Complex tumour ecosystem comprising tumour cells and its associated tumour microenvironment (TME) constantly influence the tumoural behaviour and ultimately impact therapy failure, disease progression, recurrence and poor overall survival of patients. Crosstalk between tumour cells and TME amplifies the complexity by creating metabolic changes such as hypoxic environment and nutrient fluctuations. These changes in TME initiate stem cell-like programmes in cancer cells, contribute to tumoural heterogeneity and increase tumour robustness. Recent studies demonstrate the multifaceted role of autophagy in promoting fibroblast production, stemness, cancer cell survival during longer periods of dormancy, eventual growth of metastatic disease and disease resistance. Recent ongoing studies examine autophagy/mitophagy as a powerful survival strategy in response to environmental stress including nutrient deprivation, hypoxia and environmental stress in TME. It prevents irreversible senescence, promotes dormant stem-like state, induces epithelial–mesenchymal transition and increases migratory and invasive potential of tumour cells. The present review discusses various theories and mechanisms behind the autophagy-dependent induction of cancer stem cell (CSC) phenotype. Given the role of autophagic functions in CSC aggressiveness and therapeutic resistance, various mechanisms and studies based on suppressing cellular plasticity by blocking autophagy as a powerful therapeutic strategy to kill tumour cells are discussed.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

The term ‘autophagy’ literally translates to ‘self-eating’ and alterations to autophagy have been identified as one of the several molecular changes that occur with aging in a variety of species. Autophagy and aging, have a complicated and multifaceted relationship that has recently come to light thanks to breakthroughs in our understanding of the various substrates of autophagy on tissue homoeostasis. Several studies have been conducted to reveal the relationship between autophagy and age-related diseases. The present review looks at a few new aspects of autophagy and speculates on how they might be connected to both aging and the onset and progression of disease. Additionally, we go over the most recent preclinical data supporting the use of autophagy modulators as age-related illnesses including cancer, cardiovascular and neurodegenerative diseases, and metabolic dysfunction. It is crucial to discover important targets in the autophagy pathway in order to create innovative therapies that effectively target autophagy. Natural products have pharmacological properties that can be therapeutically advantageous for the treatment of several diseases and they also serve as valuable sources of inspiration for the development of possible new small-molecule drugs. Indeed, recent scientific studies have shown that several natural products including alkaloids, terpenoids, steroids, and phenolics, have the ability to alter a number of important autophagic signalling pathways and exert therapeutic effects, thus, a wide range of potential targets in various stages of autophagy have been discovered. In this review, we summarised the naturally occurring active compounds that may control the autophagic signalling pathways.

Life expectancy has increased exponentially in the last century accompanied by disability, poor quality of life, and all-cause mortality in older age due to the high prevalence of obesity and physical inactivity in older people. Biologically, the aging process reduces the cell’s metabolic and functional efficiency, and disrupts the cell’s anabolic and catabolic homeostasis, predisposing older people to many dysfunctional conditions such as cardiovascular disease, neurodegenerative disorders, cancer, and diabetes. In the immune system, aging also alters cells' metabolic and functional efficiency, a process known as ‘immunosenescence’, where cells become more broadly inflammatory and their functionality is altered. Notably, autophagy, the conserved and important cellular process that maintains the cell’s efficiency and functional homeostasis may protect the immune system from age-associated dysfunctional changes by regulating cell death in activated CD4+ T cells. This regulatory process increases the delivery of the dysfunctional cytoplasmic material to lysosomal degradation while increasing cytokine production, proliferation, and differentiation of CD4+ T cell-mediated immune responses. Poor proliferation and diminished responsiveness to cytokines appear to be ubiquitous features of aged T cells and may explain the delayed peak in T cell expansion and cytotoxic activity commonly observed in the ‘immunosenescence’ phenotype in the elderly. On the other hand, physical exercise stimulates the expression of crucial nutrient sensors and inhibits the mechanistic target of the rapamycin (mTOR) signaling cascade which increases autophagic activity in cells. Therefore, in this perspective review, we will first contextualize the overall view of the autophagy process and then, we will discuss how body adiposity and physical fitness may counteract autophagy in naïve CD4+ T cells in aging.

