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See detailCharacterization of chemotherapy-induced cell death in glioblastoma
Coupienne, Isabelle ULg; Fettweis, Grégory ULg; Piette, Jacques ULg

Poster (2012, December 10)

Glioblastoma constitute the most frequent and deadliest type of brain tumors. Their annual incidence is estimated between 5 and 8 cases per 100,000 people in Europe and North America. They are resistant ... [more ▼]

Glioblastoma constitute the most frequent and deadliest type of brain tumors. Their annual incidence is estimated between 5 and 8 cases per 100,000 people in Europe and North America. They are resistant to all current therapies and are associated with a very high rate of recurrence. The associated prognosis is generally very poor and most patients die within a year after diagnosis. Unfortunately, despite extensive research and use of multimodality treatments combining surgical resection, chemotherapy and radiotherapy, survival hasn’t really much improved over the last 20 years. Indeed, these tumors were shown to be characterized by a high radio- and chemo-resistance. Glioblastoma cells exhibit overexpression of the anti-apoptotic Bcl-2 family proteins and downregulation of its pro-apoptotic members, high expression of the IAPs (Inhibitors of Apoptosis Proteins) and constitutive activation of the pro-survival NF-κB pathway. Currently, the most commonly used treatment offering the best prognosis to patients consists in a combination of maximal surgical tumor resection (when feasible) with subsequent radio- and/or chemotherapy. Among the most commonly used chemotherapeutic agents, the alkylating agent temozolomide and the topoisomerase I inhibitor camptothecin occupy a central position. Therefore, in this study, the impact of both temozolomide and irinotecan (a soluble derivative of camptothecin) on glioma cell survival will be investigated. Important progress was made in the comprehension of the molecular mechanisms underlying tumor development and progression however, survival benefits conferred by the use of new drugs and therapeutic strategies are counted in months rather than years. Consequently, there is an urge to rapidly improve the efficiency of the currently used treatments. This research project consists in (i) the study of the mechanisms implicated in glioblastoma cell death induced by two chemotherapeutic agents : temozolomide and irinotecan, (ii) the identification of the mechanisms underlying the resistance of glioblastoma to these treatments, (iii) the use of pharmacological tools to interfere with those resistance strategies to enhance chemotherapy efficiency. Necrosis was, until recently, long thought to be only accidental. However, it was shown to be finely regulated by specific signalling pathways. Programmed necrosis often takes place in cells in which apoptosis cannot be properly activated and serves as a back-up cell death pathway. Previous work from our lab having already demonstrated that glioblastoma are, at least partially, apoptosis-defective, special emphasis is put on the study of necrotic parameters. Results from survival tests performed on several glioblastoma cell lines in the presence of necrotic inhibitors like necrostatin-1 (an inhibitor of the central regulator of the necrotic pathway RIP1) allows to partially overcome temozolomide and irinotecan-induced glioblastoma cell death, highlighting the role played by programmed necrosis in chemotherapy sensitivity. [less ▲]

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See detailSpatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species.
Rubio, Noemi; Coupienne, Isabelle ULg; Di Valentin, Emmanuel ULg et al

in Autophagy (2012), 8(9), 1312-24

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is ... [more ▼]

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H 2O 2 in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis. [less ▲]

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See detailSignal transduction in photodynamic therapy-mediated cell death
Coupienne, Isabelle ULg

Doctoral thesis (2011)

Les glioblastomes sont des tumeurs du cerveau comptant parmi les plus fréquentes et les plus agressives. En effet, l’espérance de vie médiane des patients est d’environ quinze mois après diagnostic ... [more ▼]

