LC3 Puncta Formation Research Articles

Treatment of Mycobacterium tuberculosis infected macrophages with rifabutin loaded β-glucan microparticles induces macroautophagy mediated bacillary killing

Tuberculosis (TB), attributable to Mycobacterium tuberculosis (M.tb), constitutes a formidable global health challenge, particularly with the proliferation of multidrug-resistant (MDR) strains. The efficacious clearance of M.tb from host cells is imperative for mitigating infection and averting disease progression. Autophagy, an intricate cellular mechanism for degrading and recycling biomolecules, plays a pivotal role in the immune response to M.tb by facilitating the degradation of intracellular pathogens through the formation of autophagosomes and their subsequent fusion with lysosomes. This study elucidates the therapeutic efficacy of DYDGP microparticles in M.tb-infected macrophages. DCFHDA staining elucidated that DYDGP significantly enhances macrophage membrane integrity and amplifies reactive oxygen species (ROS) production compared to YDGP, even in the presence of NOX-2 inhibitors. Furthermore, DYDGP promotes the biogenesis of acidic vesicular organelles and phago-lysosomal maturation, as corroborated by acridine orange and Lysotracker Red staining. Immunofluorescence and dansylcadaverine staining evidenced that DYDGP enhances autophagy induction and LC3 puncta formation in infected macrophages at both 30 min and 24 h. Protein expression analyses demonstrated elevated levels of NOX-2 and LC3, confirming autophagy induction. Antimycobacterial efficacy assessments revealed that DYDGP treatment engendered significant reductions in colony-forming units (CFUs) for H37Rv (64, 40, 19), MDR32420 (44, 35, 18), MDR32422 (44, 39, 21), and MDR32521 (38, 22, 18) at 30 min, 24 h, and 48 h, respectively. These findings accentuate DYDGP's potential to substantially attenuate M.tb burden, including in MDR strains, thereby positioning it as a promising adjunctive therapy for augmenting tuberculosis treatment.

Regulation of AMP-Activated Protein Kinase (AMPK) Subunit Expression at the Transcriptional Level by the Neutral Amino Acid Transporter SNAT2

AMP-Activated Protein Kinase (AMPK) is a key enzyme that maintains cellular energy homeostasis. AMPK promotes catabolic pathways and inhibits anabolic pathways under low-energy conditions, such as glucose deprivation. SLC38A2/SNAT2 is an amino acid transporter that imports a wide range of neutral amino acids, including glutamine and alanine. The expression of SNAT2 is down regulated in patients with metabolic diseases such as obesity, nonalcoholic steatohepatitis, and hepatocellular carcinoma. Here, we demonstrate that SNAT2 is required for LKB1-mediated activation of AMPK. Depletion of SNAT2 results in diminished activation of AMPK under conditions of glucose or serum starvation, while the induction of SNAT2 expression augments AMPK activation. SNAT2 overexpression-mediated induction of AMPK activation is abrogated by LKB1 depletion but not by CaMKK2 or TAK1 depletion. Interestingly, the mRNA levels of AMPKβ2 are comparably reduced by SNAT2 depletion, indicating regulation of AMPKβ2 at the transcriptional level. AMPKβ1 mRNA levels are also reduced to a lesser extent. Consistently, SNAT2 depletion leads to a marked reduction in AMPK-mediated autophagy induction, as evidenced by reduced conversion to LC3 II and LC3 puncta formation under nutrient-deprived conditions. Given that AMPK activation plays a pivotal role in nutrient sensing and the subsequent cellular response to low-energy levels and various stress conditions, we provide another level of regulatory mechanism for AMPK-mediated cellular energy homeostasis.

Rubiadin-1-methyl ether inhibits BECN1 transcription and Beclin1-dependent autophagy during osteoclastogenesis by inhibiting NF-κB p65 activation.

