with current therapies remains a challenge in neurooncology. Our recent work showed that embryonic stem cell (ESC)-derived astrocytes conditionally expressing genes can be used to induce apoptosis in malignant glioma cells in vitro. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene has been shown to induce apoptosis in a variety of tumor cells, including gliomas. The aim of this study was to assess the proapoptotic effects of transgenic TRAIL delivered by ESC-derived astrocytes on malignant gliomas in vivo. Malignant glioma A172 cells were used to induce heterotopic xenografts in nude mice. ESC-derived astrocytes conditionally expressing TRAIL were injected into the xenografts. TRAIL expression was documented in the malignant glioma xenografts by reverse transcription PCR and immunohistochemistry after external gene induction. A significant reduction in tumor volume occurred 48 h after a single injection (14%) and double injections (31%) in the experimental groups. Terminal dUTP nick end labeling (TUNEL) revealed abundant apoptotic tumor cells in the experimental groups. Seven days after injection, the tumor had undergone severe necrosis, with only scattered residual tumor cells at the periphery. Death receptor DR4 expression increased significantly in the experimental groups compared with controls. Our data suggest that ESC-derived astrocytes conditionally expressing TRAIL should be considered as vectors to deliver gene therapy for malignant gliomas.

Gene delivery by embryonic stem cells for malignant glioma therapy: hype or hope?

Patients with glioblastomas have a poor prognosis and new treatments paradigms are needed. The futility of current treatments lies in part on the difficulty of delivering therapeutic agents to the tumor. In this article, we review and discuss current knowledge of adjuvant treatment for glioblastomas including gene therapy and viral vectors. Additionally, this review provides an update on recent progress, which has focused on improving delivery methods including the development of new approaches such as stem cells. Particular emphasis is given to laboratory data showing the use of embryonic stem cells used as vectors to deliver pro-apoptotic genes to glioblastoma cells.

Enhanced proapoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand on temozolomide-resistant glioma cells.

OBJECT: Death receptor targeting is an attractive approach in experimental treatment for tumors such as malignant gliomas, which are resistant to radiation and chemotherapy. Among the family of cytokines referred to as death li gands, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted clinical interest. The aim of this study was to assess whether TRAIL can be used as an adjuvant to temozolomide (TMZ) for apoptosis induction in malignant glioma cell lines. METHODS: Six human malignant glioma cell lines (A172, U87, U251, T98, U343, and U373) were exposed to human (h)TRAIL, TMZ, or an hTRAIL/TMZ combined treatment. Cell viability was assayed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and phase-contrast microscopy. Cell apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and quantified using flow cytometric analysis. The apoptosis signaling cascade was studied with Western blotting. The additive effects of hTRAIL and TMZ resulted in a significant decrease in cell viability and an increased apoptotic rate. Expression of the death receptors DR5 and DR4 in two cell lines (A172 and U251) upregulated significantly when they were used in combination hTRAIL/TMZ treatment (p < 0.05 compared with baseline control), leading to activation of caspase-8 and caspase-3 (p < 0.05 compared with baseline control) and confirming an extrinsic apoptotic pathway. A cell intrinsic pathway through mitochondrial cytochrome c was not activated. CONCLUSIONS: Based on this work, one may infer that hTRAIL should be considered as an adjuvant treatment for TMZ-resistant human malignant gliomas.

Internalization and dephosphorylation of connexin43 in hypertrophied right ventricles of rats with pulmonary hypertension.

