MethodsRat neonate cardiomyocytes were cultured and treated with AGEs at different concentration. Two classic autophagy markers, microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1, were detected by western blot assay. The inhibition of RAGE and phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mTOR pathway were applied to cells, respectively.ResultsAGEs administration enhanced the expression of Beclin-1 and LC3 II in cardiomyocytes, increased the number of autophagic vacuoles and impaired the cell viability in dose-dependant manners. Also, AGEs inhibited the PI3K/Akt/mTOR pathway via RAGE. Inhibition of RAGE with RAGE antibody reduced expression of Beclin-1 and LC3 II/I and inhibited the cellular autophagy, accompanied by the reactivation of PI3K/Akt/mTOR pathway in cultured cells. Notably, the presence of inhibition of PI3K/Akt/mTOR pathway abolished the protective effect of RAGE inhibition on cardiomyocytes.ConclusionThis study provides evidence that AGEs induces cardiomyocyte autophagy by, at least in part, inhibiting the PI3K/Akt/mTOR pathway via RAGE.Previous studies showed that the accumulation of advanced glycation end products (AGEs) induce cardiomyocyte apoptoisis, leading to heart dysfunction. However, the effect of AGEs on another cell death pathway, autophagy, in cardiomyocytes remains unknown.

Curcumin is a hydrophobic polyphenol derived from the herb Curcumalonga and its wide spectrum of pharmacological activities has been widely studied. It has been reported that Curcumin can induce autophagy through inhibition of the Akt-mTOR pathway. However, the effect of Curcumin on lysosome remains largely elusive. In this study, we first found that Curcumin treatment enhances autophagic flux in both human colon cancer HCT116 cells and mouse embryonic fibroblasts (MEFs). Moreover, Curcumin treatment promotes lysosomal function, evidenced by the increased lysosomal acidification and enzyme activity. Second, Curcumin is capable of suppressing the mammalian target of rapamycin (mTOR). Interestingly, Curcumin fails to inhibit mTOR and to activate lysosomal function in Tsc2−/−MEFs with constitutive activation of mTOR, indicating that Curcumin-mediated lysosomal activation is achieved via suppression of mTOR. Third, Curcumin treatment activates transcription factor EB (TFEB), a key nuclear transcription factor in control of autophagy and lysosome biogenesis and function, based on the following observations: (i) Curcumin directly binds to TFEB, (ii) Curcumin promotes TFEB nuclear translocation; and (iii) Curcumin increases transcriptional activity of TFEB. Finally, inhibition of autophagy and lysosome leads to more cell death in Curcumin-treated HCT116 cells, suggesting that autophagy and lysosomal activation serves as a cell survival mechanism to protect against Curcumin-mediated cell death. Taken together, data from our study provide a novel insight into the regulatory mechanisms of Curcumin on autophagy and lysosome, which may facilitate the development of Curcumin as a potential cancer therapeutic agent.

Physapubescin B, a steroidal compound extracted from the plant Physalis pubescens L. (Solanaceae), has been reported to possess anti-cancer potential, whereas the molecular mechanism remains elusive. In this study, we first demonstrated that physapubescin B induced autophagy in human cancer cells based on the evidence that physapubescin B increased lipidation of microtubule-associated protein 1 light chain 3 (LC3) as well as number of GFP-LC3 puncta. We further examined the molecular mechanisms and found that physapubescin B enhanced the autophagic flux through promotion of reactive oxygen species (ROS)-mediated suppression of mammalian target of rapamycin complex I (mTORC1), the key negative regulator of autophagy. Additionally, excessive ROS caused by physapubescin B also induced p53-dependent apoptotic cell death. Furthermore, we provided evidence that inhibition of autophagy either by a chemical inhibitor or gene silencing promoted physapubescin B-induced apoptotic cell death, indicating that autophagy serves as a cell survival mechanism to protect cell death. Thus, our data provide a clue that inhibition of autophagy would serve as a novel strategy for enhancing the anti-cancer potential of physapubescin B.

No abstract

Phthalate ester plasticizers are used to improve the plasticity and strength of plastics. One of the most widely used and studied, di-2-ethylhexyl phthalate (DEHP), has been labeled as an endocrine disruptor. The major and toxic metabolic derivative of DEHP, mono-2-ethylhexyl phthalate (MEHP), is capable of interfering with mitochondrial function, but its mechanism of action on mitophagy remains elusive. Here, we report that MEHP exacerbates cytotoxicity by amplifying the PINK1-Parkin-mediated mitophagy pathway. First, MEHP exacerbated mitochondrial damage induced by low-dose CCCP via increased reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (MMP), and enhanced fragmentation in mitochondria. Second, co-exposure to MEHP and CCCP (“MEHP-CCCP”) induced robust mitophagy. Mechanistically, MEHP-CCCP stabilized PINK1, increased the level of phosphorylated ubiquitin (pSer 65-Ub), and led to Parkin mitochondrial translocation and activation. Third, MEHP-CCCP synergistically caused more cell death, while inhibition of mitophagy, either through chemical or gene silencing, reduced cell death. Finally and importantly, co-treatment with N-acetyl cysteine (NAC) completely counteracted the effects of MEHP-CCCP, suggesting that mitochondrial ROS played a vital role in this process. Our results link mitophagy and MEHP cytotoxicity, providing an insight into the potential roles of endocrine disrupting chemicals (EDCs) in human diseases such as Parkinson's disease.