As an active substance isolated from the root of Morinda officinalis How., rubiadin-1-methyl ether (RBM), can improve osteoporosis due to its inhibition on osteoclastogenesis. Autophagy plays a key role in osteoclastogenesis. Our research aims to explore the relationship between RBM, autophagy, and osteoclastogenesis. Our results showed that RBM not only inhibited the differentiation level of osteoclasts and the proliferation ability of osteoclast precursors (OCPs), but also repressed the autophagic activity in OCPs (LC3 transformation and the number of autophagosomes observed by transmission electron microscopy). However, RBM-inhibited osteoclast differentiation and OCP autophagy (LC3 transformation and LC3-puncta formation) could be reversed by the application of TAT-Beclin1. Moreover, RBM administration reduced RANKL-induced p65 phosphorylation and p65 nuclear translocation in OCPs. In addition, the addition of RBM inhibited Beclin1 protein level and BECN1 (the gene form of Beclin1) mRNA level in OCPs increased by RANKL. Importantly, the reduction in the expression of BECN1 and Beclin1, LC3 transformation, and osteoclastic differentiation in OCPs caused by RBM were reversed by p65 overexpression. In conclusion, RBM may reduce the transcription of BECN1 by inhibiting the activation of nuclear factor kappa B (NF-κB) p65, thereby inhibiting Beclin1-dependent autophagy and RANKL-induced osteoclastogenesis.

Bisphenol A-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression by Syk activation

The widely used plasticizer bisphenol A (BPA) is known as an endocrine-disrupting chemical (EDC). Many studies have shown that BPA contributes to diseases involving immune system alterations, but the underlying mechanisms have yet to be elucidated. We previously reported that BPA at concentration of 100 μM caused human B cell death in accordance with an increase in nuclear factor (erythroid-derived 2)-like 2(Nrf2) expression. Autophagy is a cellular process that degraded and recycles cytoplasmic constituents. Here, we investigated whether BPA induces autophagy through Nrf2, which is associated with regulation of B cell death using human WiL2-NS lymphoblast B cells. Then, cell viability was assessed by various assays using trypan blue, MTT or Celltiter glo luminescent substrate and DAPI. When WiL2-NS cells were treated with BPA, cell viability was decreased and LC3 autophagy cargo protein/puncta was increased. BPA-induced autophagy was confirmed by the modification of LC3 puncta formation or autophagy flux turnover with the treatment of hydroxychloroquine(HCQ), NH4Cl and PI3K inhibitors including 3-methyladenine(3-MA), LY294002 and wortmannin. BPA treatment increased the expression of autophagy-related gene(Atg)7 and Beclin1 as well as Nrf2 induced by the production of reactive oxygen species (ROS). The inhibition of autophagy with siAtg7 or siBeclin1 and Nrf2 depletion aggravated BPA-induced cell death. BPA enhanced the bound of Nrf2 to the specific region on Beclin1 and Atg7 promoter. Spleen tyrosine kinase(Syk) activity was enhanced in response to BPA treatment. Bay61–3606, Syk inhibitor, decreased LC3 and the expression of Atg7 and Beclin1, leading to the increase of BPA-induced B cell death. The results suggest that BPA-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression.

Lactate protects against oxidative stress-induced retinal degeneration by activating autophagy.

Age-related macular degeneration is a common cause of blindless among the aged, which can mainly be attributed to oxidative stress and dysregulated autophagy in retinal pigment epithelium cells. Lactate was reported to act as a signaling molecule and exerted beneficial effect against oxidative stress. This study aims to investigate the protective effect of lactate against oxidative stress-induced retinal degeneration. Here, H2O2-induced oxidative stress cell model and sodium iodate-induced mice retinal degeneration model were established. It was found that H2O2 inhibited cell viability in ARPE-19cells and sodium iodate induced deterioration of retinal pigment epithelium as well as apoptosis in retina. Pretreatment with lactate alleviated oxidative stress-induced cell death and retinal degeneration. Molecularly, lactate activated autophagy by up-regulating the ratio of LC3II/I, increased formation of LC3 puncta and autophagic vacuole. Further, lactate prevented H2O2-induced mitochondrial fission and maintained mitochondrial function by alleviating H2O2-induced mitochondrial membrane potential disruption and intracellular ROS generation. In contrast, application of 3-methyladenine, an inhibitor of autophagy, effectively weakened the protective effect of lactate against oxidative stress in vivo and in vitro. Taken together, all data in this study indicate that lactate protects against oxidative stress-induced retinal degeneration and preserves mitochondrial function by activating autophagy.

