Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.     

IBP-mediated suppression of autophagy promotes growth and metastasis of breast cancer cells via activating mTORC2/Akt/FOXO3a signaling pathway

Interferon regulatory factor-4 binding protein (IBP) is a novel upstream activator of Rho GTPases. Our previous studies have shown that ectopic expression of IBP was correlated with malignant behaviors of human breast cancer cells, and invasive human breast cancer had high expression of IBP that promoted the proliferation of these cells. However, it remains unknown whether autophagy inhibition contributes to IBP-mediated tumorigenesis. In this study, we for the first time, reported that upregulation of IBP expression significantly suppressed the autophagy of breast cancer cells, and downregulation of IBP expression markedly induced autophagy of these cells. Further investigation revealed that IBP effectively counteracted autophagy by directly activating mammalian target of rapamycin complex 2 (mTORC2) and upregulating phosphorylation of Akt on ser473 and FOXO3a on Thr32. Moreover, IBP-mediated suppression of autophagy was dependent on mTORC2/Akt/FOXO3a signaling pathway. Finally, our results demonstrated that IBP-mediated breast cancer cell growth in vitro and in vivo was strongly correlated with suppression of mTORC2-dependent autophagy. These findings suggest that the anti-autophagic property of IBP has an important role in IBP-mediated tumorigenesis, and IBP may serve as an attractive target for treatment of breast cancer.     

Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.     

Patulin induces pro-survival functions via autophagy inhibition and p62 accumulation

Patulin (PAT) is one of the most common mycotoxins found in moldy fruits. Skin contact is one of the most likely exposure routes of PAT. Investigation of dermal toxicity of PAT is clearly needed and has been highlighted by WHO. In the present study, using human keratinocyte HaCaT cells as a model, we found that treatment with PAT caused an increased autophagosome accumulation. Measurements of autophagic flux demonstrated that the accumulation of autophagosomes by PAT was not directly due to enhanced autophagosome formation but due to inhibition of autophagosome degradation. Reductions in the activities of the lysosomal enzymes cathepsin B and cathepsin D by PAT might contribute to this inhibitory effect. Consistent with this, inhibition of autophagosome degradation by PAT resulted in accumulation of p62 that functioned as a pro-survival signal. The pro-survival function of p62 was found to be attributed to reactive oxygen species-mediated cytoprotective endoplasmic reticulum (ER) stress response. ER stress exerted cytoprotective effect via extracellular signal-regulated kinase1/2-dependent B-cell CLL/lymphoma 2-associated agonist of cell death inhibitory phosphorylation. Given the critical role of autophagy and its substrate p62 in carcinogenesis, our findings may have important implications in PAT-induced skin carcinogenesis.     

Survival or death: disequilibrating the oncogenic and tumor suppressive autophagy in cancer

Autophagy (macroautophagy) is an evolutionarily conserved lysosomal degradation process, in which a cell degrades long-lived proteins and damaged organelles. Recently, accumulating evidence has revealed the core molecular machinery of autophagy in carcinogenesis; however, the intricate relationship between autophagy and cancer continue to remain an enigma. Why does autophagy have either pro-survival (oncogenic) or pro-death (tumor suppressive) role at different cancer stages, including cancer stem cell, initiation and progression, invasion and metastasis, as well as dormancy? How does autophagy modulate a series of oncogenic and/or tumor suppressive pathways, implicated in microRNA (miRNA) involvement? Whether would targeting the oncogenic and tumor suppressive autophagic network be a novel strategy for drug discovery? To address these problems, we focus on summarizing the dynamic oncogenic and tumor suppressive roles of autophagy and their relevant small-molecule drugs, which would provide a new clue to elucidate the oncosuppressive (survival or death) autophagic network as a potential therapeutic target.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Autophagy triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.     

The BECN1-USP19 axis plays a role in the crosstalk between autophagy and antiviral immune responses

Macroautophagy/autophagy is a conserved intracellular degradation system that traffics substrates including protein aggregates, defunct or disused organelles and invading pathogens to lysosomes via double-membrane vesicles called autophagosomes. BECN1/Beclin 1 functions as a key protein in autophagy initiation and progression; however, the role of BECN1 in innate immunity has not been fully investigated. Recently, we have found that USP19 affects the ubiquitination of BECN1, hence promoting the formation of autophagosomes and inhibiting DDX58/RIG-I-mediated type I interferon signaling.     

Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.     

Absence of autophagy promotes apoptosis by modulating the ROS-dependent RLR signaling pathway in classical swine fever virus-infected cells

A growing number of studies have demonstrated that both macroautophagy/autophagy and apoptosis are important inner mechanisms of cell to maintain homeostasis and participate in the host response to pathogens. We have previously reported that a functional autophagy pathway is trigged by infection of classical swine fever virus (CSFV) and is required for viral replication and release in host cells. However, the interplay of autophagy and apoptosis in CSFV-infected cells has not been clarified. In the present study, we demonstrated that autophagy induction with rapamycin facilitates cellular proliferation after CSFV infection, and that autophagy inhibition by knockdown of essential autophagic proteins BECN1/Beclin 1 or MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) promotes apoptosis via fully activating both intrinsic and extrinsic mechanisms in CSFV-infected cells. We also found that RIG-I-like receptor (RLR) signaling was amplified in autophagy-deficient cells during CSFV infection, which was closely linked to the activation of the intrinsic apoptosis pathway. Moreover, we discovered that virus infection of autophagy-impaired cells results in an increase in copy number of mitochondrial DNA and in the production of reactive oxygen species (ROS), which plays a significant role in enhanced RLR signaling and the activated extrinsic apoptosis pathway in cultured cells. Collectively, these data indicate that CSFV-induced autophagy delays apoptosis by downregulating ROS-dependent RLR signaling and thus contributes to virus persistent infection in host cells.