Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4BC74A; ATG4DC144A) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4BC74A-expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts.
ATG4B; ATG4D; erythropoiesis; electron microscopy; autophagosome; amphisome; mitochondrion
Autophagy is a complex, multi-step and biologically important pathway mediated by autophagosomes and autolysosomes. Accurately dissecting and detecting different stages of autophagy is important to elucidate its molecular mechanism and thereby facilitate the discovery of pharmaceutical molecules. We herein reported a small-molecule synthetic probe, Zn-G4, which is only fluorescent upon starvation- or chemical agent-induced autophagy within the autolysosome or possible the late endosome/lysosome networks. The probe can be detected by one-photon microscopy, which gives a high signal-to-noise ratio readout of autophagic activity. The pH gradient-independent fluorescence can be detected both in live and prestained fixed cells. Moreover, the fluorescent recording can be used to quantify autophagic activity at a single point without transfection or false positive signals due to protein aggregation. Furthermore, autophagy-induced fluorescence in autolysosomes can also be detected by two-photon microscopy, suggesting potential applications in deep tissue and in vivo. In conclusion, we have developed a sensitive and specific autolysosomal probe that can be used for monitoring autophagy during later stages along with quantitative assays together with widely used early markers or microtubule-associated protein 1 light chain 3 (LC3)-based probes.
autophagy; autolysosome; ZnSalen; optical imaging; probe
Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.
ubiquitin proteasome system; noncanonical autophagy; BAG3; BECN1; crosstalk
The phagophore (also called isolation membrane) elongates and encloses a portion of cytoplasm, resulting in formation of the autophagosome. After completion of autophagosome formation, the outer autophagosomal membrane becomes ready to fuse with the lysosome for degradation of enclosed cytoplasmic materials. However, the molecular mechanism for how the fusion of completed autophagosomes with the lysosome is regulated has not been fully understood. We discovered syntaxin 17 (STX17) as an autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). STX17 has a hairpin-type structure mediated by two transmembrane domains, each containing glycine zipper motifs. This unique transmembrane structure contributes to its specific localization to completed autophagosomes but not to phagophores. STX17 interacts with SNAP29 and the lysosomal SNARE VAMP8, and all of these proteins are required for autophagosome–lysosome fusion. The late recruitment of STX17 to completed autophagosomes could prevent premature fusion of the lysosome with unclosed phagophores.
autophagosome; syntaxin 17; SNARE; glycine zipper motif; hairpin-type structure
The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3pdKO/mVps34pdKO) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes.
VPS34; PIK3C3; autophagy; aberrant autophagosome; podocyte vacuolization; proteinuria; glomerulosclerosis
Hepatocellular carcinoma (HCC) is among the most lethal and prevalent cancers in the human population. The initiation and progression of HCC is closely associated with chronic liver inflammation. Recent research indicates that nonhomologous end joining (NHEJ), one of the DNA repair mechanisms, autophagy and senescence are all involved in the pathogenesis of HCC induced by carcinogens or oxidative stress. DNA repair proteins including XRCC6/KU70 and XRCC5/KU80 are the critical NHEJ factors that play pivotal roles in genome-maintenance issues such as DNA replication and repair, telomere maintenance and chromosomal instability. Our studies indicate that a deficiency of toll-like receptor 4 (TLR4)-mediated immune activities results in a decreased expression of XRCC5 and XRCC6 in response to insult by the carcinogen diethylnitrosamine (DEN). This effect causes a failure in DNA repair, and promotes the transformation of precancerous hepatocytes and HCC development. Ectopic expression of XRCC6 protects against HCC initiation and progression by restoring the cellular senescent response and activation of immune networks, which induces an effective autophagic degradation, removes the accumulated reactive oxygen species (ROS), decreases DNA damage, attenuates proliferation, and promotes programmed cell death in TLR4-deficient livers. Our work indicates that repairing DNA damage by XRCC6 reverses TLR4-deficiency-worsened HCC development via restoring immunity to support senescence and autophagy in liver cells.
