Characterization of cannabinoid receptors expressed in Ewing sarcoma TC-71 and A-673 cells as potential targets for anti-cancer drug development.

AIMS: Characterizing cannabinoid receptors (CBRs) expressed in Ewing sarcoma (EWS) cell lines as potential targets for anti-cancer drug development. MAIN METHODS: CBR affinity and function were examined by competitive binding and G-protein activation, respectively. Cannabinoid-mediated cytotoxicity and cell viability were evaluated by LDH, and trypan blue assays, respectively. KEY FINDINGS: qRT-PCR detected CB1 (CB1R) and CB2 receptor (CB2R) mRNA in TC-71 cells. However, binding screens revealed that CBRs expressed exhibit atypical properties relative to canonical receptors, because specific binding in TC-71 could only be demonstrated by the established non-selective CB1/CB2R radioligand [3H]WIN-55,212-2, but not CB1/CB2R radioligand [3H]CP-55,940. Homologous receptor binding demonstrated that [3H]WIN-55,212-2 binds to a single site with nanomolar affinity, expressed at high density. Further support for non-canonical CBRs expression is provided by subsequent binding screens, revealing that only 9 out of 28 well-characterized cannabinoids with high affinity for canonical CB1 and/or CB2Rs were able to displace [3H]WIN-55,212-2, whereas two ligands enhanced [3H]WIN-55,212-2 binding. Five cannabinoids producing the greatest [3H]WIN-55,212-2 displacement exhibited high nanomolar affinity (Ki) for expressed receptors. G-protein modulation and adenylyl cyclase assays further indicate that these CBRs exhibit distinct signaling/functional profiles compared to canonical CBRs. Importantly, cannabinoids with the highest affinity for non-canonical CBRs reduced TC-71 viability and induced cytotoxicity in a time-dependent manner. Studies in a second EWS cell line (A-673) showed similar atypical binding properties of expressed CBRs, and cannabinoid treatment produced cytotoxicity. SIGNIFICANCE: Cannabinoids induce cytotoxicity in EWS cell lines via non-canonical CBRs, which might be a potential therapeutic target to treat EWS.

Gene Expression and Data Analysis Pipeline Using Cancer BioPortal in the Classroom.

At institutions with an emphasis on authentic research experiences as an integral part of the biology curriculum, COVID created a huge challenge for course instructors whose learning objectives were designed for such experiences. Moving such laboratory experiences online when remote learning became necessary has resulted in a new model for CUREs that utilizes free online databases to provide not only a novel research experience for students, but also the opportunity to engage in big data analysis. Cancer BioPortal (cBioPortal) is an open-access collective cancer research resource for storing and exploring clinical, genomic, proteomic, and transcriptomic data. cBioPortal eliminates the computational barrier of interpreting complex genomic data by providing easily understandable visualization that can be interpreted and translated into relevant biological insights. Because no prior computational knowledge is required, cBioPortal is an ideal educational tool for either in-person or distance learning environments. We developed a pedagogical approach, video tutorials, and data analysis workflows centered on using cBioPortal. Pedagogically, students develop an initial research outline that is continually updated and graded throughout the project. Progress during the project or course is assessed by a series of student presentations that are 5 to 15 minutes in length and are aimed at explaining the approach used in data acquisition, interpretation of the data, and relevance to the initial hypothesis. While cancer-specific, this analysis platform appeals to a wide range of classes and student interests. Further, the project has been successfully done both as an independent research experience and as part of a virtual class-based research project.

Mitoxantrone repression of astrocyte activation: relevance to multiple sclerosis.

