Background: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and 2 lysosomal proteins, the NPC1 (Niemann–Pick C1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. Methods: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells. To establish whether Wnt5a also protects against cholesterol accumulation in human vascular smooth muscle cells, we used a CRISPR/Cas9 guided nuclease approach to generate human vascular smooth muscle cells knockout for Wnt5a. Results: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 (mechanistic target of rapamycin complex 1) kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum. Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the endoplasmic reticulum, and promoted atherosclerosis. Conclusions: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

In recent years, interest in Cannabis sativa L. has been rising, as legislation is moving in the right direction. This plant has been known and used for thousands of years for its many active ingredients that lead to various therapeutic effects (pain management, anti-inflammatory, antioxidant, etc.). In this report, our objective was to optimize a method for the extraction of cannabinoids from a clone of Cannabis sativa L. #138 resulting from an agronomic test (LaFleur, Angers, FR). Thus, we wished to identify compounds with anticancer activity on human pancreatic tumor cell lines. Three static maceration procedures, with different extraction parameters, were compared based on their median inhibitory concentration (IC₅₀) values and cannabinoid extraction yield. As CBD emerged as the molecule responsible for inducing apoptosis in the human pancreatic cancer cell line, a CBD-rich cannabis strain remains attractive for therapeutic applications. Additionally, while gemcitabine, a gold standard drug in the treatment of pancreatic cancer, only triggers cell cycle arrest in G0/G1, CBD also activates the cell signaling cascade to lead to programmed cell death. Our results emphasize the potential of natural products issued from medicinal hemp for pancreatic cancer therapy, as they lead to an accumulation of intracellular superoxide ions, affect the mitochondrial membrane potential, induce G1 cell cycle arrest, and ultimately drive the pancreatic cancer cell to lethal apoptosis.

The extraction of bioactive compounds from medicinal plants requires methods which are as diverse as the chemical nature of the compounds themselves. In this study, a 96-well microplate was used where solvent mixtures spanning wide ranges of selectivity and polarity were tested with the objective of extracting a broad range bioactive compounds from plant material. Microplate wells were filled with plant material and the solvents and their mixtures were added. The obtained extracts were assessed in terms of their total antioxidant activity, oxygen radical absorbance capacity and effects on cell viability. An aqueous extract, generally used by traditional therapists, was also included in the study. The results showed that the extracts using methanol with acetic acid (0.1%, v:v), chloroform/ethanol, butanol/DMF, butanol/acetonitrile, ethylene glycol with acetic acid (0.1%, v:v), MTBE/DMSO, ethylene glycol, pentane/ethanol (v:v), ethanol, DMF, DMF with acetic acid (0.1%, v:v), DMSO, DMSO with acetic acid (0.1%, v:v) and THF had a higher antioxidant activity than the aqueous extract. Extracts with greater antioxidant activity than the aqueous extract were obtained largely from solvent mixtures with the exception of ethanol, DMF, DMSO and THF. The antioxidant activity obtained in TEAC varied between 1474.1±4.4 and 3183.0±16.0 μmol TE/g dry extract respectively for aqueous and THF extracts; in ORAC between 1727.7±8.4 and 2683.5±11.7 μmol TE/g dry extract for aqueous and DMSO acetic acid 1%, respectively, with mean ±SEM. In TEAC the THF extract had the highest antioxidant potential with 3183.0±16.0 μmol TE / g dry extract. The DMSO acetic acid (0.1%, v:v) extract had the highest antioxidant potential in ORAC with 2683.5±11.7 μmol TE / g dry extract. Cell viability test using β-pancreatic cells showed that only the acidified methanol extract was toxic after one hour of incubation. After 24 hours, cell viability was less than 70% for extracts using butanol/acetonitrile, MTBE/DMF, acidified methanol, pentane/ethanol and acidified DMF.