Autophagic Death Research Articles

Autophagy activator-loaded bicomponent peptide nanocarriers for phototherapy-triggered immunity enhancement against metastatic breast cancer

Mild autophagy accompanied with immunogenic cell death (ICD) effect destructs immune-associated antigens, weakening the immune response against tumor growth. To address this dilemma, we develop a peptide-based bicomponent nanocarrier with encapsulation of a cellular hyperautophagy activator (STF-62247) for near-infrared (NIR) photo/immunotherapy to eliminate primary and metastatic breast tumors. The electrostatic-driven nanodrug (PPNPs@STF) with active-targeting and efficient endosomal escape can induce specific ICD effect upon NIR laser irradiation, and trigger autophagy to a mild activation state. Notably, the simultaneously released STF-62247 precisely promotes autophagy to an overactivated state, resulting in autophagic death of tumor cells and further boosting ICD-related antigen presentation. More importantly, the combined photo/immunotherapy of PPNPs@STF not only inhibits tumor cell proliferation, but also promotes dendritic cells (DCs)-associated immune response. In 4 T1 tumor-bearing mice, PPNPs@STF effectively inhibits growth of primary and distant tumors, and suppresses lung metastasis with a minimized side effect. This study provides a hyperautophagy activator-assisted strategy that can enhance ICD-based antitumor immune response for the treatment of metastatic breast cancer.

Vanillin Mitigates the MPTP-Induced α-Synucleinopathy in a Mouse Model of Parkinson's Disease: Insights into the Involvement of Wnt/β-Catenin Signaling.

The abnormal aggregation of α-synuclein (α-syn) in the substantia nigra pars compacta (SNpc) region of the brain is characteristic of Parkinson's disease (PD), leading to the selective demise of neurons. Modifications in the post-translational processing of α-syn, phosphorylation at Ser129 in particular, are implicated in α-syn aggregation and are considered key hallmarks of PD. Furthermore, dysregulated Wnt/β-catenin signaling, influenced by glycogen synthase kinase-3 beta (GSK-3β), is implicated in PD pathogenesis. Inhibition of GSK-3β holds promise in promoting neuroprotection by enhancing the Wnt/β-catenin pathway. In our previous study utilizing 1-methyl-4-phenylpyridinium (MPP+)-administered differentiated SH-SY5Y cells and a PD mouse model, we explored Vanillin's neuroprotective properties and related mechanisms against neuronal loss induced by MPP+/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. In the current study, we elucidated the mitigating effects of Vanillin on motor impairments, P-Ser129-α-syn expression, Wnt/β-catenin signaling, and autophagic neuron death induced by MPTP in a mouse model of PD by performing motor function tests, western blot analysis and immunostaining. Our results show that Vanillin effectively modulated the motor dysfunctions, GSK-3β expression, and activity, activated the Wnt/β-catenin signaling, and reduced autophagic neuronal demise in the MPTP-lesioned mice, highlighting its neuroprotective effects. These findings underscore the complex interplay between α-syn pathology, GSK-3β, Wnt/β-catenin signaling, and autophagic-cell death in PD pathogenesis. Targeting these pathways, particularly with Vanillin, can be a promising therapeutic strategy for restoring dopaminergic (DA-ergic) neuronal homeostasis and slowing the progression of PD. Further research is crucial to resolving existing disputes and translating these discoveries into effective therapeutic interventions for PD patients.

Simvastatin exerts neuroprotective effects post-stroke by ameliorating endoplasmic reticulum stress and regulating autophagy/apoptosis balance through pAMPK/LC3B/ LAMP2 axis