Ageing is characterised by the accumulation of molecular and cellular damage through time, leading to a decline in physical and mental abilities. Currently, society has experienced a rapid increase in life expectancy, which has led to an increase in age-associated diseases. Therefore, it is crucial to study the process of ageing to guarantee the best conditions in the final stages of life. In recent years, interest has increased in a myokine known as irisin, which is secreted during physical exercise. This polypeptide hormone is produced by various organs, mainly muscle, and once it is released into the blood, it performs a wide variety of functions that are involved in metabolic control and may be relevant during some of the diseases associated with ageing. The aim of this review is to highlight the recent studies of irisin, such as its mechanism of expression, blood release, distribution, tissue target and participation in various cellular metabolic reactions and the relationship with key anti-ageing pathways such as adenosine monophosphate-activated protein kinase, silent information regulator T 1, autophagy and telomerase. In conclusion, irisin is a key player during the ageing process and it could be a novel target molecule for the therapeutic approach to boost longevity pathways. However, more research will be necessary to use this promising hormone for this gain.

The intestinal bulb is a simple dilatation in the anterior part of the intestine of agastric fish. This study was conducted on 18 adult specimens of molly fish (Poecilia sphenops) and demonstrated the presence of an intestinal bulb. The intestinal epithelium was composed of enterocytes covered with microvilli, many mucous goblet cells, and enteroendocrine cells. Numerous intraepithelial lymphocytes, neutrophils, plasma cells, dendritic cells, stem cells, rodlet cells, and macrophages were identified in the epithelial layer. Interestingly, this study recorded the process of autophagy and formation of autophagosomes, multivesicular bodies, and dense bodies. The intestinal epithelium extended into the intestinal gland that consisted of simple columnar epithelium, mucous cells, stem cells, enteroendocrine cells, and basal cells. These glands opened to the lumen of the bulb and were surrounded by a network of telocytes. Moreover, immunohistochemistry revealed that the intestinal epithelium expressed APG5, myostatin, TGF-β, IL-1β, NF-κB, Nrf2, and SOX9. Leukocytes in the lamina propria-submucosa expressed APG5. The inflammatory cells in the connective tissue showed strong immunoreactivity to myostatin and TGF-β. The smooth muscular layer also expressed myostatin. Both IL-1β and NF-κB showed immunoreactivity in macrophages in the lamina propria-submucosa. Stem cells expressed Sox-9 and telocytes expressed NF-κB and SOX9; while astrocytes in the tunica muscularis expressed GFAP. The high frequency of immune cells in the intestinal bulb suggested an immune role of this organ. This is the first study demonstrating the absence of the stomach and its replacement with an intestinal bulb in molly fish, and consequently, this species could be reclassified as agastric fish according to this study.

Lithium is an inhibitor of glycogen synthase kinase 3 beta, which is traditionally used in the treatment of bipolar disorders and has antitumor effects. The aim of the current study was to determine if lithium salt causes autophagy and apoptosis in skin melanoma cells to enhance cell death. Light microscopy, transmission electron microscopy, immunohistochemistry, and immunofluorescence were used to study the mechanism of action of lithium carbonate in B16 melanoma cells in vivo. Proliferating cell nuclear antigen immunofluorescence assay revealed that the proliferation of B16 melanoma cells was suppressed by lithium treatment for 7 days. Electron microscopy demonstrated a significant increase in the number of autophagic vacuoles in lithium-treated cells relative to control. In addition, levels of autophagy markers LC3 beta and LAMP1 found in lithium-treated tumor xenografts were higher than levels of these markers in the control tumors. Lithium induced caspase-3 expression and apoptotic cell death in tumor cells. Thus, lithium carbonate is the compound that inhibits cell proliferation and stimulates cell death in melanoma cells through induction of autophagy and apoptosis. Stimulation of autophagy by lithium could contribute to the development of autophagic cell death in tumor cells.