TAT-Beclin 1 represses the carcinogenesis of DUSP4-positive PTC by enhancing autophagy.

DUSP4 is a pro-tumorigenic molecule of papillary thyroid carcinoma (PTC). DUSP4 also exists as an autophagic regulator. Moreover, DUSP4, as a negative regulator of MAPK, can prevent Beclin 1 from participating in autophagic response. This study aimed to explore whether TAT-Beclin 1, a recombinant protein of Beclin 1, could inhibit the tumorigenesis of DUSP4-positive PTC by regulating autophagy. First, we divided PTC tissues into three groups according to DUSP4 expression levels by immunohistochemical analyses, and evaluated the relationship between autophagic molecules (Beclin 1 and LC3II) and DUSP4 using Western blotting assays. After overexpression of DUSP4 by lentiviral transduction, the in vitro and in vivo roles of TAT-Beclin 1 on DUSP4-overexpressed PTC cells were assessed (including autophagic activity, cell survival and function, and tumor growth). The roles of TAT-Beclin 1 in the survival of DUSP4-silenced PTC cells were also evaluated. Our results showed that the expression levels of autophagic proteins decreased with the increase of DUSP4 expression in PTC tissues. In PTC cells, DUSP4 overexpression-inhibited autophagic activity (including Beclin 1 expression, LC3 conversion rate and LC3-puncta formation) and -promoted cell proliferation and migration were reversed by TAT-Beclin 1 administration. In vivo assays also showed that DUSP4-overexpressed PTC cells had stronger tumorigenic ability and weaker autophagic activity, which was blocked by TAT-Beclin 1 administration. TAT-Beclin 1, as an autophagic promoter, could repress the carcinogenesis of DUSP4-positive PTC, which implies that the use of TAT-Beclin 1 for the PTC patients' treatment might be determined according to the DUSP4 level in their tumors.

Cathelicidin LL-37 Activates Human Keratinocyte Autophagy through the P2X₇, Mechanistic Target of Rapamycin, and MAPK Pathways

Human cathelicidin LL-37 is a multifunctional antimicrobial peptide that exhibits antimicrobial and immunomodulatory activities. LL-37 regulates skin barrier function and was recently reported to activate autophagy in macrophages. Because autophagy deficiency is associated with skin diseases characterized by a dysfunctional epidermal barrier, we hypothesized that LL-37 might regulate the skin barrier through autophagy modulation. We showed that LL-37 activated autophagy in human keratinocytes and three-dimensional skin equivalent models as indicated by increases in LC3 puncta formation, decreases in p62, and autophagosome and autolysosome formation. LL-37‒induced autophagy was suppressed by P2X7 receptor, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 inhibitors, suggesting that the P2X7, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 pathways are involved. Moreover, LL-37 enhanced the phosphorylation of adenosine monophosphate‒activated protein kinase and unc-51-like kinase 1. In addition, LL-37‒mediated autophagy involves the mechanistic target of rapamycin and MAPK pathways. Interestingly, the LL-37‒induced distribution of tight junction proteins and improvement in the tight junction barrier were inhibited in autophagy-deficient keratinocytes and keratinocytes and skin models treated with autophagy inhibitors, indicating that the LL-37‒mediated tight junction barrier is associated with autophagy activation. Collectively, these findings suggest that LL-37 is a potential therapeutic target for skin diseases characterized by dysfunctional autophagy and skin barriers.