DNA damage repair; hepatocellular carcinoma; KU70; selective autophagy; senescence
When the levels of intracellular amino acids are high, RRAG GTPases recruit MTORC1 to lysosomes and promote its activation. We found that RRAGs also recruit specific MTORC1 substrates to the lysosomal surface, thus facilitating MTORC1-mediated phosphorylation and regulation. In particular, active RRAGs interact with the transcription factor EB (TFEB), the master regulator of a gene network that promotes lysosomal biogenesis and autophagy. Redistribution to lysosomes is critical for MTORC1-dependent inactivation of TFEB under nutrient-rich conditions. Therefore, RRAGs play a critical role coordinating nutrient availability and cellular clearance.
autophagy; lysosomes; MITF; RRAG; MTORC1; TFEB
During development in the thymus, each T lymphocyte is equipped with one, essentially unique, T cell receptor (TCR)-specificity. Due to its random nature, this process inevitably also leads to the emergence of potentially dangerous T lymphocytes that may recognize ‘self.’ Nevertheless, autoimmune tissue destruction, the cause of diseases such as multiple sclerosis and diabetes, is the exception rather than the rule. This state of immunological self-tolerance is to a large degree based upon a process called ‘negative selection’: prior to joining the circulating lymphocyte pool, immature T cells test their receptor on self-antigens within the thymic microenvironment, and TCR engagement at this immature stage elicits an apoptotic suicide program. We now find evidence that macroautophagy supports the tolerogenic presentation of self-antigens in the thymus.
thymus; central tolerance; MHC class II; antigen presentation; promiscuous gene expression; thymic epithelium
We have elucidated a novel mechanism through which the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex can be recruited to the PAS in mammalian cells, through the interaction between BECN1 and the vacuole membrane protein 1 (VMP1), an integral autophagosomal membrane protein. This interaction involves the binding between the C-terminal 20 amino acids of the VMP1 hydrophilic domain, which we have named the VMP1 autophagy-related domain (VMP1-AtgD), and the BH3 domain of BECN1. The association between these two proteins allows the formation of the autophagy-specific PtdIns3K complex, which activity favors the generation of phosphatidylinositol-3-phosphate (PtdIns3P) and the subsequent association of the autophagy-related (ATG) proteins, including ATG16L1, with the phagophore membranes. Therefore, VMP1 regulates the PtdIns3K activity on the phagophore membrane through its interaction with BECN1. Our data provide a novel model describing one of the key steps in phagophore assembly site (PAS) formation and autophagy regulation, and positions VMP1 as a new interactor of the autophagy-specific PtdIns3K complex in mammalian cells.
VMP1; BECN1; phosphatidylinositol 3-kinase; autophagy; pancreatitis
As a central controller of cell growth, mechanistic target of rapamycin (MTOR) affects an array of biological processes, in particular protein synthesis, autophagy and cardiac homeostasis. Conflicting findings have been seen with regard to the role of MTOR signaling and autophagy in cardiac and adipocyte function under metabolic syndrome. AKT, an essential insulin-signaling molecule upstream of MTOR, participates in the regulation of glucose homeostasis and cardiac metabolism. Akt2 knockout may rescue against high-fat diet-disrupted autophagy flux, en route to cardioprotection. Thus, inhibition of MTOR may serve as a possible avenue to retard pathological cardiac hypertrophy via rescuing interrupted autophagic flux.
obesity; autophagy; Akt; MTOR; cardiac function
Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes coloaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), also decreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK.
autophagy; fluorescence multiwell plate reader; Lyso Tracker Red; Mito Tracker Green; mitochondrial permeability transition; mitophagy
Autophagy has emerged as a significant innate immune response to pathogens. Typically, autophagosomes deliver their contents to lysosomes for degradation. Some pathogens such as Salmonella enterica serovar Typhimurium succumb to autophagy and are transported to lysosomes for degradation. Yet, many professional pathogens, including Legionella pneumophila and Burkholderia cenocepacia, subvert this pathway exploiting autophagy to their advantage.