Streptococcus agalactiae (S. agalactiae) infection is a significant cause of mastitis, resulting in loss of cellular homeostasis and tissue damage. Autophagy plays an essential function in cell survival, defense, and the preservation of cellular homeostasis, and is often part of the response to pathogenic challenge. However, the effect of autophagy induced by S. agalactiae in bovine mammary epithelial cells (bMECs) is mainly unknown. So in this study, an intracellular S. agalactiae infection model was established. Through evaluating the autophagy-related indicators, we observed that after S. agalactiae infection, a significant quantity of LC3-I was converted to LC3-II, p62 was degraded, and levels of Beclin1 and Bcl2 increased significantly in bMECs, indicating that S. agalactiae induced autophagy. The increase in levels of LAMP2 and LysoTracker Deep Red fluorescent spots indicated that lysosomes had participated in the degradation of autophagic contents. After autophagy was activated by rapamycin (Rapa), the amount of p-Akt and p-mTOR decreased significantly, whilst the amount of intracellular S. agalactiae increased significantly. Whereas the autophagy was inhibited by 3-methyladenine (3MA), the number of intracellular pathogens decreased. In conclusion, the results demonstrated that S. agalactiae could induce autophagy through PI3K/Akt/mTOR pathway and utilize autophagy to survive in bMECs.

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy that develops in or around the throat, larynx, nose, sinuses and mouth, and is mostly treated with a combination of chemo- and radiotherapy (RT). The main goal of RT is to kill enough of the cancer cell population, whilst preserving the surrounding normal and healthy tissue. The mechanisms by which conventional photon RT achieves this have been extensively studied over several decades, but little is known about the cell death pathways that are activated in response to RT of increasing linear energy transfer (LET), including proton beam therapy and heavy ions. Here, we provide an up-to-date review on the observed radiobiological effects of low- versus high-LET RT in HNSCC cell models, particularly in the context of specific cell death mechanisms, including apoptosis, necrosis, autophagy, senescence and mitotic death. We also detail some of the current therapeutic strategies targeting cell death pathways that have been investigated to enhance the radiosensitivity of HNSCC cells in response to RT, including those that may present with clinical opportunities for eventual patient benefit.

Multiple myeloma (MM) is a deadly, incurable malignancy in which antibody-secreting plasma cells (PCs) become neoplastic. Previous studies have shown that the PC niche plays a role cancer progression. Bone marrow (BM) cores from MM and a premalignant condition known as monoclonal gammopathy of unknown significance (MGUS) patients were analyzed with confocal and transmission electron microscopy. The BM aspirates from these patients were used to generate 3D PC cultures. These in vitro cultures were then assayed for the molecular, cellular, and ultrastructural hallmarks of dysfunctional PC at days 1 and 5. In vivo, evidence of PC endoplasmic reticulum stress was found in both MM and MGUS BM; however, evidence of PC autophagy was found only in MM BM. Analysis of in vitro cultures found that MM PC can survive and maintain a differentiated phenotype over an unprecedented 5 days, had higher levels of paraprotein production when compared to MGUS-derived cultures, and showed evidence of PC autophagy as well. Increased fibronectin deposition around PC associated with disease severity and autophagy dysregulation was also observed. 3D cultures constructed from BM aspirates from MGUS and MM patients allow for long-term culture of functional PC while maintaining their distinct morphological phenotypes.

This study investigates the mechanism by which maternal protein restriction induces hepatic autophagy-related gene expression in the offspring of rats. Pregnant Sprague-Dawley rats were fed either a control diet (C, 18 % energy from protein) or a low-protein diet (LP, 8·5 % energy from protein) during gestation, followed by the control diet during lactation and post-weaning. Liver tissue was collected from the offspring at postnatal day 38 and divided into four groups according to sex and maternal diet (F-C, F-LP, M-C and M-LP) for further analysis. Autophagy-related mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. In addition, chromatin immunoprecipitation (ChIP) was performed to investigate the interactions between transcription factors and autophagy-related genes. Protein levels of p- eukaryotic translation initiation factor 2a and activating transcription factor 4 (ATF4) were increased only in the female offspring born to dams fed the LP diet. Correlatively, the mRNA expression of hepatic autophagy-related genes including Map1lc3b, P62/Sqstm1, Becn1, Atg3, Atg7 and Atg10 was significantly greater in the F-LP group than in the F-C group. Furthermore, ChIP results showed greater ATF4 and C/EBP homology protein (CHOP) binding at the regions of a set of autophagy-related genes in the F-LP group than in the F-C group. Our data demonstrated that a maternal LP diet transcriptionally programmed hepatic autophagy-related gene expression only in female rat offspring. This transcriptional programme involved the activation of the eIF2α/ATF4 pathway and intricate regulation by transcription factors ATF4 and CHOP.