Statins have evident neuroprotective role in acute ischemic stroke(AIS). The pleiotropic effect by which statin exerts neuroprotective effects, needs to be explored for considering it as one of the future adjunctive therapies in AIS. Endoplasmic reticulum(ER) assists cellular survival by reducing protein aggregates during ischemic conditions. ER-stress mediated apoptosis and autophagy are predominant reasons for neuronal death in AIS. Statin exerts both anti-apoptotic and anti-autophagic effect in neurons under ischemic stress. Although the influence of statin on autophagic neuroprotection has been reported with contradictory results. Thus, in our study we have attempted to understand its influence on autophagic protection while inhibiting upregulation of autophagic death(autosis). Previously we reported, statin can alleviate apoptosis via modulating cardiolipin mediated mitochondrial dysfunction. However, the clearance of damaged mitochondria is essential for prolonged cell survival. In our study, we tried to decipher the mechanism by which statin leads to neuronal survival by the mitophagy mediated cellular clearance. Simvastatin was administered to Sprague Dawley(SD) rats both as prophylaxis and treatment. The safety and efficacy of the statin was validated by assessment of infarct size and functional outcome. A reduction in oxidative and ER-stress were observed in both the prophylactic and treatment groups. The influence of statin on autophagy/apoptosis balance was evaluated by molecular assessment of mitophagy and cellular apoptosis. Statin reduces the post-stroke ER-stress and predominantly upregulated autophagolysosome mediated mitophagy than apoptotic cell death by modulating pAMPK/LC3B/LAMP2 axis. Based on the above findings statin could be explored as an adjunctive therapy for AIS in future.

The pro-differentiating capability of a flavonoid-rich extract of Citrus bergamia juice prompts autophagic death in THP-1 cells

Acute myeloid leukemia (AML) is a hematologic neoplasm, characterized by a blockage of differentiation and an unconstrained proliferation of immature myeloid cells. Recently, the survival of leukemia patients has increased thanks to the use of differentiating agents, though these may cause serious side effects. Hence, the search for safer differentiating compounds is necessary. Our aim was to assess the pro-differentiating effects of a flavonoid-rich extract of bergamot juice (BJe) in human monocytic leukemia THP-1 cells, an in vitro AML model. For the first time, we showed that treatment with BJe induced differentiation of THP-1 cells, changes in cell morphology and increased expression of differentiation-associated surface antigens CD68, CD11b and CD14. Moreover, BJe enhanced protein levels of autophagy-associated markers, such as Beclin-1 and LC3, as well as induced the phosphorylation of the MAPKs JNK, ERK and p38, hence suggesting a potential mechanism underlying its antiproliferative effects. Indeed, parallel experiments highlighted that BJe was able to hamper THP-1 cell growth. In conclusion, our study suggests that BJe induces the differentiation of THP-1 cells and reduces their proliferation, highlighting its potential in differentiation therapy of AML.

Synthesis of novel 4-substituted isatin Schiff base derivatives as potential autophagy inducers and evaluation of their antitumour activity.

Induction of autophagic death in cancer cells is one of the promising strategies for the development of anti-cancer therapeutics. In the present study, we designed and synthesized a series of isatin Schiff base derivatives containing thioether structures. After discovering the highly active target compound H13 (IC50 = 4.83μM) based on in vitro antiproliferation, we also found it had a high safety against normal cells HEK293 with CC50 of 69.01μM, indicating a sufficient therapeutic window. In addition, to provide reference for subsequent studies, a model was successfully constructed by Sybyl software. Preliminary mechanistic studies suggested that H13-induced apoptosis may be closely related to ROS accumulation and mitochondrial dysfunction. Subsequent studies revealed that H13 inhibited cell proliferation by inducing cellular autophagy mainly through blocking signal of the PI3K/AKT/mTOR pathway. Altogether, these results suggested that H13 was potentially valuable as a lead compound.

Immune suppressive drugs negatively regulate the CD8+T cells function by acetyltransferase p300 induced canonical and non-canonical autophagy

Macroautophagy, the mainly regulated form of autophagy, maintains the cellular homeostasis and degrades the transported cargoes. It is initiated by the protein kinase complex regulating by two signals pathway Mammalian target of rapamycin complex 1 (mTORC1)-Adenosine 5‘ monophosphate activated protein kinase (AMPK)-Unc 51 like kinase 1(ULK1) and ULK1-PI3K- phosphatidylinositol 3-phosphate (PI3P). Currently, autolysosomes are accumulated during the aging process of CD8+T cells in vitro and may participate in inducing death sensitization of senescent cells. The main mechanism of aplastic anemia, a hyperimmune disease, is the T cells subsets imbalance such as CD8+T cells abnormal activation and hyperfunction. Therefore, the role of autophagy in the CD8+T cells and supposed whether some immunosuppress drugs induced the cells autophagic death to treat the hyperimmune diseases were focused. It was decided found that the acetyltransferase p300 obviously increased in the aplastic anemia patients and was related with the severity of disease. Previous studies have reported that canonical autophagy is regulated by the mTORC1-p300 axis. p300 is a critical bridge in the p300-VPS34 axis mediated non-canonical autophagy. There is the deficiency of autophagy and acetylation in the CD8+T cells. The expression of p300 also decreased notably after the immunosuppressive drugs therapy. Our findings provide a framework for understanding how immunosuppressive drugs effect on the AA autophagy deficiency mechanism and proved that immunosuppressive drugs negatively regulated the function of CD8+T cells by p300-mediated canonical autophagy pathway and non-canonical autophagy pathway.

