Exploring Lin28 proteins: Unravelling structure and functions with emphasis on nervous system malignancies.

Cancer and stem cells share many characteristics related to self-renewal and differentiation. Both cell types express the same critical proteins that govern cellular stemness, which provide cancer cells with the growth and survival benefits of stem cells. LIN28 is an example of one such protein. LIN28 includes two main isoforms, LIN28A and LIN28B, with diverse physiological functions from tissue development to control of pluripotency. In addition to their physiological roles, LIN28A and LIN28B affect the progression of several cancers by regulating multiple cancer hallmarks. Altered expression levels of LIN28A and LIN28B have been proposed as diagnostic and/or prognostic markers for various malignancies. This review discusses the structure and modes of action of the different LIN28 proteins and examines their roles in regulating cancer hallmarks with a focus on malignancies of the nervous system. This review also highlights some gaps in the field that require further exploration to assess the potential of targeting LIN28 proteins for controlling cancer.

LIN28 expression and function in medulloblastoma.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment modalities are not completely effective and can lead to severe neurological and cognitive adverse effects. In addition to urgently needing better treatment approaches, new diagnostic and prognostic biomarkers are required to improve the therapy outcomes of MB patients. The RNA-binding proteins, LIN28A and LIN28B, are known to regulate invasive phenotypes in many different cancer types. However, the expression and function of these proteins in MB had not been studied to date. This study identified the expression of LIN28A and LIN28B in MB patient samples and cell lines and assessed the effect of LIN28 inhibition on MB cell growth, metabolism and stemness. LIN28B expression was significantly upregulated in MB tissues compared to normal brain tissues. This upregulation, which was not observed in other brain tumors, was specific for the aggressive MB subgroups and correlated with patient survival and metastasis rates. Functionally, pharmacological inhibition of LIN28 activity concentration-dependently reduced LIN28B expression, as well as the growth of D283 MB cells. While LIN28 inhibition did not affect the levels of intracellular ATP, it reduced the expression of the stemness marker CD133 in D283 cells and the sphere formation of CHLA-01R cells. LIN28B, which is highly expressed in the human cerebellum during the first few months after birth, subsequently decreased with age. The results of this study highlight the potential of LIN28B as a diagnostic and prognostic marker for MB and open the possibility to utilize LIN28 as a pharmacological target to suppress MB cell growth and stemness.

T-Type Calcium Channel Inhibitors Induce Apoptosis in Medulloblastoma Cells Associated with Altered Metabolic Activity.

Medulloblastoma (MB) is the most common malignant paediatric brain tumour. In our previous studies, we developed a novel 3D assay for MB cells that was used to screen a panel of plasma membrane calcium channel modulators for their effect on the 3D growth of D341 MB cells. These studies identified T-type (CaV3) channel inhibitors, mibefradil and NNC-55-0396 (NNC) as selective inhibitors of MB cell growth. Mibefradil was originally approved for the treatment of hypertension and angina pectoris, and recently successfully completed a phase I trial for recurrent high-grade glioma. NNC is an analogue of mibefradil with multiple advantages compared to mibefradil that makes it attractive for potential future clinical trials. T-type channels have a unique low voltage-dependent activation/inactivation, and many studies suggest that they have a direct regulatory role in controlling Ca2+ signalling in non-excitable tissues, including cancers. In our previous study, we also identified overexpression of CaV3.2 gene in MB tissues compared to normal brain tissues. In this study, we aimed to characterise the effect of mibefradil and NNC on MB cells and elucidate their mechanism of action. This study demonstrates that the induction of toxicity in MB cells is selective to T-type but not to L-type Ca2+ channel inhibitors. Addition of CaV3 inhibitors to vincristine sensitised MB cells to this MB chemotherapeutic agent, suggesting an additive effect. Furthermore, CaV3 inhibitors induced cell death in MB cells via apoptosis. Supported by proteomics data and cellular assays, apoptotic cell death was associated with reduced mitochondrial membrane potential and reduced ATP levels, which suggests that both compounds alter the metabolism of MB cells. This study offers new insights into the action of mibefradil and NNC and will pave the way to test these molecules or their analogues in pre-clinical MB models alone and in combination with vincristine to assess their suitability as a potential MB therapy.

Calcium Signalling in Medulloblastoma: An In Silico Analysis of the Expression of Calcium Regulating Genes in Patient Samples.

Dysregulation in calcium signalling is implicated in several cancer-associated processes, including cell proliferation, migration, invasion and therapy resistance. Modulators of specific calcium-regulating proteins have been proposed as promising future therapeutic agents for some cancers. Alterations in calcium signalling have been extensively studied in some cancers; however, this area of research is highly underexplored in medulloblastoma (MB), the most common paediatric malignant brain tumour. Current MB treatment modalities are not completely effective and can result in several long-lasting mental complications. Hence, new treatment strategies are needed. In this study, we sought to probe the landscape of calcium signalling regulators to uncover those most likely to be involved in MB tumours. We investigated the expression of calcium signalling regulator genes in MB patients using publicly available datasets. We stratified the expression level of these genes with MB molecular subgroups, tumour metastasis and patient survival to uncover correlations with clinical features. Of particular interest was CACNA1 genes, in which we were able to show a developmentally-driven change in expression within the cerebellum, MB's tissue of origin, highlighting a potential influence on tumour incidence. This study lays a platform for future investigations into molecular regulators of calcium signalling in MB formation and progression.

Development of a High-throughput Agar Colony Formation Assay to Identify Drug Candidates against Medulloblastoma.

