Spatiotemporal roles of AMPK in PARP-1- and autophagy-dependent retinal pigment epithelial cell death caused by UVA.

BACKGROUND: Although stimulating autophagy caused by UV has been widely demonstrated in skin cells to exert cell protection, it remains unknown the cellular events in UVA-treated retinal pigment epithelial (RPE) cells. METHODS: Human ARPE-19 cells were used to measure cell viability, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial mass and lysosomal mass by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics, LC3 level, and AMPK translocation after UVA irradiation. RESULTS: We confirmed mitochondrial ROS production and DNA damage are two major features caused by UVA. We found the cell death is prevented by autophagy inhibitor 3-methyladenine and gene silencing of ATG5, and UVA induces ROS-dependent LC3II expression, LC3 punctate and TFEB expression, suggesting the autophagic death in the UVA-stressed RPE cells. Although PARP-1 inhibitor olaparib increases DNA damage, ROS production, and cell death, it also blocks AMPK activation caused by UVA. Interestingly we found a dramatic nuclear export of AMPK upon UVA irradiation which is blocked by N-acetylcysteine and olaparib. In addition, UVA exposure gradually decreases lysosomal mass and inhibits cathepsin B activity at late phase due to lysosomal dysfunction. Nevertheless, cathepsin B inhibitor, CA-074Me, reverses the death extent, suggesting the contribution of cathepsin B in the death pathway. When examining the role of EGFR in cellular events caused by UVA, we found that UVA can rapidly transactivate EGFR, and treatment with EGFR TKIs (gefitinib and afatinib) enhances the cell death accompanied by the increased LC3II formation, ROS production, loss of MMP and mass of mitochondria and lysosomes. Although AMPK activation by ROS-PARP-1 mediates autophagic cell death, we surprisingly found that pretreatment of cells with AMPK activators (A769662 and metformin) reverses cell death. Concomitantly, both agents block UVA-induced mitochondrial ROS production, autophagic flux, and mitochondrial fission without changing the inhibition of cathepsin B. CONCLUSION: UVA exposure rapidly induces ROS-PARP-1-AMPK-autophagic flux and late lysosomal dysfunction. Pre-inducing AMPK activation can prevent cellular events caused by UVA and provide a new protective strategy in photo-oxidative stress and photo-retinopathy.

Membrane Protein Modification Modulates Big and Small Extracellular Vesicle Biodistribution and Tumorigenic Potential in Breast Cancers In Vivo.

Extracellular vesicles (EVs) are released by cells to mediate intercellular communication under pathological and physiological conditions. While small EVs (sEVs; <100-200 nm, exosomes) are intensely investigated, the properties and functions of medium and large EVs (big EVs (bEVs); >200 nm, microvesicles) are less well explored. Here, bEVs and sEVs are identified as distinct EV populations, and it is determined that bEVs are released in a greater bEV:sEV ratio in the aggressive human triple-negative breast cancer (TNBC) subtype. PalmGRET, bioluminescence-resonance-energy-transfer (BRET)-based EV reporter, reveals dose-dependent EV biodistribution at nonlethal and physiological EV dosages, as compared to lipophilic fluorescent dyes. Remarkably, the bEVs and sEVs exhibit unique biodistribution profiles, yet individually promote in vivo tumor growth in a syngeneic immunocompetent TNBC breast tumor murine model. The bEVs and sEVs share mass-spectrometry-identified tumor-progression-associated EV surface membrane proteins (tpEVSurfMEMs), which include solute carrier family 29 member 1, Cd9, and Cd44. tpEVSurfMEM depletion attenuates EV lung organotropism, alters biodistribution, and reduces protumorigenic potential. This study identifies distinct in vivo property and function of bEVs and sEVs in breast cancer, which suggest the significant role of bEVs in diseases, diagnostic and therapeutic applications.

Multiresolution Imaging Using Bioluminescence Resonance Energy Transfer Identifies Distinct Biodistribution Profiles of Extracellular Vesicles and Exomeres with Redirected Tropism.

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP-specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.

Delivery of nitric oxide with a nanocarrier promotes tumour vessel normalization and potentiates anti-cancer therapies.

Abnormal tumour vasculature has a significant impact on tumour progression and response to therapy. Nitric oxide (NO) regulates angiogenesis and maintains vascular homeostasis and, thus, can be delivered to normalize tumour vasculature. However, a NO-delivery system with a prolonged half-life and a sustained release mechanism is currently lacking. Here we report the development of NanoNO, a nanoscale carrier that enables sustained NO release to efficiently deliver NO into hepatocellular carcinoma. Low-dose NanoNO normalizes tumour vessels and improves the delivery and effectiveness of chemotherapeutics and tumour necrosis factor-related, apoptosis-inducing, ligand-based therapy in both primary tumours and metastases. Furthermore, low-dose NanoNO reprogrammes the immunosuppressive tumour microenvironment toward an immunostimulatory phenotype, thereby improving the efficacy of cancer vaccine immunotherapy. Our findings demonstrate the ability of nanoscale NO delivery to efficiently reprogramme tumour vasculature and immune microenvironments to overcome resistance to cancer therapy, resulting in a therapeutic benefit.

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics.

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer-encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell-to-cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel "information capsule" for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV-based multiomics for cancer diagnostics.

Tracking Extracellular Vesicles Delivery and RNA Translation Using Multiplexed Reporters.

Elucidating extracellular vesicle (EV; e.g., exosomes, microvesicles) delivery and translation of its RNA cargo with an accurate spatiotemporal resolution is critical in helping understand EV's role under normal and pathological conditions. We here describe a multiplexed fluorescent and bioluminescent reporter strategy to simultaneously monitor and quantify EV delivery, as well as EV-RNA translation in EV-recipient cells.