In vitro anti-breast cancer studies of LED red light therapy through autophagy.

LED red light has been reported to have many health benefits. The present study was conducted to characterise anti-proliferation properties of four LED red light wavelengths (615, 630, 660 and 730 nm) against non-triple negative (MCF-7) and triple negative (MDA-MB-231) breast cancer-origin cell lines. It has been shown by MTT assay that at 24 h post-exposure time point, only LED red light with wavelength 660 nm possessed anti-proliferative effects against both cell lines with 40% reduction of cell viability. The morphology of LED 660 nm irradiated cells was found flatten with enlarged cell size, typical characteristic of cell senescent. Indications of autophagy activities following the irradiation have been provided by acridine orange staining, showing high presence of acidic vesicle organelles (AVOs). In addition, high LC3-II/LC3-I to LC3 ratio has been observed qualitatively in Western blot analysis indicating an increase number of autophagosomes formation in LED 660 nm irradiated cells compared to control cells. Electron dense bodies observed in these cells under TEM micrographs provided additional support to the above observations, leading to the conclusion that LED 660 nm irradiation promoted anti-proliferative activities through autophagy in breast cancer-origin cells. These findings have suggested that LED 660 nm might be developed and be employed as an alternative cancer treatment method in future.

AIE-Based Theranostic Agent: In Situ Tracking Mitophagy Prior to Late Apoptosis To Guide the Photodynamic Therapy.

Photodynamic therapy (PDT) takes advantage of reactive oxygen species (ROS) to trigger the apoptosis for cancer therapy. Given that cell apoptosis is a form of programmed cell death involved with multiple suborganelles and cancer cells are more sensitive to ROS than normal cells, early confirmation of the apoptosis induced by ROS would effectively avoid overtreatment. Herein, we highlight an aggregation-induced emission (AIE)-based theranostic agent (TPA3) to in situ dynamically track mitophagy prior to late apoptosis. TPA3 showed high specificity to autophagy vacuoles (AVs), of which appearance is the signature event of mitophagy during early apoptosis and delivered photocytotoxicity to cancer cells and skin cancer tumors in nude mice under irradiation of white light. Furthermore, in situ monitoring of the dynamical mitophagy process involved with mitochondria, AVs, and lysosomes was performed for the first time under confocal microscopy, providing a real-time self-monitoring system for assessing the curative effect prior to late apoptosis. This fluorescence imaging guided PDT witness great advances for applying in the clinical application.

UVA-induced photoaging inhibits autophagic degradation by impairing lysosomal function in dermal fibroblasts.

Autophagy has been associated with a variety of diseases especially aging. Human dermal fibroblasts (HDFs) can internalize and then degrade elastin, collagen and advanced glycation end products (AGEs) in lysosomes, which plays prominent roles in extracellular matrix homeostasis and AGEs removal in the dermis. Although autophagy has been reported to be decreased in photoaged fibroblasts, the underlying mechanism and its relevance to photoaging remain elusive. Here, we showed that GFP-LC3 puncta per cell, LC3Ⅰ/Ⅱ conversion and p62 expression were significantly increased, whereas beclin1 expression was not altered in UVA-induced photoaged fibroblasts compared with non-photoaged control. Moreover, autophagic flux was not significantly affected by chloroquine treatment, but was remarkably induced by rapamycin treatment in photoaged fibroblasts, suggesting that UVA-induced photoaging might inhibit autophagy at the degradation stage. Further lysosomal function studies demonstrated that degradation of formed autophagosomes, LC3Ⅱprotein and DQ-Green BSA was all dramatically decreased in photoaged fibroblasts. LysoSensor yellow/blue DND 160 staining and flow cytometry assays demonstrated that photoaging obviously attenuated lysosomal acidification. Also, decreased expression of cathepsin B, L and D was found in photoaged fibroblasts. These data suggest that lowered lysosomal acidity and decreased cathepsins expression might contribute to the inhibition of autophagic degradation, which might be crucial in the development of photoaging through impairing intracellular degradation.

Autophagy in Xenopus laevis rod photoreceptors is independently regulated by phototransduction and misfolded RHOP23H.

We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHOP23H), suggesting that autophagy may play a role in degrading the mutant RHO and/or be involved in photoreceptor cell death. To further examine autophagy in normal and diseased rods, we generated transgenic Xenopus laevis tadpoles expressing the dually fluorescent autophagy marker mRFP-eGFP-LC3 in rods, which changes from green to yellow and finally red as autophagic structures develop and mature. Using transgenic lines with constitutive and inducible expression, we determined the time-course of autophagy in rod photoreceptors: autophagosomes last for 6 to 8 hours before fusing with lysosomes, and acidified autolysosomes last for about 28 hours before being degraded. Autophagy was diurnally regulated in normal rods, with more autophagic structures generated during periods of light, and this regulation was non-circadian. We also found that more autophagosomes were produced in rods expressing the misfolding RHOP23H mutant. The RHO chromophore absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes RHO folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased RHO misfolding or phototransduction, we used CRISPR/Cas9 to knock out the RPE65 and GNAT1 genes, which are essential for chromophore biosynthesis and phototransduction respectively. Both knockouts suppressed light-induced autophagy, indicating that although light and misfolded rhodopsin can both induce autophagy in rods, light-induced autophagy is not due to misfolding of RHO, but rather due to phototransduction. Abbreviations: CYCS: cytochrome c; bRHOP23H: bovine RHOP23H; Cas9: CRISPR associated protein 9; dpf: days post-fertilization; eGFP: enhanced green fluorescent protein; GNAT1: guanine nucleotide-binding protein G(t) subunit alpha-1 aka rod alpha-transducin; HSPA1A/hsp70: heat shock protein of 70 kilodaltons; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1A/1B light chain 3; mRFP: monomeric red fluorescent protein; RHO: rhodopsin; RP: retinitis pigmentosa; RPE65: retinal pigment epithelium-specific 65 kDa protein: sfGFP: superfolding GFP; sgRNA: single guide RNA; WGA: wheat germ agglutinin; RHOp: the Xenopus laevis RHO.2.L promoter.

