Autophagy-dependent Cell Death Research Articles

Autophagy-Dependent Ferroptosis in Cancer.

Significance: Autophagy is a self-degrading process that determines cell fate in response to various environmental stresses. In contrast to autophagy-mediated cell survival, the signals, mechanisms, and effects of autophagy-dependent cell death remain obscure. The discovery of autophagy-dependent ferroptosis provides a paradigm for understanding the relationship between aberrant degradation pathways and excessive lipid peroxidation in driving regulated cell death. Recent Advances: Ferroptosis was originally described as an autophagy-independent and iron-mediated nonapoptotic cell death. Current studies reveal that the level of intracellular autophagy is positively correlated with ferroptosis sensitivity. Selective autophagic degradation of proteins (e.g., ferritin, SLC40A1, ARNTL, GPX4, and CDH2) or organelles (e.g., lipid droplets or mitochondria) promotes ferroptosis by inducing iron overload and/or lipid peroxidation. Several upstream autophagosome regulators (e.g., TMEM164), downstream autophagy receptors (e.g., HPCAL1), or danger signals (e.g., DCN) are selectively required for ferroptosis-related autophagy, but not for starvation-induced autophagy. The induction of autophagy-dependent ferroptosis is an effective approach to eliminate drug-resistant cancer cells. Critical Issues: How different organelles selectively activate autophagy to modulate ferroptosis sensitivity is not fully understood. Identifying direct protein effectors of ferroptotic cell death remains a challenge. Future Directions: Further understanding of the molecular mechanics and immune consequences of autophagy-dependent ferroptosis is critical for the development of precision antitumor therapies.

Targeting regulated cell death with plant natural compounds for cancer therapy: A revisited review of apoptosis, autophagy-dependent cell death, and necroptosis.

Regulated cell death (RCD) refers to programmed cell death regulated by various protein molecules, such as apoptosis, autophagy-dependent cell death, and necroptosis. Accumulating evidence has recently revealed that RCD subroutines have several links to many types of human cancer; therefore, targeting RCD with pharmacological small-molecule compounds would be a promising therapeutic strategy. Moreover, plant natural compounds, small-molecule compounds synthesized from plant sources, and their derivatives have been widely reported to regulate different RCD subroutines to improve potential cancer therapy. Thus, in this review, we focus on updating the intricate mechanisms of apoptosis, autophagy-dependent cell death, and necroptosis in cancer. Moreover, we further discuss several representative plant natural compounds and their derivatives that regulate the above-mentioned three subroutines of RCD, and their potential as candidate small-molecule drugs for the future cancer treatment.

Copper-dependent autophagic degradation of GPX4 drives ferroptosis

ABSTRACT Ferroptosis is a type of iron-dependent regulated cell death characterized by unrestricted lipid peroxidation and membrane damage. Although GPX4 (glutathione peroxidase 4) plays a master role in blocking ferroptosis by eliminating phospholipid hydroperoxides, the regulation of GPX4 remains poorly understood. Here, we report an unexpected role for copper in promoting ferroptotic cell death, but not cuproptosis, by inducing macroautophagic/autophagic degradation of GPX4. Copper chelators reduce ferroptosis sensitivity but do not inhibit other types of cell death, such as apoptosis, necroptosis, and alkaliptosis. Conversely, exogenous copper increases GPX4 ubiquitination and the formation of GPX4 aggregates by directly binding to GPX4 protein cysteines C107 and C148. TAX1BP1 (Tax1 binding protein 1) then acts as an autophagic receptor for GPX4 degradation and subsequent ferroptosis in response to copper stress. Consequently, copper enhances ferroptosis-mediated tumor suppression in a mouse model of pancreatic cancer tumor, whereas copper chelators attenuate experimental acute pancreatitis associated with ferroptosis. Taken together, these findings provide new insights into the link between metal stress and autophagy-dependent cell death. Abbreviations: CALCOCO2, calcium binding and coiled-coil domain 2; GPX4, glutathione peroxidase 4; MAP1LC3A/B, microtubule associated protein 1 light chain 3 alpha/beta; MPO, myeloperoxidase; NCOA4, nuclear receptor coactivator 4; OPTN, optineurin; PDAC, pancreatic ductal adenocarcinoma; RIPK1, receptor interacting serine/threonine kinase 1; ROS, reactive oxygen species; SLC40A1, solute carrier family 40 member 1; SQSTM1, sequestosome 1; TAX1BP1, Tax1 binding protein 1; TEPA, tetraethylenepentamine; TM, tetrathiomolybdate.

