Promotion of cytoplasmic vacuolation-mediated cell death of human prostate cancer PC-3 cells by oxidative stress induced by daucusol, a new guaiane-type sesquiterpenoid from Daucus carota L.

We investigated the antitumor activity of daucusol (DS) derived from Daucus carota L. in PC-3, A549 and HeLa cell lines by the MTT assay. Optical microscopy revealed that exposure of PC-3 cells to DS resulted in cytoplasmic vacuolation. Flow cytometry analysis of the phase of the cell cycle did not reveal a sub-G1 peak, and no caspase-dependent activation was observed after DS treatment. The levels of endoplasmic reticulum (ER) stress biomarkers, LC3B-II and ubiquitinated proteins were increased. It was also observed that oxidative stress played an important role in the activation of the cytoplasmic vacuolation-mediated cell-death pathway. In vivo, DS inhibited tumor growth in nude mice by 39.13% compared to the vehicle. Protein expression in the tumor tissue was consistent with their expression in vitro. Our findings indicate that DS induced cytoplasmic vacuolation-mediated death in PC-3 cells by triggering oxidative stress and suggest that targeting this pathway could serve as a novel therapeutic approach for prostate cancer

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression

NFκB-Mediated Expression of Phosphoinositide 3-Kinase δ Is Critical for Mesenchymal Transition in Retinal Pigment Epithelial Cells

Epithelial mesenchymal transition (EMT) plays a vital role in a variety of human diseases including proliferative vitreoretinopathy (PVR), in which retinal pigment epithelial (RPE) cells play a key part. Transcriptomic analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was up-regulated in human RPE cells upon treatment with transforming growth factor (TGF)-β2, a multifunctional cytokine associated with clinical PVR. Stimulation of human RPE cells with TGF-β2 induced expression of p110δ (the catalytic subunit of PI3Kδ) and activation of NFκB/p65. CRISPR-Cas9-mediated depletion of p110δ or NFκB/p65 suppressed TGF-β2-induced fibronectin expression and activation of Akt as well as migration of these cells. Intriguingly, abrogating expression of NFκB/p65 also blocked TGF-β2-induced expression of p110δ, and luciferase reporter assay indicated that TGF-β2 induced NFκB/p65 binding to the promoter of the PIK3CD that encodes p110δ. These data reveal that NFκB/p65-mediated expression of PI3Kδ is essential in human RPE cells for TGF-β2-induced EMT, uncovering hindrance of TGF-β2-induced expression of p110δ as a novel approach to inhibit PVR

Idelalisib inhibits experimental proliferative vitroretinopathy

Proliferative vitreoretinopathy (PVR) is a fibrotic eye disease that develops after rhegmatogenous retinal detachment surgery and open-globe traumatic injury. Idelalisib is a specific inhibitor of phosphoinositide 3-kinase (PI3K) δ. While PI3Kδ is primarily expressed in leukocytes, its expression is also considerably high in retinal pigment epithelial (RPE) cells, which play a crucial part in the PVR pathogenesis. Herein we show that GeoMx Digital Spatial Profiling uncovered strong expression of fibronectin in RPE cells within epiretinal membranes from patients with PVR, and that idelalisib (10 μM) inhibited Akt activation, fibronectin expression and collagen gel contraction induced by transforming growth factor (TGF)-β2 in human RPE cells. Furthermore, we discovered that idelalisib at a vitreal concentration of 10 μM, a non-toxic dose to the retina, prevented experimental PVR induced by intravitreally injected RPE cells in rabbits assessed by experienced ophthalmologists using an indirect ophthalmoscope plus a + 30 D fundus lens, electroretinography, optical coherence tomography and histological analysis. These data suggested idelalisib could be harnessed for preventing patients from PVR