Regulatory roles of miR-22/Redd1-mediated mitochondrial ROS and cellular autophagy in ionizing radiation-induced BMSC injury.

Ionizing radiation (IR) response has been extensively investigated in BMSCs with an increasing consensus that this type of cells showed relative radiosensitivity in vitro analysis. However, the underlying mechanism of IR-induced injury of BMSCs has not been elucidated. In current study, the regulatory role of miR-22/Redd1 pathway-mediated mitochondrial reactive oxygen species (ROS) and cellular autophagy in IR-induced apoptosis of BMSCs was determined. IR facilitated the generation and accumulation of mitochondrial ROS, which promoted IR-induced apoptosis in BMSCs; meanwhile, cellular autophagy activated by IR hold a prohibitive role on the apoptosis program. The expression of miR-22 significantly increased in BMSCs after IR exposure within 24 h. Overexpression of miR-22 evidently accelerated IR-induced accumulation of mitochondrial ROS, whereas attenuated IR stimulated cellular autophagy, thus advancing cellular apoptosis. Furthermore, we verified Redd1 as a novel target for miR-22 in rat genome. Redd1 overexpression attenuated the regulatory role of miR-22 on mitochondrial ROS generation and alleviated the inhibitive role of miR-22 on cell autophagy activated by IR, thus protecting BMSCs from miR-22-mediated cell injury induced by IR exposure. These results confirmed the role of miR-22/Redd1 pathway in the regulation of IR-induced mitochondrial ROS and cellular autophagy, and subsequent cellular apoptosis.

α-Synuclein impairs ferritinophagy in the retinal pigment epithelium: Implications for retinal iron dyshomeostasis in Parkinson's disease.

Retinal degeneration is prominent in Parkinson's disease (PD), a neuromotor disorder associated with aggregation of α-synuclein (α-syn) in the substantia-nigra (SN). Although α-syn is expressed in the neuroretina, absence of prominent aggregates suggests altered function as the likely cause of retinal pathology. We demonstrate that α-syn impairs ferritinophagy, resulting in the accumulation of iron-rich ferritin in the outer retina in-vivo and retinal-pigment-epithelial (RPE) cells in-vitro. Over-expression of Rab1a restores ferritinophagy, suggesting that α-syn impairs lysosomal function by disrupting the trafficking of lysosomal hydrolases. Surprisingly, upregulation of ferritin in RPE cells by exogenous iron in-vitro stimulated the release of ferritin and α-syn in exosomes, suggesting that iron overload due to impaired ferritinophagy or other cause(s) is likely to initiate prion-like spread of α-syn and ferritin, creating retinal iron dyshomeostasis and associated cytotoxicity. Since over-expression of α-syn is a known cause of PD, these results explain the likely cause of PD-associated retinal degeneration.

Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress.

Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC's stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H2DCFDA live staining, respectively. Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of ATG7 abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation.

Autophagic flux in glioblastoma cells.

To establish metabolic context for radiation sensitivity by measuring autophagic flux in two different glioblastoma (GBM) cell lines. Clonogenic survival curve analysis of U87 or U251 cells exposed to γ radiation, fast neutrons, a mixed energy neutron beam (METNB) or Auger electrons from a gadolinium neutron capture (GdNC) reaction suggested other factors, beyond a defective DNA damage response, contribute to cell death of U251 cells. Altered tumor metabolism (autophagy) was hypothesized as a factor in U251 cells' clonogenic response. Each of the four different radiation modalities induced an increase in the number of autophagosomes in both U87 and U251 cells. Changes in the number of autophagosomes can be explained by either induction of autophagy or alterations in autophagic flux so autophagic flux was assayed by p62 immunoblotting or in engineered GBM cells that stably express an autophagy marker protein, LC3B-eGFP-mCherry. Perturbations in later stages of autophagy in U251 cells corresponded with radiation sensitivity of U251 cells irradiated with 10 Gy γ rays. Establishment of altered autophagic flux is a useful biomarker for metabolic stress and provided metabolic context for radiation sensitization to 10 Gy γ rays. These results provide strong evidence for the usefulness of managing tumor cell metabolism as a tool for the enhancement of radiation therapy.