Assessment of antiproliferative and toxic effects of a peptide from Momordica dioica using in vitro and in vivo studies

Abstract: Inflammation occurs during a cascade reaction to cause damage to the tissues. Increased oxidant and cytokine expression were observed in the damaged tissues. Inflammatory bowel disease (IBD) may be a chronic and lethal disease of inflammation in gastro enteric tissue characterized by intestinal inflammation. The objective of the study was to investigate the toxicological effects of Peptide extract from Momordica dioica in treating colitis conditions. The isolated protein was digested with trypsin enzyme with an E:S ratio of 1:100 and the hydrolyzed peptides were analyzed using LCMS. The peak obtained at 678.67 Da at 17mins showed hydrophobic peptide and MALDI-TOF analysis which showed similar peaks at 3388.7Da. The results from the MTT assay showed the IC50 value of the peptide extract at 100µg/ml. Similarly, the acridine orange staining showed decreased green fluorescent nuclei cells than red fluorescent acidic vesicular organelles indicating autophagy-dependent cell death. The peptide extract displayed a cell viability effect on Colo-205 cells at 100 μg/ml when compared to the control. In the acute toxicity test, the mice were orally receiving peptide extract at a dose of 50-250 mg/kg BW for 14 days. The significant adverse effects were evident at a dose of 250mg/kg BW indicating that the LD50 value is lesser than 250 mg/kg.

Endoplasmic reticulum stress regulates autophagic response that is involved in Saikosaponin a-induced liver cell damage

Saikosaponin a (Ssa) is an active ingredient of the Chinese herbal plant Radix Bupleuri (RB) and has severe hepatotoxicity. However, biomolecular mechanisms involved in Ssa-induced hepatotoxicity are not yet entirely clear. Previous studies reported that Ssd (an isomer of Ssa) as a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor can induce autophagy in apoptotic defective cells, leading to autophagy-dependent cell death. Therefore, we speculate that endoplasmic reticulum (ER) stress and autophagy may also play an important role in Ssa-induced hepatocyte death. This study aimed to explore the connection between ER stress and autophagy and Ssa-induced hepatotoxicity. Experiments in vitro showed that the cell viability of L-02 cells in the Ssa treatment group decreased, the level of autophagy marker LC3-II/LC3-I and Beclin1 increased, the level of p62 decreased, the colocalization of autophagosome and lysosome increased, and the cell viability was significantly increased after the application of autophagy inhibitors 3-MA. In addition, SSa can induce ER stress in L-02 cells in vitro. Further studies demonstrated that SSa activated the PERK/eIF2α/ATF4/CHOP pathway, IRE1-TRAF2 pathway, ATF6 pathway, and AMPK/mTOR pathway associated with ER stress. Application of ER stress inhibitors 4-PBA can significantly down-regulate the level of autophagy and improve cell viability. Results of in vivo experiments showed that treatment with 150 and 300 mg/kg Ssa significantly elevated the liver/body weight ratio and caused histological injury in mice liver. Furthermore, Ssa treatment induced significantly downregulated p62 expression but upregulated LC3-II, CHOP, and GRP78 expression in mice livers. Taken together, our results showed that SSa can activate endoplasmic reticulum stress, promote toxic autophagy, and then induce cell death. We revealed an alternative mechanism involving autophagy and ERs, by which Ssa induced hepatotoxicity.

The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38MAPK and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death.

Extracellular SQSTM1 exacerbates acute pancreatitis by activating autophagy-dependent ferroptosis

Acute pancreatitis (AP) is an abdominal inflammatory disease initiated by damaged pancreatic acinar cells and developed by systemic inflammation. SQSTM1 (sequestosome 1) has an intracellular function in mediating substrate degradation during macroautophagy/autophagy, and it can be released by macrophages and monocytes to trigger lethal inflammation during bacterial infection. Here, we report that extracellular SQSTM1 acts as a mediator of AP by enhancing the sensitivity to autophagy-dependent ferroptotic cell death. Serum SQSTM1 is elevated in AP patients as well as in mice that have cerulein-induced AP. The administration of SQSTM1-neutralizing antibodies protects against experimental AP in mice. Mechanistically, recombinant SQSTM1 protein (rSQSTM1) increases AGER (advanced glycosylation end-product specific receptor)-dependent ACSL4 (acyl-CoA synthetase long chain family member 4) expression, leading to polyunsaturated fatty acid production for autophagosome formation and subsequent ferroptosis. The rSQSTM1-elicited pathological responses during AP are attenuated in mice with the conditional deletion of Ager in the pancreas. These findings may provide not only new insights into the mechanism of autophagy-dependent cell death, but also suggest that targeting the extracellular SQSTM1 pathway is a potential strategy for the treatment of AP.

