RESUMO
Kidney cancer is one of the top ten cancer diagnosed worldwide and its incidence has increased the last 20 years. Clear Cell Renal Cell Carcinoma (ccRCC) are characterized by mutations that inactivate the von Hippel-Lindau (VHL) tumor suppressor gene and evidence indicated alterations in metabolic pathways, particularly in glutamine metabolism. We previously identified a small molecule, STF-62247, which target VHL-deficient renal tumors by affecting late-stages of autophagy and lysosomal signaling. In this study, we investigated ccRCC metabolism in VHL-deficient and proficient cells exposed to the small molecule. Metabolomics profiling using 1H NMR demonstrated that STF-62247 increases levels of glucose, pyruvate, glycerol 3-phosphate while glutamate, asparagine, and glutathione significantly decreased. Diminution of glutamate and glutamine was further investigated using mass spectrometry, western blot analyses, enzymatic activities, and viability assays. We found that expression of SLC1A5 increases in VHL-deficient cells treated with STF-62247, possibly to stimulate glutamine uptake intracellularly to counteract the diminution of this amino acid. However, exogenous addition of glutamine was not able to rescue cell viability induced by the small molecule. Instead, our results showed that VHL-deficient cells utilize glutamine to produce fatty acid in response to STF-62247. Surprisingly, this occurs through oxidative phosphorylation in STF-treated cells while control cells use reductive carboxylation to sustain lipogenesis. We also demonstrated that STF-62247 stimulated expression of stearoyl-CoA desaturase (SCD1) and peripilin2 (PLIN2) to generate accumulation of lipid droplets in VHL-deficient cells. Moreover, the carnitine palmitoyltransferase 1A (CPT1A), which control the entry of fatty acid into mitochondria for ß-oxidation, also increased in response to STF-62247. CPT1A overexpression in ccRCC is known to limit tumor growth. Together, our results demonstrated that STF-62247 modulates cellular metabolism of glutamine, an amino acid involved in the autophagy-lysosome process, to support lipogenesis, which could be implicated in the signaling driving to cell death.
RESUMO
BACKGROUND: Community pharmacists play an important role in the wellness of patients, families and friends affected by prescription and illicit opioid drugs. They are key partners of the Community Based Naloxone (CBN) Program in Alberta and similar programs across other Canadian jurisdictions. This publicly funded program is an evidence-based response to the opioid overdose crisis, facilitating access to and distribution of naloxone kits through pharmacies. The study aimed to describe Alberta community pharmacists' practices, training, comfort levels and views in dispensing naloxone kits through the CBN program and detail potential perceived barriers to program participation. METHODS: The study was conducted as a cross-sectional online survey of Alberta pharmacists. Data collected from the survey were descriptive and evaluated using Microsoft Excel. Fisher exact tests were used to study the associations in responses among several demographic characteristics and related to dispensing practices, pharmacists' beliefs and perceived barriers. RESULTS: A total of 255 responses were included in the final analysis, with 89.8% of pharmacists replying "yes" to CBN program participation. Pharmacists on average were "comfortable" dispensing naloxone to patients for varying indications, with 85% reporting always providing education when dispensing naloxone to an individual for the first time. About 41% of pharmacists reported no barriers to the program, with the most common perceived barriers being lack of time (29%), demand (20%) and funding (19%). CONCLUSION: Most community pharmacists who responded to the survey participate in the CBN program in Alberta. They held positive beliefs about their role in screening patients for the risk of opioid overdose and are confident in their abilities to recommend and educate on naloxone kits. Proactive screening appeared lower, however, and dispensing kits were potentially variable. Addressing factors such as time, funding for services and demand may help further pharmacist uptake and success of the program. Can Pharm J (Ott) 2021;154:xx-xx.
