Screening, Identification And Application Of Autophagy Modulators In The Treatment Of Cancer And Diabetic Cardiomyopathy

AutopHagy is a major intracellular catabolic pathway involved in protein and organelles degradation. There is great interest in identifying compounds that modulate autopHagy because they may have applications in the treatment of major diseases including cancer, inflammation and neurodegenerative diseases. LC3 is shown to selectively incorporate into autopHagosomes, thus serving as a unique bona fide marker of autopHagosomes in mammals. We present a straightforward selection system for autopHagy modulators which combines the sensitivity of the GFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the nonautopHagosome associated GFP-LC3-I form, flow cytometry can be used to measure total fluorescence of saponin-extracted CHO-GFP-LC3 cells as a measure of the levels of autopHagosome associated GFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to quantify the changes in autopHagic flux. By utilizing this system, we screened a compound library composed of different natural products and identified several as either autopHagy activators or inhibitors. AutopHagy-induced cancer cell death has become a novel signaling target for development of cancer therapeutic drugs. We hypothesized that the most potent autopHagy activator represented by XY1 and ZH33 might have antitumor activity. XY1 treatment induces apoptosis in melanoma cells B16-F10 more potently than that in normal hepatocytes AML-12. Studies of its mechanism revealed that XY1 selectively causes ROS accumulation in B16-F10 cells, and this effect is associated with corresponding increases in a series of key components in ER stress-mediated apoptosis pathway. PHarmacological inhibition of ROS by NAC attenuates XY1 induced autopHagy and apoptosis, indicating an up-stream role of ROS in XY1 induced autopHagy. In vivo study also showed the efficacy of XY1 in a B16-F10 xenograft mouse model. ZH33 also exhibites good antitumor activity against human hepatoma cells Hepg2. ZH33 also triggeres ROS release in Hepg2 cells. Further studies showed that ZH33 mediates its antitumor effects through autopHagic cell death which is apoptosis independent. Together our studies provid an autopHagy modulator selection system and by utilizing this system we obtain several autopHagy modulators which may be useful for treatment of autopHagy related diseases. Meanwhile we present two potential anticancer agents and by demonstrating their molecular mechanisms we raise the possibility of targeting autopHagy pathway to eliminate cancer cells.Diabetic cardiomyopathy (DCM) is one of the leading causes of increased morbidity and mortality in the diabetic population. However, little information about the effect of autopHagy on type 2 diabetic cardiomyopathy has been available. We established mouse type 2 DCM model using streptozoticin (STZ) combined with high fat diet. Glycolysis pathways and glucose intake are inhibited in diabetic hearts, along with activated autopHagic flux indicated by augmented autopHagy signaling pathways and decreased autopHagy cargo proteins including P62, NBR1 and Nix. Over activated autopHagy in diabetic cardiomyocytes promotes the degradation of ERBB family signaling proteins via ACK1-P62 axis. Lacking of ERBB signaling pathways makes cardiomyocytes more susceptible to diabetic stress induced apoptosis. PHarmacological inhibition of autopHagy by 3-MA and chloroquine (CQ) attenuates DCM indicated by improved metabolic characteristics, preserved heart structure and improved myocardial contractility.In conclusion, our results highlight the intervention of autopHagy as therapeutic strategies in the treatment of cancer and DCM. We provided several novel autopHagy modulators which may represent candidate therapeutic agents for autopHagy related diseases.