autophagosomes; bacteria; lysosome fusion; Anaplasma; Legionella; Burkholderia; Francisella
Acidic pH is an important feature of tumor microenvironment and a major determinant of tumor progression. We reported that cancer cells upregulate autophagy as a survival mechanism to acidic stress. Inhibition of autophagy by administration of chloroquine (CQ) in combination anticancer therapies is currently evaluated in clinical trials. We observed in 3 different human cancer cell lines cultured at acidic pH that autophagic flux is not blocked by CQ. This was consistent with a complete resistance to CQ toxicity in cells cultured in acidic conditions. Conversely, the autophagy-inhibiting activity of Lys-01, a novel CQ derivative, was still detectable at low pH. The lack of CQ activity was likely dependent on a dramatically reduced cellular uptake at acidic pH. Using cell lines stably adapted to chronic acidosis we could confirm that CQ lack of activity was merely caused by acidic pH. Moreover, unlike CQ, Lys-01 was able to kill low pH-adapted cell lines, although higher concentrations were required as compared to cells cultured at normal pH conditions. Notably, buffering medium pH in low pH-adapted cell lines reverted CQ resistance. In vivo analysis of tumors treated with CQ showed that accumulation of strong LC3 signals was observed only in normoxic areas but not in hypoxic/acidic regions. Our observations suggest that targeting autophagy in the tumor environment by CQ may be limited to well-perfused regions but not achieved in acidic regions, predicting possible limitations in efficacy of CQ in antitumor therapies.
autophagy; chloroquine; tumor acidosis; cancer therapy; pH
PDCD4 (programmed cell death protein 4), a suppressor of gene transcription and translation, plays a crucial inhibitory role in several types of human tumors. However, its underlying mechanisms remain unclear. Autophagy, an evolutionarily conserved catabolic process, maintains cellular homeostasis under stress conditions such as starvation and plays a crucial role in tumor initiation and progression. We report here that PDCD4 inhibits autophagy in multiple cell types both in vitro and in vivo, which in turn contributes to its tumor suppressor activity. Importantly, PDCD4 inhibits the expression of an essential autophagy related gene, ATG5 and the formation of an ATG12–ATG5 complex, and its ma3 domains are required for PDCD4-mediated inhibition of autophagy. Unlike most tumor suppressors that act as positive or dual regulators of autophagy, our findings indicate that PDCD4 negatively regulates autophagy by targeting ATG5, which provides a novel mechanism of tumor suppression.
PDCD4; tumor suppressor; autophagy; ATG5; proliferation
LC3 is a marker protein that is involved in the formation of autophagosomes and autolysosomes, which are usually characterized and monitored by fluorescence microscopy using fluorescent protein-tagged LC3 probes (FP-LC3). FP-LC3 and even endogenous LC3 can also be incorporated into intracellular protein aggregates in an autophagy-independent manner. However, the dynamic process of LC3 associated with autophagosomes and autolysosomes or protein aggregates in living cells remains unclear. Here, we explored the dynamic properties of the two types of FP-LC3-containing puncta using fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The FRAP data revealed that the fluorescent signals of FP-LC3 attached to phagophores or in mature autolysosomes showed either minimal or no recovery after photobleaching, indicating that the dissociation of LC3 from the autophagosome membranes may be very slow. In contrast, FP-LC3 in the protein aggregates exhibited nearly complete recovery (more than 80%) and rapid kinetics of association and dissociation (half-time < 1 sec), indicating a rapid exchange occurs between the aggregates and cytoplasmic pool, which is mainly due to the transient interaction of LC3 and SQSTM1/p62. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we provide a convenient and useful FRAP approach to distinguish autophagosomes from LC3-involved protein aggregates in living cells. Using this approach, we find the FP-LC3 puncta that adjacently localized to the phagophore marker ATG16L1 were protein aggregate-associated LC3 puncta, which exhibited different kinetics compared with that of autophagic structures.
autophagosome; protein aggregate; inclusion body; LC3; FRAP; FRET
We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.
isothermal titration calorimetry; liposome flotation assays; multi-angle laser light scattering; PROPPIN; small unilamellar vesicle
Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.