Metronomic doses and drug schematic combination response tested within chambered coverslips for the treatment of breast cancer cells (JIMT-1).

Low-dose metronomic (LDM) chemotherapy is an alternative to conventional chemotherapy and is the most frequently used approach in low dose chemotherapy regimens. The selection of patients, drug dosages, and dosing intervals in LDM is empirical. In this study, we systematically examined the schedule-dependent interaction of drugs on a breast cancer cell line (BCC) cultured in chambered coverslips. The LDM studies were combined with cell staining in order to better characterize different cell states and cell death modes, including caspase-dependent apoptosis, caspase-independent cell death and autophagy-dependent cell death. Microscope images were examined using the Fiji Trainable Weka Segmentation plugin to analyse cell area in 7500 images showing different modes of cell death. Paclitaxel combined with LDM chemotherapy demonstrated a reduction in the area covered by live cells. In contrast, there was an induction of high levels of cell death due to caspase-dependent apoptosis.

Leveraging diverse cell-death patterns to predict the prognosis and drug sensitivity of triple-negative breast cancer patients after surgery

BackgroundPostoperative progression and chemotherapy resistance is the major cause of treatment failure in patients with triple-negative breast cancer (TNBC). Currently, there is a lack of an ideal predictive model for the progression and drug sensitivity of postoperative TNBC patients. Diverse programmed cell death (PCD) patterns play an important role in tumor progression, which has the potential to be a prognostic and drug sensitivity indicator for TNBC after surgery. Materials and methodsTwelve PCD patterns (apoptosis, necroptosis, pyroptosis, ferroptosis, cuproptosis, entotic cell death, netotic cell death, parthanatos, lysosome-dependent cell death, autophagy-dependent cell death, alkaliptosis, and oxeiptosis) were analyzed for model construction. Bulk transcriptome, single-cell transcriptome, genomics, and clinical information were collected from TCGA-BRCA, METABRIC, GSE58812, GSE21653, GSE176078, GSE75688, and KM-plotter cohorts to validate the model. ResultsThe machine learning algorithm established a cell death index (CDI) with a 12-gene signature. Validated in five independent datasets, TNBC patients with high CDI had a worse prognosis after surgery. Two molecular subtypes of TNBC with distinct vital biological processes were identified by an unsupervised clustering model. A nomogram with high predictive performance was constructed by incorporating CDI with clinical features. Furthermore, CDI was associated with immune checkpoint genes and key tumor microenvironment components by integrated analysis of bulk and single-cell transcriptome. TNBC patients with high CDI are resistant to standard adjuvant chemotherapy regimens (docetaxel, oxaliplatin, etc.); however, they might be sensitive to palbociclib (an FDA-approved drug for luminal breast cancer). ConclusionGenerally, we established a novel CDI model by comprehensively analyzing diverse cell death patterns, which can accurately predict clinical prognosis and drug sensitivity of TNBC after surgery. A user-friendly website was created to facilitate the application of this prediction model (https://tnbc.shinyapps.io/CDI_Model/).

Radiation-induced Cell Death and Its Mechanisms.

With rapid technical advances, ionizing radiation has been put into wider application in ordinary living, with the worst cytological effect on the human body being cell death. Moreover, according to the Nomenclature Committee on Cell Death, the method of radiation-induced cell death, usually classified as interphase and proliferative death, undergoes more detailed classifications oriented by its molecular mechanism. Elaborating its mode and molecular mechanism is crucial for the protection and treatment of radiation injury, as well as the radiotherapy and recovery of tumors. Varying with the changes of the radiation dose and the environment, the diverse targets and pathways of ionizing radiation result in various cell deaths. This review focuses on classifications of radiation-induced cell death and its molecular mechanism. We also examine the main characteristics of ionizing radiation-induced cell death. The modes of radiation-induced cell death can be classified as apoptosis, necrosis, autophagy-dependent cell death, pyroptosis, ferroptosis, immunogenic cell death, and non-lethal processes. Once the dose is high enough, radiation effects mostly appear as destructiveness (“destructiveness” is used to describe a situation in which cells do not have the opportunity to undergo a routine death process, in which case high-dose radiation works like a physical attack). This breaks up or even shatters cells, making it difficult to find responses of the cell itself. Due to diversities concerning cell phenotypes, phases of cell cycle, radiation dose, and even cellular subregions, various methods of cell death occur, which are difficult to identify and classify. Additionally, the existence of common initial activation and signaling molecules among all kinds of cell deaths, as well as sophisticated crossways in cellular molecules, makes it more laborious to distinguish and classify various cell deaths.