Les glioblastomes sont des tumeurs du cerveau comptant parmi les plus fréquentes et les plus agressives. En effet, l’espérance de vie médiane des patients est d’environ quinze mois après diagnostic. Malgré d’intenses recherches et l’utilisation de traitements combinant la chirurgie, la radiothérapie et la chimiothérapie, le pronostic des patients n’a guère évolué depuis une vingtaine d’années. Ces tumeurs sont caractérisées par la présence de diverses altérations génétiques et, généralement, par une activation constitutive du facteur de transcription NF-κB. Étant un régulateur majeur de l’apoptose et de la survie cellulaire, ce facteur pourrait jour un rôle central dans la résistance des glioblastomes aux diverses thérapies. Au cours de ce travail, nous avons étudié les effets de l’inhibition du NF-κB sur la mort cellulaire des glioblastomes induite en réponse à un traitement par PDT, dont l’efficacité repose sur la production d’espèces réactives de l’oxygène suite à la présence simultanée d’un photosensibilisateur, de lumière et d’oxygène. Ainsi, nos résultats montrent que les cellules dans lesquelles le NF-κB est inhibé présentent une sensibilité accrue au traitement par rapport aux cellules non-inhibées. De plus, cette augmentation de mort cellulaire est majoritairement due à de la nécrose. Le traitement par PDT conduit également à l’apparition d’autophagie cytoprotectrice dont l’inhibition améliore l’efficacité du traitement. Nous nous sommes ensuite focalisés sur la mort par nécrose et avons démontré l’implication de la kinase RIP3 dans la mort nécrotique induite par PDT. Il est apparu que l’oxygène singulet produit au cours du traitement était à l’origine de la nécrose RIP3-dépendante. Cependant, les intermédiaires transductionnels et la manière dont le NF-κB exerce ses effets anti-nécrotiques demeurent encore inconnus. Enfin, afin de mieux comprendre le rôle joué par RIP3 dans la mort induite par PDT, nous avons utilisé la lignée d’ostéosarcome U2OS, déficiente en cette protéine, et au sein de laquelle nous en avons restauré l’expression. De manière surprenante, les cellules sauvages se sont révélées plus sensibles au traitement que les U2OS exprimant RIP3. De plus, la lignée RIP3-U2OS montre un taux d’apoptose supérieur à celui de son homologue sauvage. Cette divergence peut en partie s’expliquer par une induction d’autophagie plus importante au sein des RIP3-U2OS. Celle-ci, pouvant être un mécanisme pro-survie, peut leur conférer une protection contre la mort cellulaire en facilitant l’élimination des organelles endommagés. Enfin, nous avons déterminé que les cellules sauvages présentent un taux de nécrose plus important que les cellules RIP3-U2OS en réponse au traitement. Nos résultats ont dont permis de prouver (i) que l’inhibition du NF-κB améliorait la sensibilité des glioblastomes à la mort cellulaire induite par un traitement par 5-ALA-PDT, (ii) que celle-ci était, dans ce cas, majoritairement de la nécrose RIP3-dépendante et (iii) de mettre au jour un rôle pour RIP3 dans la régulation de l’apoptose et de l’autophagie induites par PDT. [less ▲]

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See detailRIP3 Expression Induces a Death Profile Change in U2OS Osteosarcoma Cells After 5-ALA-PDT
Coupienne, Isabelle ULg; Fettweis, Grégory ULg; Piette, Jacques ULg

in Lasers in Surgery and Medicine (2011), 43(7), 557564

Background and Objective The receptor-interacting protein 3 (RIP3) has recently been outlined as a key necrosis mediator but is also thought to participate in the regulation of apoptosis. The aim of this ... [more ▼]

Background and Objective The receptor-interacting protein 3 (RIP3) has recently been outlined as a key necrosis mediator but is also thought to participate in the regulation of apoptosis. The aim of this study is to compare the cell death profile induced by 5-aminolevulic acid (5-ALA)-mediated photodynamic therapy (PDT) in the RIP3-deficient cell line U2OS and in U2OS cells in which the expression of RIP3 was restored. Materials and Methods RIP3-expressing U2OS cells (RIP3-U2OS) were obtained after transfection and antibiotic selection. Wild type and RIP3-U2OS cells were treated by 5-ALA-PDT. Overall cell viability was evaluated and different parameters characteristic of apoptosis, autophagy, and necrosis were studied. Results Surprisingly, the survival of RIP3-U2OS cells was higher compared to that of the wild type cells. In addition, RIP3-U2OS cell death was decreased by a zVAD-fmk pre-treatment. A higher cleavage of caspase-3, 7, 8, 9, and PARP was also detected in these cells, pointing out to the activation of caspase-dependent apoptosis. In parallel, a thrust of autophagy was clearly identified in the RIP3-U2OS cells. Conversely, RIP3-U2OS exhibited a lower level of necrosis than the wild types. Interestingly, necrostatin-1 efficiently decreased necrosis level in RIP3-U2OS but not in wild type cells. Conclusion Expression of RIP3 in U2OS cells led to a better survival but also to a death profile change in response to PDT. The apoptotic and autophagic pathways were clearly up-regulated compared to the RIP3-deficient wild type cells. However, induction of necrosis was weaker in the RIP3-U2OS cells. In this context, autophagy is likely to play a protective role against PDT-induced cell death and to allow a better survival of RIP3-U2OS cells. This work also highlights the important role played by RIP3 in the apoptotic pathway, although the modalities are still widely unknown. [less ▲]

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See detailNF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy.
Coupienne, Isabelle ULg; Bontems, Sébastien ULg; Dewaele, M. et al

in Biochemical Pharmacology (2011)

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases ... [more ▼]

