Antiproliferative and apoptotic effect of LY2090314, a GSK-3 inhibitor, in neuroblastoma in vitro.

BACKGROUND: Neuroblastoma (NB) is a devastating disease. Despite recent advances in the treatment of NB, about 60% of high-risk NB will have relapse and therefore long-term event free survival is very minimal. We have reported that targeting glycogen synthase kinase-3 (GSK-3) may be a potential strategy to treat NB. Consequently, investigating LY2090314, a clinically relevant GSK-3 inhibitor, on NB cellular proliferation and may be beneficial for NB treatment. METHODS: The effect of LY2090314 was compared with a previously studied GSK-3 inhibitor, Tideglusib. Colorimetric, clonogenic, and live-cell image confluency assays were used to study the proliferative effect of LY2090314 on NB cell lines (NGP, SK-N-AS, and SH-SY-5Y). Western blotting and caspase glo assay were performed to determine the mechanistic function of LY2090314 in NB cell lines. RESULTS: LY2090314 treatment exhibited significant growth reduction starting at a 20 nM concentration in NGP, SK-N-AS, and SH-SY-5Y cells. Western blot analysis indicated that growth suppression was due to apoptosis as evidenced by an increase in pro-apoptotic markers cleaved PARP and cleaved caspase-3 and a reduction in the anti-apoptotic protein, survivin. Further, treatment significantly reduced the level of cyclin D1, a key regulatory protein of the cell cycle and apoptosis. Functionally, this was confirmed by an increase in caspase activity. LY2090314 treatment reduced the expression levels of phosphorylated GSK-3 proteins and increased the stability of ß-catenin in these cells. CONCLUSIONS: LY2090314 effectively reduces growth of both human MYCN amplified and non-amplified NB cell lines in vitro. To our knowledge, this is the first study to look at the effect of LY2090314 in NB cell lines. These results indicate that GSK-3 may be a therapeutic target for NB and provide rationale for further preclinical analysis using LY2090314.

Perpetuating stigma or reducing risk? Perspectives from naloxone consumers and pharmacists on pharmacy-based naloxone in 2 states.

OBJECTIVES: Little is known about attitudes of pharmacists and consumers to pharmacy naloxone. We examined perceptions and experiences of pharmacy naloxone from people with opioid use disorder, patients taking chronic opioids for pain, caregivers of opioid users, and pharmacists from 2 early pharmacy naloxone adopter states: Massachusetts and Rhode Island. DESIGN: Eight focus groups (4 per state) were held in October to December 2015. SETTING AND PARTICIPANTS: Participants were recruited from pharmacies, health clinics, and community organizations; pharmacists were recruited from professional organizations and pharmacy colleges. OUTCOME MEASURES: Focus groups were led by trained qualitative researchers using a topic guide, and recorded and transcribed for analysis. Five analysts developed and applied a coding scheme to transcripts. Thematic analysis involved synthesis of coded data and connections between key themes, with comparisons across the groups. RESULTS: Sixty-one participants included patients with chronic pain (n = 15), people with opioid use disorders (n = 19), caregivers (n = 16), and pharmacists (n = 11). A majority of pharmacists had dispensed naloxone to patients; a minority of all consumer participants had obtained pharmacy naloxone. Four themes emerged: consumer fear of future consequences if requesting naloxone; pharmacists' concerns about practice logistics related to naloxone; differing perceptions of how opioid safety is addressed in the pharmacy; and solutions to addressing these barriers. Whereas consumer groups differed in awareness of naloxone and availability at pharmacies, all groups expressed support for the pharmacist's role and preferences for a universal offer of naloxone based on clear criteria. CONCLUSION: Pharmacies complement community naloxone provision to patients and caregivers. To overcome stigma of naloxone receipt, increased public awareness of naloxone and pharmacist training about naloxone and addiction are required. Pharmacists should offer naloxone via universal opt-out strategies-where all patients meeting evidence-based criteria are offered naloxone-rather than targeted or opt-in strategies-where only patients perceived as high risk or patients who request it are offered naloxone.