Atg10; Atg12; Atg3; Atg7; Atg8; E1 enzyme; E2 enzyme; ubiquitin-like protein
MicroRNAs (miRNAs) form a class of ~21 nucleotide (nt) RNAs that post-transcriptionally repress partially complementary messenger RNAs. miRNA-mediated silencing is critical for control of many key biological processes such as tumorigenesis, neuronal synaptic plasticity and defense against bacteria and viruses. Thus, unsurprisingly, miRNA biogenesis, abundance and action are under refined feedback control that is only beginning to be experimentally uncovered. We recently discovered that DICER1 and EIF2C/AGO are targeted for degradation by autophagy as miRNA-free entities by the selective autophagy receptor CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2/nuclear dot protein, 52 kDa). Strikingly, autophagy establishes a checkpoint for continued loading of miRNA, and this checkpoint is required for maintenance of miRNA abundance and proper miRNA activity. This newfound role for autophagy in miRNA biology suggests that human diseases exhibiting misregulated autophagy may be interdependent with defects in miRNA-mediated regulation of gene networks.
autophagy; DICER; microRNA; NDP52
Autophagy defends the mammalian cytosol against bacterial invasion. Efficient bacterial engulfment by autophagy requires cargo receptors that bind (a) homolog(s) of the ubiquitin-like protein Atg8 on the phagophore membrane. The existence of multiple ATG8 orthologs in higher eukaryotes suggests that they may perform distinct functions. However, no specific role has been assigned to any mammalian ATG8 ortholog. We recently discovered that the autophagy receptor CALCOCO2/NDP52, which detects cytosol-invading Salmonella enterica serovar Typhimurium (S. Typhimurium), preferentially binds LC3C. The CALCOCO2/NDP52-LC3C interaction is essential for cell-autonomous immunity against cytosol-exposed S. Typhimurium, because cells lacking either protein fail to target bacteria into the autophagy pathway. The selectivity of CALCOCO2/NDP52 for LC3C is determined by a novel LC3C interacting region (CLIR), in which the lack of the key aromatic residue of canonical LIRs is compensated by LC3C-specific interactions. Our findings provide a new layer of regulation to selective autophagy, suggesting that specific interactions between autophagy receptors and the ATG8 orthologs are of biological importance.
autophagy; Salmonella; ATG8/LC3; LC3C; NDP52; LIR
The lysosome is a key subcellular organelle that receives and degrades macromolecules from endocytic, secretory and autophagic pathways. Lysosomal function is thus critical for an efficient autophagic process. However, the molecular mechanisms mediating lysosomal function upon autophagic induction are largely unknown. Our laboratory recently discovered that upon autophagy activation, the lysosome is activated, and this functional activation is dependent on MTORC1 suppression, suggesting that MTORC1 exerts a suppressive effect on lysosomal function. Therefore, data from our study demonstrate that MTORC1 exerts a dual inhibitory effect on autophagy, blocking autophagy not only at the initiation stage via suppression of the ULK1 complex, but also at the degradation stage via inhibition of lysosomal function. We think that understanding the negative regulatory effect of MTORC1 on lysosomal function expands the functional scope of MTORC1 in autophagy regulation, and offers new clues for developing novel interventional strategies in autophagy- and lysosome-related diseases.
autophagy; lysosome; MTORC1; autophagosome; fusion
Several years ago, an explosion of research into pathogens and autophagy showed that viruses have a wide variety of relationships to this conserved homeostatic pathway. Often, autophagy acts as a host defense mechanism, degrading viruses before they can escape the host cell, and, as such, autophagy is suppressed or avoided by those viruses. A subset of viruses, however, induces and subverts the autophagic machinery to promote their own replication. Many of these viruses inhibit the degradative step in the autophagic pathway, presumably to prevent degradation of cytosolic virions before they exit the cell. Recently, we published a study showing that poliovirus (PV), a well-studied model virus, induces true autophagic degradation. The remainder of our study provided surprising clues about the role of autophagy in promoting virus production. The purpose of this punctum is to discuss the significance of our findings to a general understanding of the autophagic pathway and its relationship to a common class of cellular pathogens.