Rab11a promotes the malignant progression of ovarian cancer by inducing autophagy.

Rab11a is a novel identified tumorigenic factor involved in different cancers. This study aimed to assess the biological function of Rab11a in ovarian cancer (OC). GEPIA database and real-time PCR were used to determine Rab11a expression in OC tissues and normal ovarian tissues. CCK-8, cell cycle, wound healing, transwell, and enzyme linked immunosorbent assay were used to detect the effects of Rab11a knockdown or overexpression on the proliferation, migration, and invasion of OC cells. Western blot analysis of autophagy-related markers and immunofluorescence staining of LC3 were performed to determine autophagy induction in Rab11a-silenced or overexpressed OC cells. Moreover, autophagy inhibitor 3-MA was employed to clarify the effects of Rab11a-regulated autophagy on the malignant phenotypes of OC cells. The mRNA level of Rab11a was increased in tumor tissues from OC patients as compared to the normal ovarian tissues. Knockdown of Rab11a in OVCAR-3 cells inhibited the growth of OC cells and led to cell cycle arrest, accompanied by reduced expression of PCNA and Cyclin D1. Rab11a deficiency suppressed migration and invasion of OC cells, accompanied by decreased secretion of MMP-2 and MMP-9. Silence of Rab11a impeded autophagy induction, as evidenced by decreased LC3 puncta formation, reduced abundance of LC3II and Beclin1, and increased p62 protein expression. In contrast, the ectopic expression of Rab11a in A2780 cells exerted opposite effects. Interestingly, autophagy inhibitor 3-MA abolished the effects of Rab11a overexpression on autophagy, proliferation, migration, and invasion. Rab11a promotes the malignant phenotypes of OC cells by inducing autophagy.

Beclin1- and Atg13-dependent autophagy activation and morroniside have synergistic effect on osteoblastogenesis.

Morroniside is known to improve osteoporosis by promoting osteoblastogenesis. The activation of PI3K/Akt/mTOR signaling is a significant mechanism in morroniside-promoted osteoblastogenesis. It is well known that protective autophagy is an important factor in osteoblastogenesis. However, the activation of mTOR signaling can inhibit autophagy. This study aimed to investigate the relationship between mTOR signaling and autophagy in morroniside-regulated osteoblastogenesis. In this study, we investigated the effect of morroniside on the autophagic activity (LC3 conversion rate, LC3-puncta formation, and autophagosome number) of differentiated osteoblast precursors (MC3T3-E1 cells). Then, we identified the roles of mTOR knockdown in morroniside-regulated alterations of autophagy and osteogenic parameters in MC3T3-E1 cells. Next, mTOR knockdown and overexpression were used to observe the roles of mTOR in morroniside-regulated alterations of autophagic molecules (Atg7, Atg13, and Beclin1). Subsequently, the additional value of the above autophagic molecules on morroniside-regulated osteogenic parameters in MC3T3-E1 cells was analyzed based on lentiviral transduction. Finally, combined with morroniside and TAT-Beclin1, the roles of Beclin1 upregulation in the in vivo effects of morroniside was investigated. Our experimental data showed that morroniside promoted both the mTOR activity and autophagy in MC3T3-E1 cells. Morroniside-upregulated autophagic activity and Atg13 or Beclin1 protein level in MC3T3-E1 cells were enhanced by mTOR knockdown. Furthermore, Morroniside-upregulated Atg13 and Beclin1 expression was reversed by mTOR overexpression. Importantly, autophagy upregulation with overexpression of the autophagic gene, Atg13 or BECN1 (gene form of Beclin1), significantly promoted osteoblastogenesis regulated by morroniside. The promotional effect of morroniside on bone microarchitecture, bone mass, and bone parameters (including trabecular bone area and OCN expression in trabecular bone) in ovariectomized (OVX) mice was enhanced by TAT-Beclin1 administration. In conclusion, the autophagy-enhancing drugs related to Beclin1 or Atg13 may be an effective adjuvant therapy in the treatment of osteoporosis with morroniside.