Mitoxantrone has been approved by the FDA for the treatment of multiple sclerosis (MS). However, the mechanisms by which mitoxantrone modulates MS are largely unknown. Activated astrocytes produce nitric oxide (NO), TNF-α, and IL-1ß, molecules which can be toxic to central nervous system (CNS) cells including oligodendrocytes, thus potentially contributing to the pathology associated with MS. MCP-1 is a chemokine believed to modulate the migration of monocytes to inflammatory lesions present in the CNS of MS patients. IL-12 and IL-23 have been demonstrated to play critical roles in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, by contributing to the development of CD4(+) T cell lineages termed Th1 and Th17, respectively. The current study demonstrates that mitoxantrone inhibits lipopolysachharide (LPS) induction of NO, TNF-α, IL-1ß, and MCP-1 production by primary astrocytes. Mitoxantrone also inhibited IL-12 and IL-23 production by these cells. Furthermore, mitoxantrone suppressed the expression of C-reactive protein (CRP). Finally, we demonstrate that mitoxantrone suppressed LPS induction of NF-κB DNA-binding activity, suggesting a novel mechanism by which mitoxantrone suppresses the expression of proinflammatory molecules. Collectively, these studies demonstrate that mitoxantrone represses astrocyte production of potentially cytotoxic molecules, as well as molecules capable of altering T-cell phenotype. These in vitro studies suggest mechanisms by which mitoxantrone may modulate inflammatory diseases including MS.

Resveratrol effects on astrocyte function: relevance to neurodegenerative diseases.

Inflammatory molecules have been implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis. Resveratrol is an anti-fungal compound found in the skins of red grapes and other fruits and nuts. We examined the ability of resveratrol to inhibit lipopolysaccharide (LPS)-induced production of inflammatory molecules from primary mouse astrocytes. Resveratrol inhibited LPS-induced production of nitric oxide (NO); the cytokines tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), and IL-6; and the chemokine monocyte chemotactic protein-1 (MCP-1), which play critical roles in innate immunity, by astrocytes. Resveratrol also suppressed astrocyte production of IL-12p40 and IL-23, which are known to alter the phenotype of T cells involved in adaptive immunity. Finally resveratrol inhibited astrocyte production of C-reactive protein (CRP), which plays a role in a variety of chronic inflammatory disorders. Collectively, these studies suggest that resveratrol may be an effective therapeutic agent in neurodegenerative diseases initiated or maintained by inflammatory processes.

Neuron-specific enolase-cre mouse line with cre activity in specific neuronal populations.

To establish genetic tools for conditional gene deletion in mouse neurons, we generated two independent neuron-specific enolase (Nse)-cre transgenic lines. The transgenic line termed Nse-cre(CK1) showed cre activity in most neuronal regions in the nervous system, while the Nse-cre(CK2) line exhibited a unique cre activity that has not been reported in other cre transgenic lines. Nse-cre(CK2) cre activity was detectable from embryogenesis and mostly restricted to neuronal regions. In postnatal brain, the Nse-cre(CK2) line exhibited cre activity limited to differentiated neurons in the cerebral cortex and hippocampus. Cre activity was assayed in several internal organs and sporadic activity was limited to the kidney and testis. We conclude that these cre lines will be useful for studying loss of gene function in specific neuronal populations.

Transgenic mice expressing lipoprotein lipase in adipose tissue. Absence of the proximal 3'-untranslated region causes translational upregulation.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and adipocyte metabolism. Defects in LPL can lead to hypertriglyceridemia and the subsequent development of atherosclerosis. The mechanisms of regulation of this enzyme are complex and may occur at multiple levels of gene expression. Because the 3'-untranslated region (UTR) is involved in LPL translational regulation, transgenic mice were generated with adipose tissue expression of an LPL construct either with or without the proximal 3'-UTR and driven by the aP2 promoter. Both transgenic mouse colonies were viable and expressed the transgene, resulting in a 2-fold increase in LPL activity in white adipose tissue. Neither mouse colony exhibited any obvious phenotype in terms of body weight, plasma lipids, glucose, and non-esterified fatty acid levels. In the mice expressing hLPL with an intact 3'-UTR, hLPL mRNA expression approximately paralleled hLPL activity. However in the mice without the proximal 3'-UTR, hLPL mRNA was low in the setting of large amounts of hLPL protein and LPL activity. In previous studies, the 3'-UTR of LPL was critical for the inhibitory effects of constitutively expressed hormones, such as thyroid hormone and catecholamines. Therefore, these data suggest that the absence of the 3'-UTR results in a translationally unrepressed LPL, resulting in a moderate overexpression of adipose LPL activity.