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-kappaB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-kappaB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a physiological compound widely used in PDT as well as in tumor photodetection (PDD). Our results show that inhibition of NF-kappaB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-kappaB inhibition. We found that apoptosis was induced by PDT but in an incomplete manner and that, unexpectedly, NF-kappaB inhibition reduced its level. Oppositely PDT mainly induces necrosis in glioblastoma cells and NF-kappaB is found to have anti-necrotic functions in this context. The autophagic flux was also enhanced as a result of 5-ALA-PDT and we demonstrate that stimulation of autophagy acts as a pro-survival mechanism confering protection against PDT-mediated necrosis. These data point out that 5-ALA-PDT has an interesting potential as a mean to treat glioblastoma and that inhibition of NF-kappaB renders glioblastoma cells more sensitive to the treatment. [less ▲]

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See detailStudy of the combined effect of 5-ALA-based photodynamic therapy and NF-kappaB inhibition on human glioblastoma cell survival
Coupienne, Isabelle ULg; Fettweis, Grégory ULg; Piette, Jacques ULg

Poster (2011, January)

Glioblastoma constitute the most frequent and deadliest type of brain tumors in human adults. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most ... [more ▼]

Glioblastoma constitute the most frequent and deadliest type of brain tumors in human adults. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-kappaB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-kappaB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)–based photodynamic therapy (PDT). Our results show that inhibition of NF-kappaB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-kappaB inhibition. We found that PDT mainly induced necrosis in glioblastoma cells and NF-kappaB was found to have anti-necrotic functions in this context. In the second part of this study, we examined the role of the kinase RIP3, recently identified as a key effector of the necrotic pathway, in 5-ALA-PDT-induced necrosis and studied whether NF-kappaB interfered in RIP3-dependent necrosis induction. [less ▲]

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See detail5-ALA-PDT induces RIP3-dependent necrosis in glioblastoma
Coupienne, Isabelle ULg; Fettweis, Grégory ULg; Rubio-Romero, Noemi ULg et al

in Photochemical & Photobiological Sciences (2011)

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are resistant to all current therapies and are associated with a high rate of recurrence. Glioblastoma were ... [more ▼]

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are resistant to all current therapies and are associated with a high rate of recurrence. Glioblastoma were previously shown to respond to treatments by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) mainly by activating a necrotic type of cell death. The receptor-interacting protein 3 (RIP3) has recently been outlined as a key mediator of this caspase-independent form of programmed cell death. In the present study, we analyzed the necrotic mechanism induced by 5-ALA-PDT in human glioblastoma cells and explored the role of RIP3 in this context. Our results show that PDT-induced necrosis is dependent on RIP3, which forms aggregates and colocalizes with RIP1 following photosensitization. We demonstrate that PDT-mediated singlet oxygen production is the cause of RIP3-dependent necrotic pathway activation. We also prove that PDT induces the formation of a pro-necrotic complex containing RIP3 and RIP1 but lacking caspase-8 and FADD, two proteins usually part of the necrosome when TNF-α is used as a stimulus. Thus, we hypothesize that PDT might lead to the formation of a different necrosome whose components, besides RIP1 and RIP3, are still unknown. In most cases, glioblastoma are characterized by a constitutive activation of NF-κB. This factor is a key regulator of various processes, such as inflammation, immune response, cell growth or apoptosis. Its inhibition was shown to further sensitize glioblastoma cells to PDT-induced necrosis, however, no difference in RIP3 upshift or aggregation could be observed when NF-κB was inhibited. [less ▲]

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See detailHow to monitor NF-kappaB activation after photodynamic therapy.
Coupienne, Isabelle ULg; Piette, Jacques ULg; Bontems, Sébastien ULg

in Methods in Molecular Biology (Clifton, N.J.) (2010), 635

The nuclear factor-kappa B (NF-kappaB) is a multipotent factor involved in many cellular processes such as inflammation, immune response and embryonic development and it can be activated by a large number ... [more ▼]

The nuclear factor-kappa B (NF-kappaB) is a multipotent factor involved in many cellular processes such as inflammation, immune response and embryonic development and it can be activated by a large number of stimuli. Consequently, this transcription factor plays a pivotal role in many natural processes but also in different pathologies. For several years, photodynamic therapy (PDT) has emerged as an attractive alternative approach for the treatment of different affections involving various forms of cancer and an increasing number of reports have highlighted the activation of the NF-kappaB following PDT treatment. Furthermore, it has been shown that the mechanism of activation of the NF-kappaB as well as its target genes depends on the nature of the photosensitizers and the cell type used. As this transcription factor is known to be a key regulator of the immune response but also controls cell survival and proliferation, it is important to assess its activation status and its impact on the target genes. In this review, we will present different techniques allowing identification of the activation status of this factor, from the degradation of its inhibitor in the cytoplasm to its ability to induce the expression of a reporter gene under the control of a target promoter. As a working model we will present results obtained from a 5-aminolevulinic acid-PDT treatment on cervix adenocarcinoma cells. [less ▲]

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