TFEB signaling attenuates NLRP3-driven inflammatory responses in severe asthma.

NLRP3-driven inflammatory responses by circulating and lung-resident monocytes are critical drivers of asthma pathogenesis. Autophagy restrains NLRP3-induced monocyte activation in asthma models. Yet, the effects of autophagy and its master regulator, transcription factor EB (TFEB), on monocyte responses in human asthma remain unexplored. Here, we investigated whether activation of autophagy and TFEB signaling suppress inflammatory monocyte responses in asthmatic individuals. Peripheral blood CD14+ monocytes from asthmatic patients (n=83) and healthy controls (n=46) were stimulated with LPS/ATP to induce NLRP3 activation with or without the autophagy inducer, rapamycin. ASC specks, caspase-1 activation, IL-1β and IL-18 levels, mitochondrial function, ROS release, and mTORC1 signaling were examined. Autophagy was evaluated by LC3 puncta formation, p62/SQSTM1 degradation and TFEB activation. In a severe asthma (SA) model, we investigated the role of NLRP3 signaling using Nlrp3-/- mice and/or MCC950 administration, and the effects of TFEB activation using myeloid-specific TFEB-overexpressing mice or administration of the TFEB activator, trehalose. We observed increased NLRP3 inflammasome activation, concomitant with impaired autophagy in circulating monocytes that correlated with asthma severity. SA patients also exhibited mitochondrial dysfunction and ROS accumulation. Autophagy failed to inhibit NLRP3-driven monocyte responses, due to defective TFEB activation and excessive mTORC1 signaling. NLRP3 blockade restrained inflammatory cytokine release and linked airway disease. TFEB activation restored impaired autophagy, attenuated NLRP3-driven pulmonary inflammation, and ameliorated SA phenotype. Our studies uncover a crucial role for TFEB-mediated reprogramming of monocyte inflammatory responses, raising the prospect that this pathway can be therapeutically harnessed for the management of SA.

Evaluation of anti-cancer potential of Excoecaria\xa0agallocha (L.) leaf extract on human cervical cancer (SiHa) cell line and assessing the underlying mechanism of action

BackgroundThe incidence of cervical cancer is increasing at an alarming rate in many countries and presently, it is the most common form of malignant cancer being reported among women in India. Development of novel approach for cervical cancer therapy, sparing healthy normal cells overcoming the limitations of prevailing therapies is of prime importance. Mangroves constitute a significant repository of medicinally important plants. Thus, in this study, we aimed to determine the anticancer activity of the mangrove Excoecaria agallocha L. leaf extracts on human cervical cancer (SiHa HPV 16+) cell line with subsequent characterization of the bioactive compounds conferring the anticancer activity and studying the probable underlying mechanism of action of the purified plant extract.ResultsThe plant extract was subjected to silica gel column chromatography and the fractions obtained were analyzed for cytotoxic activity against SiHa cells by MTT assay. One out of the three eluted fractions exhibited selective toxicity against SiHa cells with an IC50 value of 15.538 ± 0.577 µg/mL, while it had no cytotoxic effect on normal healthy human peripheral blood mononuclear cells. High-resolution liquid chromatography mass spectroscopy, coupled to electron spray ionization and diode array detection analysis, led to the structure elucidation and identification of a few pharmacologically important compounds, with Bergenin being present in the highest abundance. Fluorescence microscopy results revealed that the plant extract fraction induced LC3 puncta formation, in EGFP- SiHa cells indicating the onset of autophagy, with simultaneous stimulation of mitophagy. The plant extract also inhibited proliferation of the SiHa-smac-mCherry cells by second mitochondria-derived activator of caspase (SMAC)—induced cytochrome c dependent apoptosis, that was further confirmed with Caspase-3 activation by colorimetric assay. The GFP-dgn in SiHa cells was remarkably protected from proteasomal degradation that might upregulate the survivability of the cells significantly. Flow cytometry followed by Western blot analysis further asserted the ability of the plant extract fraction to cause cell cycle arrest of SiHa cells in the G2/M phase by significantly reducing protein expression levels of cyclin B1 and D1, decreasing Cdc2 level and simultaneously increasing p21 and p53 levels.ConclusionIt could be inferred that the aqueous extract of E. agallocha successfully decreased the proliferation of SiHa cervical cancer cells through induction of autophagy and apoptosis in a concerted manner, with simultaneous stimulation of mitophagy and G2/M phase cell cycle arrest, hinting at Bergenin being the major compound conferring the anti-cancer activity of the plant extract. Thus, isolation of the identified bioactive compounds from E. agallocha and their subsequent purification for drug development might serve as a novel medicinal approach for the treatment of cervical cancer in conjugation with existing therapeutic methods.