Staphylococcus aureus is a common pathogen of bovine mastitis which can induce autophagy and inhibit autophagy flux, resulting in intracellular survival and persistent infection. The aim of the current study was to investigate the role of p38α in the autophagy induced by intracellular S. aureus in bovine mammary epithelial cells. An intracellular infection model of MAC-T cells was constructed, and activation of p38α was examined after S. aureus invasion. Through activating/inhibiting p38α by anisomycin/SB203580, the autophagosomes, LC3 and p62 level were analyzed by immunofluorescence and western blot. To further study the detailed mechanism of p38α, phosphorylation of ULK1ser757 was also detected. The results showed that intracellular S. aureus activated p38α, and the activation developed in a time-dependent manner. Inhibition of p38α promoted intracellular S. aureus-induced autophagy flow, up-regulated the ratio of LC3 II/I, reduced the level of p62 and inhibited the phosphorylation of ULK1ser757, whereas the above results were reversed after activation of p38α. The current study indicated that intracellular S. aureus can inhibit autophagy flow by activating p38α in bovine mammary epithelial cells.

Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.

The multidrug resistance (MDR) is a widely observed phenotype that contributed to the major obstacle of impairing the outcome of cancer chemotherapy. With the aim to reverse MDR in the breast cancer cell line, the autophagy-related 7 (ATG7) small interfering RNA (siRNA) capable of downregulating the cellular autophagy level was loaded into a cationic nanostructured lipid carrier (NLC) with doxorubicin (Dox) to build a platform (NLC/D-R) for effective chemotherapy of breast cancer. Our results revealed that NLC/D-R was well-dispersed nanoparticles with satisfy protection to siRNA. In addition, NLC/D-R also exerted a sufficient drug release of both cargos under an acidic environment with high stability and biocompatibility at the physiological environment. Furthermore, NLC/D-R showed a preferable transfection profile to PEI 25k. The downregulated autophagy level in NLCF-7/Adr cells resulted in reverse of MDR and accumulated Dox retention in cells. The in vitro cytotoxicity using both cells on flat surfaces and multicellular tumor spheroid (NLCTS) model confirmed that NLC/D-R showed much elevated anticancer performance than NLC/Dox or NLC/siRNA, which suggested the synergistic effect between anti-autophagy and chemotherapy.

Skeletal muscle atrophy causes decreased physical activity and increased risk of metabolic diseases. We investigated the effects of oleamide (cis-9,10-octadecanamide) treatment on skeletal muscle health. The plasma concentration of endogenous oleamide was approximately 30 nm in male ddY mice under normal physiological conditions. When the stable isotope-labelled oleamide was orally administered to male ddY mice (50 mg/kg), the plasma concentration of exogenous oleamide reached approximately 170 nm after 1 h. Male ddY mice were housed in small cages (one-sixth of normal size) to enforce sedentary behaviour and orally administered oleamide (50 mg/kg per d) for 4 weeks. Housing in small cages decreased tibialis anterior (TA) muscle mass and the cross-sectional area of the myofibres in TA muscle. Dietary oleamide alleviated the decreases in TA muscle and resulted in plasma oleamide concentration of approximately 120 nm in mice housed in small cages. Housing in small cages had no influence on the phosphorylation levels of Akt serine/threonine kinase (Akt), mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) in TA muscle; nevertheless, oleamide increased the phosphorylation levels of the proteins. Housing in small cages increased the expression of microtubule-associated protein 1 light chain 3 (LC3)-II and sequestosome 1 (p62), but not LC3-I, in TA muscle, and oleamide reduced LC3-I, LC3-II and p62 expression levels. In C2C12 myotubes, oleamide increased myotube diameter at ≥100 nm. Furthermore, the mTOR inhibitor, Torin 1, suppressed oleamide-induced increases in myotube diameter and protein synthesis. These results indicate that dietary oleamide rescued TA muscle atrophy in mice housed in small cages, possibly by activating the phosphoinositide 3-kinase/Akt/mTOR signalling pathway and restoring autophagy flux.