Neuronal cell death mechanisms in Alzheimer's disease: An insight.

Regulated cell death (RCD) is an ordered and tightly orchestrated set of changes/signaling events in both gene expression and protein activity and is responsible for normal development as well as maintenance of tissue homeostasis. Aberrant activation of this pathway results in cell death by various mechanisms including apoptosis, necroptosis, pyroptosis, ferroptosis, and autophagy-dependent cell death. Such pathological changes in neurons alone or in combination have been observed in the pathogenesis of various neurodegenerative diseases including Alzheimer’s disease (AD). Pathological hallmarks of AD focus primarily on the accumulation of two main protein markers: amyloid β peptides and abnormally phosphorylated tau proteins. These protein aggregates result in the formation of A-β plaques and neuro-fibrillary tangles (NFTs) and induce neuroinflammation and neurodegeneration over years to decades leading to a multitude of cognitive and behavioral deficits. Autopsy findings of AD reveal massive neuronal death manifested in the form of cortical volume shrinkage, reduction in sizes of gyri to up to 50% and an increase in the sizes of sulci. Multiple forms of cell death have been recorded in neurons from different studies conducted so far. However, understanding the mechanism/s of neuronal cell death in AD patients remains a mystery as the trigger that results in aberrant activation of RCD is unknown and because of the limited availability of dying neurons. This review attempts to elucidate the process of Regulated cell death, how it gets unregulated in response to different intra and extracellular stressors, various forms of unregulated cell death, their interplay and their role in pathogenesis of Alzheimer’s Disease in both human and experimental models of AD. Further we plan to explore the correlation of both amyloid-beta and Tau with neuronal loss as seen in AD.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR

Autophagy is a biological process that maintains cellular homeostasis and regulates the internal cellular environment. Hyperactivating autophagy to trigger cell death has been a suggested therapeutic strategy for cancer treatment. Mechanistic target of rapamycin (mTOR) is a crucial protein kinase that regulates autophagy; therefore, using a structure-based virtual screen analysis, we identified lomitapide, a cholesterol-lowering drug, as a potential mTOR complex 1 (mTORC1) inhibitor. Our results showed that lomitapide directly inhibits mTORC1 in vitro and induces autophagy-dependent cancer cell death by decreasing mTOR signaling, thereby inhibiting the downstream events associated with increased LC3 conversion in various cancer cells (e.g., HCT116 colorectal cancer cells) and tumor xenografts. Lomitapide also significantly suppresses the growth and viability along with elevated autophagy in patient-derived colorectal cancer organoids. Furthermore, a combination of lomitapide and immune checkpoint blocking antibodies synergistically inhibits tumor growth in murine MC38 or B16-F10 preclinical syngeneic tumor models. These results elucidate the direct, tumor-relevant immune-potentiating benefits of mTORC1 inhibition by lomitapide, which complement the current immune checkpoint blockade. This study highlights the potential repurposing of lomitapide as a new therapeutic option for cancer treatment.

Non-coding RNAs associated with autophagy and their regulatory role in cancer therapeutics.

Cancer widely affects the world's health population and ranks second leading cause of death globally. Because of poor prognosis of various types of cancer such as sarcoma, lymphoma, adenomas etc., their high recurrence and metastasis rate and low early diagnosis rate have become concern lately. Role of autophagy in cancer progression is being studied since long. Autophagy is cell's self-degradative mechanism towards stress and has role in degradation of the cytoplasmic macromolecules which has potential to damage other cytosolic molecules. Autophagy can promote as well as inhibit tumorigenesis depending upon the associated protein combinations in cancer cells. Recent studies have shown that non-coding RNAs (ncRNAs) do not code for protein but play essential role in modulation of gene expression. At transcriptional level, different ncRNAs like lncRNAs, miRNAs and circRNAs directly or indirectly affect different stages of autophagy like autophagy-dependent and non-apoptotic cell death in cancer cells. This review focuses on the involvement of ncRNAs in autophagy and the modulation of several cancer signal transduction pathways in cancers such as lung, breast, prostate, pancreatic, thyroid, and kidney cancer.

