A Pivotal Role Of Autophagy In Anti-senescence Effect Of Caffeine: Via A2AR/SIRT3/AMPK Pathway

Cellular senescence and autophagy are two kinds of cellular response to stress.The relationship between them during stress is ambiguous. Accumulating evidenceindicates that autophagy can regulate cellular senescence and influence cellularlifespan. Caffeine has been reported to induce autophagy in yeast and mammal cells.Besides, it has also been shown to extend the life-span of yeast and attenuatesenescence. However, whether autophagy has involved in the mechanism of caffeinein attenuating cellular senescence remains unknown. In this study, the relationshipbetween the anti-senescence effect and the autophagy-inducing effect of caffeine andthe mechanism involved in the autophagy-inducing effect of caffeine will be furtherstudied.We used a free radical initiator, AAPH, to induce oxidative stress and establish amodel of oxidative stress-induced cellular senescence in human A375melanoma cells.Inhibition ratio of proliferation was determined by MTT. And ROS generation andmitochondrial membrane potential were detected by flow cytometry. AAPH wasfound to inhibit the proliferation of A375cells in a concentration and time dependentmanner. It also increased ROS generation and reduced mitochondrial membranepotential in concentration and time dependent manner. Senescence-associated (SA)β-galactosidase staining, western blotting, immunofluorescence and confocal laserscanning microscopy were used to determine the biomarkers of cellular senescence.Results showed that AAPH (1mM) treatments for48h significantly increased thepercentage of SA β-Gal positive ratio, the phosphorylation of p53, the proteinexpression of p21and the percentage of SAHFs positive cells. These results indicatedthat oxidative stress resulted by AAPH led A375cells to senescence. Based on thiscellular senescence model, we investigated the effects of caffeine on AAPH-inducedcellular senescence. Results showed that administration of caffeine significantly decreased the percentage of SA β-Gal positive staining cells in AAPH-treated A375cells. The p53phosphorylation, p21protein expression, and SAHFs positive ratiowere also down-regulated. These results all suggested that caffeine attenuatedAAPH-induced cellular senescence.We investigated whether autophagy was involved in the mechanism of caffeinein attenuating cellular senescence. Using confocal microscopy, western blotting andtransmission electron microscopy, we found that caffeine increased the number ofautophagosomes, the protein expression of Beclin1, conversion of LC3I to II and thenumber of autolysosomes. The results of confocal microscopy showed that LC3βpuncta and Mito Red overlapped to a certain extent, and the results of transmissionelectron microscopy showed that contents in autolysosomes appeared to be thedegrading mitochondria, suggesting that mitophagy was induced by caffeine in thisprogress. In the meantime, promoting autophagy by rapamycin augmented the effectsof caffeine on attenuating cellular senescence while inhibiting autophagy by3-MAweakened the anti-senescence effects of caffeine. The results above indicated thatautophagy might have contributed to the effects of caffeine on cellular senescence.Finally, we investigated the further mechanism involved in the effect of caffeineinduced autophagy. The results of western blot showed that caffeine increased theexpression of SIRT3and activation of AMPK to p-AMPK. SCH58261, a selectiveA2A adenosine receptor (A2AR) antagonist, showed stronger effects on up-regulatingexpression of SIRT3and activation of AMPK to p-AMPK when compared to caffeine.It also increased Beclin1expression and LC3II/I ratio, and decreased p53phosphorylation, p21protein expression. Meanwhile, a selective A2AR agonist CGS21680, antagonized the effects of caffeine. Furthermore, the results of confocalmicroscopy showed that SAHFs positive ratio in AAPH-treated cells weredown-regulated by SCH58261and caffeine administrations, whereas these effects ofcaffeine were inhibited by CGS21680. These data suggested that caffeine might actas an A2AR antagonist to up-regulate SIRT3expression, activate AMPK and induceautophagy. Silencing SIRT3by RNA interference blocked caffeine-inducedup-regulation of SIRT3expression. Besides, caffeine induced AMPK activation was weakened by SIRT3siRNA transfection. These might suggest that SIRT3was theupstream regulator of AMPK. Moreover, increased Beclin1expression and LC3II/Iconversion induced by caffeine were decreased significantly by siRNA transfection.The down-regulation of p53phosphorylation and p21protein expression induced bycaffeine were also abolished by SIRT3siRNA transfection. The above resultsindicated that caffeine increased SIRT3expression and subsequently activated AMPK,ultimately induced autophagy and attenuated cellular senescence in AAPH-treatedA375cells.In summary, AAPH, a free radical generator, caused oxidative stress in A375cells. The free radicals damaged mitochondria and eventually resulted in cellularsenescence. Nevertheless, the attenuation of AAPH-induced cellular senescence bycaffeine was probably related to autophagy, especially mitophagy. Furtherexperiments demonstrated that A2AR/SIRT3/AMPK pathway might be the molecularmechanisms involved in caffeine-induced autophagy.