Hexokinase 2 Is a Pivot for Lovastatin-induced Glycolysis-to-Autophagy Reprogramming in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited therapeutic options available. We have recently demonstrated that lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, suppresses TNBC cell proliferation and stemness properties in vitro and in vivo. However, the mechanisms through which lovastatin inhibits TNBC cells are not fully understood. Here, we used 1H NMR-based metabolomic profiling to investigate lovastatin-induced metabolic changes in TNBC cell line MDA-MB-231. Among the 46 metabolites identified, lactate demonstrated the highest variable importance in projection (VIP) score. Glycolysis stress test revealed that lovastatin significantly decreased the extracellular acidification rate (ECAR) in MDA-MB-231 cells. Furthermore, lovastatin treatment down-regulated the levels of glycolysis-related proteins including GLUT1, PFK1, and PKM2 in MDA-MB-231 but not non-TNBC MDA-MB-453 cells. In addition, lovastatin induced autophagy as evidenced by increased LC3 puncta formation and LC3-II/I ratio, increased AMPK phosphorylation, and decreased Akt phosphorylation. We also revealed the interaction between the glycolytic enzyme hexokinase 2 (HK2) and the mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1), an important regulator of autophagy. Further bioinformatics analysis revealed that VDAC1 was expressed at a higher level in breast cancer than normal tissues and higher level of VDAC1 predicted poorer survival outcomes in breast cancer patients. The present study suggests that lovastatin might exert anti-tumor activity by reprogramming glycolysis toward autophagy in TNBC cells through HK2-VDAC1 interaction.

Hydroxychloroquine synergizes with the PI3K inhibitor BKM120 to exhibit antitumor efficacy independent of autophagy

BackgroundThe critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer.MethodsThe antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining.ResultsHCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51.ConclusionsOur study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the “off target” effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer.

GRASP55 restricts early-stage autophagy and regulates spatial organization of the early secretory network.

ABSTRACTThere is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)­­-Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.

Roles of autophagy in relation to mitochondrial stress responses of HeLa cells to lamellarin cytotoxicity

As a promising class of bioactive marine pyrrole alkaloids, lamellarins reportedly act on multiple targets to suppress the vitality of various cancer cell lines. Nevertheless, an in-depth understanding of the molecular mechanisms governing their cytotoxicity is still in demand. Here we report that while activating intrinsic apoptosis, up to 5 μM of lamellarins and their lactam-containing analogs, azalamellarins, also induced mitochondrial stress responses and autophagy in HeLa cervical cancer cells. Detailed characterization of the mitochondria in the treated cells revealed shifted abundance of the two optic atrophy protein 1 (Opa1) isoforms, disturbed morphology, and dissipated membrane potential, leading to PTEN-induced kinase-1 (PINK1) and microtubule-associated protein 1 light chain 3-II (LC3-II) accumulation as a molecular signature of mitophagy. Furthermore, an acute treatment with lamellarins also modulated cellular autophagy flux as evidenced by elevated LC3-II levels, LC3 puncta formation, and p62 degradation. Surprisingly, clustered regularly interspaced short palindromic repeats (CRISPR)-based suppression of autophagy transiently affected the number of apoptotic cells induced by these compounds. Our findings illustrate the potential of these alkaloids for further development into prospective anti-cancer agents.