Targeting Regulated Cell Death with Pharmacological Small Molecules: An Update on Autophagy-Dependent Cell Death, Ferroptosis, and Necroptosis in Cancer.

Regulated cell death is a widely attractive subject among the topics of cancer therapy and has gained some advances for discovery of targeted anticancer drugs. In the past decade, nonapoptotic regulated cell death has been implicated in the development and therapeutic responses of a variety of human cancers. Hitherto, targeting autophagy-dependent cell death (ADCD), ferroptosis, and necroptosis with small molecules has been emerging as a hopeful strategy for the improvement of potential cancer therapy, which may have an advantage to bypass the apoptosis-resistance machinery. Thus, in this perspective, we concentrate on the key molecular insights into ADCD, ferroptosis, and necroptosis and summarize the corresponding small molecules in potential cancer therapy. Moreover, the relationships between the three subroutines and small molecules modulating the crosstalk are discussed. We believe that these inspiring findings would be advantageous to exploiting more potential targets and pharmacological small molecules in future cancer treatment.

Juxtavascular Microglia Scavenge Dying Pericytes and Vascular Smooth Muscle Cells in Diabetic Retinopathy

Diabetic retinopathy (DR) remains a prevalent complication of diabetes and a major cause of vision loss among the working population. Selective loss of pericytes and vascular smooth muscle cells (SMCs), the mural cells of the retinal blood vessels, is pathognomonic of the vasodegenerative element of diabetic retinopathy, and recent studies suggest a central role for autophagy-dependent cell death in this pathology. Our first study of archival electron micrographs from diabetic donor retina provided evidence for the involvement of autophagy in mural cell death during DR and the current report extends those observations to the fate of mural cell corpses in the vascular wall. Here we show that the efferocytosis, or phagocytic removal of dying mural cells, is carried out by a population of juxtavascular microglia (JVM). This population of microglia are well-characterised in the brain but previously unreported in the retina. We demonstrate that JVM are distinct from perivascular macrophages as they participate in the glia limitans of the retinal vasculature and constitute an integral component of the neurovascular unit of the retina. Importantly, mural cells undergoing active phagocytic engulfment appeared to represent relatively early stages in autophagy-dependent cell death, suggesting that the more degraded pericyte and SMC corpses, known as “ghosts”, have evaded efficient efferocytosis and undergone secondary necrosis. The alternative fates of mural cell corpses in the retinal vasculature may have important implications for inflammatory processes in the vasodegenerative pathology characteristic of DR.

Pericyte and Vascular Smooth Muscle Death in Diabetic Retinopathy Involves Autophagy

Diabetic retinopathy (DR) is the most common complication of diabetes and a major cause of vision loss worldwide. The premature death of the microvascular mural cells represents both a pathological hallmark of vasodegeneration in DR and a basis for therapeutic intervention to halt progression to the sight-threatening stages. Recent studies suggest that retinal microvascular mural cells, classed as pericytes in the capillaries and vascular smooth muscle cells in the larger vessels (VSMC), may undergo autophagy-dependent cell death during DR. The present investigation was undertaken to assess electron microscopic evidence for involvement of autophagy in mediation of cell death in the mural cells of the retinal vasculature, in eyes from human diabetic donors and diabetic dogs. All specimens examined showed widespread evidence of autophagosomes in processes of viable pericytes and VSMCs, and the membranous remnants of excessive autophagic activity in their “ghost cell” remnants within the vascular walls. Autophagy was termed “excessive” when it occupied the greater part of the cytoplasm in mural cell processes. This was notable in specimens from short-term diabetic donors with no evidence of basement-membrane thickening or mural cell loss, in which regions of mural cell cytoplasm filled with autophagic bodies appeared to be undergoing cytoplasmic cleavage. No equivalent evidence of autophagy was detected in the adjacent endothelial cells of the retinal vessels. We conclude that increased autophagy in the retinal pericytes and VSMCs is linked to the diabetic milieu, and over time may also act as a trigger for mural cell loss and progressive vasodegeneration.

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.

Rapalogs induce non-apoptotic, autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma.

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Sincemutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.

Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.