Mammary tissue (MT) turnover is characterized by programed cell death and remodeling which might be affected by both feeding level and animal species. Thus, twenty-four dairy goats and the same number of sheep were assigned to three homogenous sub-groups per animal species and fed the same diet in quantities which met 70% (FL70), 100% (FL100) and 130% (FL130) of their daily energy and crude protein requirements. Individual MT samples were taken by biopsy from the animals on the 30th and 60th experimental day. The results showed, in the first sampling time, a significant reduction in the mRNA abundance for selected genes involved in programed cell death in both FL 70 fed goats (STAT3 and BECN1) and sheep (CASPASE8 and BECN1) compared with the respective FL100 groups. The FL130, in comparison with the FL100, caused a significant increase in transcripts accumulation of STAT3 gene in both sampling times and CASPASE8 gene in the second sampling time in goat MT, while the opposite happened for the mRNA expression of CASPASE8 and BECN1 genes in sheep MT, but only in the first sampling time. Moreover, a significant up regulation in the mRNA levels of MMP2 gene in MT of FL130 fed sheep was observed. The FL130, in comparison with the FL70, caused an enhancement in the mRNA expression levels of BECN1, CASPASE8, BAX and STAT3 genes in goat MT only. It was also shown that apoptosis and autophagy can be affected simultaneously by the feeding level. Overfeeding affects MT programed cell death and remodeling by a completely different way in goats than sheep. In conclusion, feeding level and animal species have strong effects on both MT programed cell death (apoptosis and autophagy) and remodeling but the molecular mechanisms need further investigation.

The carpenter ant Camponotus rufipes has intracellular bacteria in bacteriocytes scattered in the midgut epithelium, which have different amounts of endosymbionts, according to the developmental stages. However, there are no detailed data about the midgut cells in adult workers. The present work aimed to evaluate the morphology and cellular events that coordinate the abundance of endosymbionts in the midgut cells in C. rufipes workers. The midgut epithelium has digestive cells, bacteriocytes, and cells with intermediate morphology. The latter is similar to bacteriocytes, due to the abundance of endosymbionts, and similar to digestive cells, due to their microvilli. The digestive and intermediate cells are rich in autophagosomes and autolysosomes, both with bacteria debris in the lumen. These findings suggest that midgut cells of C. rufipes control the endosymbiont level by the autophagy pathway.

Energy restriction (ER) has anti-ageing effects and probably protects from a range of chronic diseases including cancer, diabetes and chronic kidney disease (CKD). Specifically, ER has a positive impact on experimental kidney ageing, CKD (diabetic nephropathy, polycystic kidney disease) and acute kidney injury (nephrotoxic, ischaemia–reperfusion injury) through such mechanisms as increased autophagy, mitochondrial biogenesis and DNA repair, and decreased inflammation and oxidative stress. Key molecules contributing to ER-mediated kidney protection include adenosine monophosphate-activated protein kinase, sirtuin-1 and PPAR-γ coactivator 1α. However, CKD is a complex condition, and ER may potentially worsen CKD complications such as protein–energy wasting, bone–mineral disorders and impaired wound healing. ER mimetics are drugs, such as metformin and Na–glucose co-transporter-2 which mimic the action of ER. This review aims to provide comprehensive data regarding the effect of ER on CKD progression and outcomes.

Connexin 43 (CX43) is a component of gap junctions. The lack of functional CX43 induces oxidative stress, autophagy, and apoptosis in somatic cells. However, the role of CX43 in the early development of porcine embryos is still unknown. Thus, the aim of this study was to investigate the role of CX43, and its underlying molecular mechanisms, on the developmental competence of early porcine embryos. We performed CX43 knockdown by microinjecting dsRNA into parthenogenetically activated porcine parthenotes. The blastocyst development rate and the total number of cells in the blastocysts were significantly reduced by CX43 knockdown. Results from FITC-dextran assays showed that CX43 knockdown significantly increased membrane permeability. ZO-1 protein was obliterated in CX43 knockdown blastocysts. Mitochondrial membrane potential and ATP production were significantly reduced following CX43 knockdown. Reactive oxygen species (ROS) levels were significantly increased in the CX43 knockdown group compared to those in control embryos. Moreover, CX43 knockdown induced autophagy and apoptosis. Our findings indicate that CX43 is essential for the development and preimplantation of porcine embryos and maintains mitochondrial function, cell junction structure, and cell homeostasis by regulating membrane permeability, ROS generation, autophagy, and apoptosis in early embryos.