DUSP4 promotes the carcinogenesis of CCRCC via negative regulation of autophagic death.

DUSP4 is considered as an oncogenic gene. However, the effect of DUSP4 on the carcinogenesis of clear cell Renal cell carcinoma (CCRCC) is still unclear. In this study, DUSP4 mRNA levels were significantly increased in CCRCC tissues and cell lines. Furthermore, DUSP4 overexpression promotes the proliferation, migration, and tumorigenicity of CCRCC cells while DUSP4 silencing showed the opposite effects. Importantly, both autophagic activity (LC3 conversion rate and LC3 puncta formation) and total death level promoted by DUSP4 silencing were reversed by treatment with 3-MA in CCRCC cells. Moreover, the proliferation and migration of CCRCC cells inhibited by DUSP4 silencing were also recovered by suppression of autophagy with 3-MA. In conclusion, DUSP4 serves as an oncogenic gene in CCRCC carcinogenesis due to its inhibitory effect on autophagic death, indicating the potential value of DUSP4 in the diagnosis and treatment of CCRCC.

Pharmacological Inhibition of O-GlcNAc Transferase Promotes mTOR-Dependent Autophagy in Rat Cortical Neurons.

O-GlcNAc transferase (OGT) is a ubiquitous enzyme that regulates the addition of β-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of target proteins. Autophagy is a cellular process of self-digestion, in which cytoplasmic resources, such as aggregate proteins, toxic compounds, damaged organelles, mitochondria, and lipid molecules, are degraded and recycled. Here, we examined how three different OGT inhibitors, alloxan, BXZ2, and OSMI-1, modulate O-GlcNAcylation in rat cortical neurons, and their autophagic effects were determined by immunoblot and immunofluorescence assays. We found that the treatment of cortical neurons with an OGT inhibitor decreased O-GlcNAcylation levels and increased LC3-II expression. Interestingly, the pre-treatment with rapamycin, an mTOR inhibitor, further increased the expression levels of LC3-II induced by OGT inhibition, implicating the involvement of mTOR signaling in O-GlcNAcylation-dependent autophagy. In contrast, OGT inhibitor-mediated autophagy was significantly attenuated by 3-methyladenine (3-MA), a blocker of autophagosome formation. However, when pre-treated with chloroquine (CQ), a lysosomotropic agent and a late-stage autophagy inhibitor, OGT inhibitors significantly increased LC3-II levels along with LC3 puncta formation, indicating the stimulation of autophagic flux. Lastly, we found that OGT inhibitors significantly decreased the levels of the autophagy substrate p62/SQSTM1 while increasing the expression of lysosome-associated membrane protein 1 (LAMP1). Together, our study reveals that the modulation of O-GlcNAcylation by OGT inhibition regulates mTOR-dependent autophagy in rat cortical neurons.

Soybean lectin induces autophagy through P2RX7 dependent activation of NF-κB-ROS pathway to kill intracellular mycobacteria