virus; amphisome; acidification; degradation; secretion
Autophagy and senescence share a number of characteristics, which suggests that both responses could serve to collaterally protect the cell from the toxicity of external stress such as radiation and chemotherapy and internal forms of stress such as telomere shortening and oncogene activation. Studies of oncogene activation in normal fibroblasts as well as exposure of tumor cells to chemotherapy have indicated that autophagy and senescence are closely related but not necessarily interdependent responses; specifically, interference with autophagy delays but does not abrogate senescence. The literature relating to this topic is inconclusive, with some reports appearing to be consistent with a direct relationship between autophagy and senescence and others indicative of an inverse relationship. Before this question can be resolved, additional studies will be necessary where autophagy is clearly inhibited by genetic silencing and where the temporal responses of both autophagy and senescence are monitored, preferably in cells that are intrinsically incapable of apoptosis or where apoptosis is suppressed. Understanding the nature of this relationship may provide needed insights relating to cytoprotective as well as potential cytotoxic functions of both autophagy and senescence.
Helicobacter pylori is a Gram-negative pathogen that colonizes the gastric epithelium of 50–60% of the world’s population. Approximately one-fifth of the infected individuals manifest severe diseases such as peptic ulcers or gastric cancer. H. pylori infection has proven difficult to cure despite intensive antibiotic treatment. One possible reason for the relatively high resistance to antimicrobial therapy is the ability of H. pylori to reside inside host cells. Although considered by most as an extracellular pathogen, H. pylori can invade both gastric epithelial cells and immunocytes to some extent. The intracellular survival of H. pylori has been implicated in its ability to persist in the stomach, evade host immune responses and resist eradication by membrane-impermeable antibiotics. Interestingly, recent evidence suggests that macroautophagy, a cellular self-degradation process characterized by the formation of double-membraned autophagosomes, plays an important role in determining the intracellular fate of H. pylori. Detailed understanding of the interaction between H. pylori and host cell autophagic processes is anticipated to provide novel insights into the molecular mechanisms of macroautophagy and H. pylori pathogenesis, opening new avenues for the therapeutic intervention of autophagy-related and H. pylori-related disorders.
Helicobacter pylori; autophagy; infection; bacteria; VacA; intracellular survival; antibiotic resistance; LC3-associated phagocytosis; pathogenesis
Small molecules with the potential to initiate different types of programmed cell death could be useful ‘adjunct therapy’ where current anticancer modalities fail to generate significant activity due to a defective apoptotic machinery or resistance of cancer cells to the specific death mechanism induced by that treatment. The current study identified silibinin, for the first time, as one such natural agent, having dual efficacy against colorectal cancer (CRC) cells. First, silibinin rapidly induced oxidative stress in CRC SW480 cells due to reactive oxygen species (ROS) generation with a concomitant dissipation of mitchondrial potential (ΔΨm) and cytochrome c release leading to mild apoptosis as a biological effect. However, with increased exposure to silibinin, cytoplasmic vacuolization intensified within the cells followed by sequestration of the organelles, which inhibits the further release of cytochrome c. Interestingly, this decrease in apoptotic response correlated with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Mechanistic studies revealed that silibinin strongly inhibited PIK3CA-AKT–MTOR but activated MAP2K1/2-MAPK1/3 pathways for its biological effects. Corroborating these effects, endoplasmic reticulum stress was generated and glucose uptake inhibition as well as energy restriction were induced by silibinin, thus, mimicking starvation-like conditions. Further, the cellular damage to tumor cells by silibinin was severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipid and protein synthesis, suggesting that silibinin harbors a deadly ‘double-edged sword’ against CRC cells thereby further advocating its clinical effectiveness against this malignancy.
colorectal cancer; silibinin; autophagy; oxidative stress; energy restrictions