Medulloblastoma (MB) is the most common malignant childhood brain cancer. High-risk MB tumours have a high incidence of metastasis and result in poor patient survival. Drug screens, commonly used to identify potential novel therapeutic agents against MB, focus on 2D cell proliferation and viability assays given that these assays are easily adaptable to high-throughput regimes. However, 2D models fail to address invasive characteristics that are crucial to MB metastasis and are thus not representative of tumour growth in vivo. In this study, we developed a 3D 384-well agar colony formation assay using MB cells of molecular subgroup 3 that is associated with the highest level of metastasis. Two fluorescence substrates, resazurin and glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) that measure cell viability via distinct mechanisms were used to assess the growth of MB cells in the agar matrix. The assay was optimised for seeding density, growth period, substrate incubation time and homogeneity of the fluorescent signals within individual wells. Our data demonstrate the feasibility to multiplex the two fluorescent substrates without detectable signal interference. This assay was validated by assessing the concentration-dependent effect of two commonly used chemotherapeutic agents clinically used for MB treatment, vincristine and lomustine. Subsequently, a panel of plasma membrane calcium channel modulators was screened for their effect on the 3D growth of D341 MB cells, which identified modulators of T-type voltage gated and ORAI calcium channels as selective growth modulators. Overall, this 3D assay provides a reproducible, time and cost-effective assay for high-throughput screening to identify potential drugs against MB.

Comparative In Vitro Toxicology of Novel Cytoprotective Short-Chain Naphthoquinones.

Short-chain quinones (SCQs) have been identified as potential drug candidates against mitochondrial dysfunction, which largely depends on the reversible redox characteristics of the active quinone core. We recently identified 11 naphthoquinone derivatives, 1-11, from a library of SCQs that demonstrated enhanced cytoprotection and improved metabolic stability compared to the clinically used benzoquinone idebenone. Since the toxicity properties of our promising SCQs were unknown, this study developed multiplex methods and generated detailed toxicity profiles from 11 endpoint measurements using the human hepatocarcinoma cell line HepG2. Overall, the toxicity profiles were largely comparable across different assays, with simple standard assays showing increased sensitivity compared to commercial toxicity assays. Within the 11 naphthoquinones tested, the L-phenylalanine derivative 4 consistently demonstrated the lowest toxicity across all assays. The results of this study not only provide useful information about the toxicity features of SCQs but will also enable the progression of the most promising drug candidates towards their clinical use.

EMT signaling: potential contribution of CRISPR/Cas gene editing.

Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.

Stability of Meropenem and Piperacillin/Tazobactam with Heparin in Various Peritoneal Dialysis Solutions.

BACKGROUND: Infections caused by ceftazidime-resistant Pseudomonas and extended-spectrum beta-lactamase (ESBL)-producing gram-negative bacteria are increasing worldwide. Meropenem and piperacillin/tazobactam (PIP/TZB) are recommended for the treatment of peritoneal dialysis-associated peritonitis (PDAP) caused by ceftazidime-resistant Pseudomonas and other resistant gram-negative bacteria. Patients may also receive intraperitoneal heparin to prevent occlusion of their catheters. However, the stability of meropenem or PIP/TZB, in combination with heparin, in different types of peritoneal dialysis (PD) solutions used in clinical practice is currently unknown. Therefore, we investigated the stability of meropenem and PIP/TZB, each in combination with heparin, in different PD solutions. METHODS: A total of 15 PD bags (3 bags for each type of PD solution) containing meropenem and heparin and 24 PD bags (3 bags for each type of PD solution) containing PIP/TZB and heparin were prepared and stored at 4°C for 168 hours. The same bags were stored at 25°C for 3 hours followed by 10 hours at 37°C. An aliquot withdrawn before storage and at defined time points was analyzed for the concentration of meropenem, PIP, TZB, and heparin using high-performance liquid chromatography. Samples were also analysed for particle content, pH and color change, and the anticoagulant activity of heparin. RESULTS: Meropenem and heparin retained more than 90% of their initial concentration in 4 out of 5 types of PD solutions when stored at 4°C for 168 hours, followed by storage at 25°C for 3 hours and then at 37°C for 10 hours. Piperacillin/tazobactam and heparin were found to be stable in all 8 types of PD solutions when stored under the same conditions. Heparin retained more than 98% of its initial anticoagulant activity throughout the study period. No evidence of particle formation, color change, or pH change was observed at any time under the storage conditions employed in the study. CONCLUSIONS: This study provides clinically important information on the stability of meropenem and PIP/TZB, each in combination with heparin, in different PD solutions. The use of meropenem-heparin admixed in pH-neutral PD solutions for the treatment of PDAP should be avoided, given the observed suboptimal stability of meropenem.

Stability of Cefazolin in Polyisoprene Elastomeric Infusion Devices.

PURPOSE: The aim was to investigate the stability of cefazolin in elastomeric infusion devices. METHODS: Elastomeric devices (Infusor LV) that contain cefazolin (3 g/240 mL and 6 g/240 mL) were prepared and stored at 4°C for 72 hours and then at 35°C for 12 hours, followed by 25°C for 12 hours. An aliquot was withdrawn at predefined time points and analyzed for the concentration of cefazolin. Samples were also assessed for changes in pH, solution color, and particle content. FINDINGS: Cefazolin retained acceptable chemical and physical stability over the studied storage period and conditions. IMPLICATIONS: These findings will allow the administration of cefazolin by the Infusor LV elastomeric device in the outpatient and remote settings.