Background: Host-directed therapy is considered a novel anti-tuberculosis strategy in tackling the tuberculosis burden through autophagy induction by various inducers to curtail the growth of intracellular Mycobacterium tuberculosis.Methods: In this study, we investigated the anti-tubercular role of soybean lectin, a lectin isolated from Glycine max (Soybean). Effect of SBL on intracellular mycobacterial viability through autophagy and the mechanism involved in differentiated THP-1 cells was studied using different experimental approaches.Results: We initially performed a time kinetic experiment with the non-cytotoxic dose of SBL (20 μg/ml) and observed autophagy induction after 24 h of treatment. Abrogation of autophagy in the presence of 3-MA and an increase in LC3 puncta formation upon Baf-A1 addition elucidated the specific effect on autophagy and autophagic flux. SBL treatment also led to autophagy induction in mycobacteria infected macrophages that restricted the intracellular mycobacterial growth, thus emphasizing the host defensive role of SBL induced autophagy. Mechanistic studies revealed an increase in P2RX7 expression, NF-κB activation and reactive oxygen species generation upon SBL treatment. Inhibition of P2RX7 expression suppressed NF-κB dependent ROS level in SBL treated cells. Moreover, SBL induced autophagy was abrogated in the presence of either different inhibitors or P2RX7 siRNA, leading to the reduced killing of intracellular mycobacteria.Conclusion: Taken together, these results conclude that SBL induced autophagy exerts an anti-mycobacterial effect in P2RX7-NF-κB dependent manner through the generation of ROS.General significance: This study has provided a novel anti-mycobacterial role of SBL, which may play an important role in devising new therapeutic interventions.

Enteromorpha compressa extract induces anticancer activity through apoptosis and autophagy in oral cancer.

Marine algae are an auspicious source of innovative bioactive compounds containing possible therapeutic agents against mammalian cancers. However, the mechanism by which bioactive algal compounds exhibit anticancer activity against oral squamous cell carcinoma (OSCC) is scant. The main objective of the current study was to explore the properties of the Enteromorpha compressa solvent extracts that induced autophagy and apoptosis with reference to their potent phytochemical and antioxidant properties. The presence of bioactive compounds were confirmed by UV and FT-IR spectroscopy. The free radical scavenging activity were analyzed by evaluating H2O2, DPPH, superoxide and hydroxyl activity. The anticancer activities of the extracts were investigated by employing clonogenic and scratch assay. The apoptosis potential was evaluated by DAPI and MMP by Rh123 fluorescence assay. Moreover, the CAT, SOD, GPX, APX, and GR activities were measured. The autophagy potential was evaluated by LC3 puncta formation, acridine orange in addition to LysoTracker staining. The present investigation revealed that the methanolic extract of E. compressa elicited robust free radical scavenging activity that discerns its antiproliferative potency. Moreover, the methanolic algal extract boosted intrinsic apoptosis against OSCC by downregulating protective antioxidant enzymes. Furthermore, it also revealed induction of autophagy to promote cell death in oral cancer cells. The presence of novel bioactive compounds in E. compressa has uncovered possible therapeutic value against OSCC by modulating antioxidant defense system, apoptosis and autophagy that could be used to explore very competent algal candidates for the development of potential alternative anticancer drugs.

Abscisic Acid-Induced Autophagy Selectively via MAPK/JNK Signalling Pathway in Glioblastoma

As a widely known plant hormone, Abscisic acid plays an important role in the progress of planting cell and their stress response. Recently, we reported that ABA might play an anti-cancer role in glioma tissues. In the present study, the molecular mechanism of ABA anti-cancer was further explored in glioblastoma cells. By measuring LC3 puncta formation and conversion in glioblastoma cells, inhibiting the autophagic pathway, targeting the essential autophagic modulator beclin 1 with RNA interference, and analysing cellular morphology via transmission electron microscopy, we found that ABA-treated glioblastoma cells exhibited the features of autophagy. Specifically, ABA-induced autophagy in glioblastoma cells was mediated by the MAPK/JNK signalling pathway rather than the PI3K/AKT/mTOR axis. Moreover, the inhibition or knockdown of JNK specifically blocked ABA-induced autophagic cell death. ABA-induced autophagy was further confirmed in tumour-bearing mice and was accompanied by the inhibition of glioma growth in vivo. This report is the first to describe autophagy induced by ABA and mediated by the MAPK/JNK pathway in human cancer cells and tumour-bearing mice. These results may shed some light in new therapeutic strategies of glioma.