BACKGROUND: Altered expression and distribution of gap junctions might provide substrates for abnormal conduction and arrhythmogenesis in the heart, but little is known about the regulation of gap junctions under pathological conditions. The organization and phosphorylation state of connexin43 (Cx43) in ventricular hypertrophy will be investigated. METHODS AND RESULTS: Right ventricular (RV) hypertrophy was induced in rats by treatment with monocrotaline. Subcellular Cx43 distribution was assessed by immunoconfocal and electron microscopy. Immunolabeling of Cx43 was confined to the intercalated disks in the normal ventricular myocytes of control rats, but hypertrophied RV cells from monocrotaline-treated rats showed dispersion of Cx43 immunolabeling over the cell surface and in the cytoplasm; cytoplasmic Cx43 was increased by approximately 7-fold (n=15). The Cx43 internalization was confirmed by the double staining of monocrotaline-treated RV tissues for Cx43/wheat germ agglutinin (WGA) and Cx43/zonula occludens protein-1 (ZO-1). Electron microscopy of hypertrophied RVs showed an increase in annular gap junctions immunolabeled with Cx43. Immunoblotting revealed a significant increase in non-phosphorylated Cx43 in hypertrophied RVs (by approximately 5-fold, n=8) without changes in the total amount of Cx43. The accumulation of non-phosphorylated Cx43 in hypertrophied RVs was also recognized by immunoconfocal-microscopy with an isoform-specific antibody. CONCLUSION: Ventricular hypertrophy is associated with the dephosphorylation of Cx43 and its translocation from the intercalated disks to intracellular pools, suggesting accelerated gap junction degradation.

Apoptosis in human glioblastoma cells produced using embryonic stem cell-derived astrocytes expressing tumor necrosis factor-related apoptosis-inducing ligand.

OBJECT: Embryonic stem (ES) cell-derived astrocytes have several theoretical and practical advantages as gene therapy vectors in the treatment of malignant gliomas. The aim of this study was to test the proapoptotic effects of ES cell-derived astrocytes expressing transgenic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human malignant glioma cells. METHODS: Mouse ES cells containing a doxycycline-inducible transgene were engineered with human TRAIL (hTRAIL) and then directed to differentiate into astrocytes. The ES cell-derived-TRAIL-expressing astrocytes were cocultured with human malignant glioma cells. Reverse transcriptase polymerase chain reaction, immunocytochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and flow cytometry were used to quantify results. In vitro coculture of ES cell-derived astrocytes expressing hTRAIL with A172 human malignant glioma cells after doxycycline induction caused a significant decrease in cell viability from 85 +/- 2% at baseline to 8 +/- 2% posttreatment (p < 0.001). Labeling with apoptotic markers showed that cell death occurred by means of apoptosis. A significant increase in apoptotic rate (88 +/- 3%) from baseline (4 +/- 2%) was found in A172 cells after doxycycline induction (p < 0.005). This effect was superior to the apoptotic rate seen after treatment with recombinant TRAIL (57 +/- 2%). A decrease in cell viability and an increase in the apoptotic rate were not found in TRAIL-expressing-ES cell-derived astrocytes after induction with doxycycline or in A172 cells exposed to doxycycline alone. CONCLUSIONS: Engineering of transgenic hTRAIL by using ES cell-derived astrocytes induced apoptosis in human malignant glioma cells while sparing nontumor astrocytes. The apoptotic effects of transgenic hTRAIL are greater than those of recombinant hTRAIL. Analysis of these results suggests that hTRAIL-expressing-ES cell-derived astrocytes should be considered in the development of new in vivo strategies to treat malignant human gliomas.

Embryonic stem cell-derived astrocytes: a novel gene therapy vector for brain tumors.

OBJECT: For gene therapy strategies currently in clinical trials, viral vectors are used to deliver transgenes directly to normal and tumor cells within the central nervous system (CNS). The use of viral vectors is limited by several factors. The aim of this study was to assess whether embryonic stem cell (ESC)-derived astrocytes expressing a doxycyclineinducible transgene can be used as a vector for gene therapy. METHODS: The authors generated a pure population of ESC-derived astrocytes carrying a transgene, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), inserted in the chromosome under the control of a highly regulated doxycycline-inducible expression system. Fully differentiated ESC-derived astrocytes were stereotactically transplanted in the mouse brain, and then cell migration and transgene expression were studied. RESULTS: The ESC-derived astrocytes started to migrate from the transplant site 48 hours after the procedure. They were found to have migrated throughout the brain tissue by 6 weeks. Transplanted ESC-derived astrocytes expressed the TRAIL transgene after doxycycline induction throughout the duration of the experiment. Teratoma formation was not observed in long-term experiments (12 weeks). CONCLUSIONS: These data show that ESC-derived astrocytes can be used as delivery vectors for CNS tumors. This technique might have a major impact on the treatment of patients with malignant gliomas and a wide spectrum of other neurological diseases.

