Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

Arsenic trioxide and neuroblastoma cytotoxicity

The majority of aggressive forms of the childhood tumor neuroblastoma can with current treatment protocols not be cured and possess a major challenge in pediatric oncology. After initial rounds of chemotherapy, surgery and irradiation, which in most cases result in tumor regression, these aggressive neuroblastomas relapse and frequently develop drug resistance. As approximately 50% of the children with neuroblastoma have an aggressive form, there is a compelling demand for new treatment strategies. Arsenic trioxide has the capacity to kill multidrug-resistant neuro-blastoma cells in vitro and in vivo and the drug is currently being evaluated in clinical trials. In this report we discuss the background to the use of arsenic trioxide in cancer therapy and the currently known mechanisms by which arsenic trioxide kills human neuroblastoma cells.     

Population genetics of Plasmodium resistance genes in Anopheles gambiae: no evidence for strong selection

Anopheles mosquitoes are the primary vectors for malaria in Africa, transmitting the disease to more than 100 million people annually. Recent functional studies have revealed mosquito genes that are crucial for Plasmodium development, but there is presently little understanding of which genes mediate vector competence in the wild, or evolve in response to parasite-mediated selection. Here, we use population genetic approaches to study the strength and mode of natural selection on a suite of mosquito immune system genes, CTL4, CTLMA2, LRIM1, and APL2 (LRRD7), which have been shown to affect Plasmodium development in functional studies. We sampled these genes from two African populations of An. gambiae s.s., along with several closely related species, and conclude that there is no evidence for either strong directional or balancing selection on these genes. We highlight a number of challenges that need to be met in order to apply population genetic tests for selection in Anopheles mosquitoes; in particular the dearth of suitable outgroup species and the potential difficulties that arise when working within a closely-related species complex.     

Combination of arsenic trioxide and BCNU synergistically triggers redox-mediated autophagic cell death in human solid tumors

Arsenic trioxide (As₂O₃) is an effective treatment for relapsed or refractory acute promyelocytic leukemia (APL). After the discovery of As₂O₃ as a promising treatment for APL, several studies investigated the use of As₂O₃ as a single agent in the treatment of solid tumors; however, its therapeutic efficacy is limited. Thus, the systematic study of the combination of As₂O₃ with other clinically used chemotherapeutic drugs to improve its therapeutic efficacy in treating human solid tumors is merited. In this study, we demonstrate for the first time, using isobologram analysis, that As₂O₃ exhibits a synergistic interaction with N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU). The synergistic augmentation of the cytotoxicity of As₂O₃ with BCNU is in part through the autophagic cell death machinery in human solid tumor cells. As₂O₃ and BCNU in combination produce enhanced cytotoxicity via the depletion of reduced glutathione (GSH) and augmentation of reaction oxygen species (ROS) production. Further analysis indicated that the extension of GSH depletion by this combined regimen occurs through the inhibition of the catalytic activity of glutathione reductase. Blocking ROS production with antioxidants or ROS scavengers effectively inhibits cell death and autophagy formation, indicating that redox-mediated autophagic cell death involves the synergism of As₂O₃ with BCNU. Taken together, this is the first evidence that BCNU could help to extend the therapeutic spectrum of As₂O₃. These findings will be useful in designing future clinical trials of combination chemotherapy with As₂O₃ and BCNU, with the potential for broad use against a variety of solid tumors.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.     

Fruit fly behavior in response to chemosensory signals

An important question in contemporary sensory neuroscience is how animals perceive their environment and make appropriate behavioral choices based on chemical perceptions. The fruit fly Drosophila melanogaster exhibits robust tastant and odor-evoked behaviors. Understanding how the gustatory and olfactory systems support the perception of these contact and volatile chemicals and translate them into appropriate attraction or avoidance behaviors has made an unprecedented contribution to our knowledge of the organization of chemosensory systems. In this review, I begin by describing the receptors and signaling mechanisms of the Drosophila gustatory and olfactory systems and then highlight their involvement in the control of simple and complex behaviors. The topics addressed include feeding behavior, learning and memory, navigation behavior, neuropeptide modulation of chemosensory behavior, and I conclude with a discussion of recent work that provides insight into pheromone signaling pathways.