Decreased vagal control over heart rate in rats with right-sided congestive heart failure: downregulation of neuronal nitric oxide synthase.

BACKGROUND: Parasympathetic drive is attenuated in heart failure, and resulting autonomic imbalance may increase the risk of sudden cardiac death. The anatomic site(s) and molecular mechanisms underlying this parasympathetic withdrawal are unknown. METHODS AND RESULTS: We examined the effects of pre- and post-ganglionic vagal nerve stimulation (VS) and acetylcholine (ACh) application on the heart rate of rats with right-sided congestive heart failure (CHF) induced by monocrotaline. Heart rate reduction in response to pre-ganglionic VS in CHF rats in vivo was significantly less than in controls. The suppression of spontaneous beating of isolated right atria including the whole sinoatrial (SA) node in response to post-ganglionic VS was significantly attenuated in CHF rats as well. In contrast, ACh application to the right atria resulted in a significantly larger suppression of spontaneous beating in CHF rats than controls. Proteins of neuronal nitric oxide synthase (nNOS) in the right atria were significantly decreased, whereas muscarinic (M2) receptor was significantly increased in CHF rats compared with controls. CONCLUSIONS: Both pre-and post-ganglionic vagal nerve functions are diminished in CHF rats, whereas M2 receptor-mediated regulation of the SA node is upregulated. Downregulation of nNOS may be involved in this parasympathetic withdrawal.

Overexpression of calpastatin by gene transfer prevents troponin I degradation and ameliorates contractile dysfunction in rat hearts subjected to ischemia/reperfusion.

Calpain is a Ca(2+)-activated neutral protease that supposedly plays a key role in myocardial dysfunction following ischemia/reperfusion, by degrading certain proteins involved in the contraction mechanism. It is possible that overexpression of calpastatin, an endogenous calpain inhibitor, lessens contractile dysfunction in the heart after reperfusion by preventing cardiac troponin I (TnI) degradation. This claim is tested by overexpression of human calpastatin (hCS) in rat hearts ex vivo using an adenovirus vector; the hearts were transplanted heterotopically into the abdomens of recipient rats to allow expression of hCS. On the fourth day after surgery, the hearts were excised and perfused in vitro to study their recovery from 30 min of global ischemia, which was followed by 60 min of reperfusion. The peak recovery of the left ventricular developed pressure (LVDP), and the values of its first derivative (max dP/dt, min dP/dt) in the hCS-overexpressed hearts were 88.9 +/- 4.8%, 90.8 +/- 9.2% and 106.4 +/- 9.8%, respectively; these values were all significantly greater than in the control hearts transfected with LacZ alone (51.4 +/- 6.9%, 52.6 +/- 8.1% and 54.7 +/- 6.6%, P < 0.05). In western blot analysis of ventricular myocardial samples (at 60-min reperfusion) using a monoclonal anti-TnI antibody, two bands corresponding to intact TnI (30 kDa) and TnI fragments (27 kDa) were distinguished. The fraction of 27-kDa TnI (percent of total TnI immunoreactivity) in hCS-overexpressed hearts was significantly less than the controls (5.7 +/- 2.7% vs. 18.1 +/- 3.2%, P < 0.05), implying a protective action of hCS against TnI degradation. These results suggest that adenovirus-mediated overexpression of hCS in the heart could be a novel biological means to minimize myocardial stunning by ischemia/reperfusion.