The Autophagic and Apoptotic Death of Forebrain Neurons of Rats with Global Brain Ischemia Is Diminished by the Intranasal Administration of Insulin: Possible Mechanism of Its Action.

Insulin is a promising neuroprotector. To better understand the mechanism of insulin action, it was important to show its ability to diminish autophagic neuronal death in animals with brain ischemic and reperfusion injury. In forebrain ischemia and reperfusion, the number of live neurons in the hippocampal CA1 region and frontal cortex of rats decreased to a large extent. Intracerebroventricular administration of the autophagy and apoptosis inhibitors to ischemic rats significantly increased the number of live neurons and showed that the main part of neurons died from autophagy and apoptosis. Intranasal administration of 0.5 IU of insulin per rat (before ischemia and daily during reperfusion) increased the number of live neurons in the hippocampal CA1 region and frontal brain cortex. In addition, insulin significantly diminished the level of autophagic marker LC3B-II in these forebrain regions, which markedly increased during ischemia and reperfusion. Our studies demonstrated for the first time the ability of insulin to decrease autophagic neuronal death, caused by brain ischemia and reperfusion. Insulin administered intranasally activated the Akt-kinase (activating the mTORC1 complex, which inhibits autophagy) and inhibited the AMP-activated protein kinase (which activates autophagy) in the hippocampus and frontal cortex of rats with brain ischemia and reperfusion.

The ameliorative effects of chrysin on bortezomib-induced nephrotoxicity in rats: Reduces oxidative stress, endoplasmic reticulum stress, inflammation damage, apoptotic and autophagic death

AimBortezomib is a proteasome inhibitor antineoplastic agent that was the first to be approved for cancer treatment. One of bortezomib's most prominent dose-limiting effects is nephrotoxicity; the underlying mechanism is believed to be oxidative stress. Chrysin is a compound found actively in honey and many plant species and stands out with its antioxidant properties. The present study aimed to determine the ameliorative effects of chrysin in bortezomib-induced nephrotoxicity. Material-methodThirty-five male Wistar rats were divided into control, BTZ, CHR, BTZ + CHR25, and BTZ + CHR50. Biochemical, molecular, Western blot, and histological methods analyzed renal function indicators, oxidative stress, endoplasmic reticulum stress, inflammation, apoptosis, and damage pathways. ResultsChrysin decreased oxidative stress by reducing oxidants (MDA) and increasing antioxidants (SOD, CAT, Gpx, GSH, Nrf-2, HO-1, NQO1). Chrysin reduced endoplasmic reticulum stress by decreasing ATF-6, PERK, IRE1, and GRP-78 levels. Chrysin reduced inflammation damage by inhibiting the NF-κB pathway. Chrysin exhibited protective properties against apoptotic damage by decreasing Bax and Caspase-3 levels and increasing Bcl-2 levels. In addition, chrysin improved renal function and structural integrity and exhibited healing properties against toxic damage in tissue structure. ConclusionOverall, chrysin exhibited an ameliorative effect against bortezomib-induced nephrotoxicity.

Neuronal autosis is Na+/K+-ATPase alpha 3-dependent and involved in hypoxic-ischemic neuronal death

Macroautophagy (hereafter called autophagy) is an essential physiological process of degradation of organelles and long-lived proteins. The discovery of autosis, a Na+/K+-ATPase (ATP1)-dependent type of autophagic cell death with specific morphological and biochemical features, has strongly contributed to the acceptance of a pro-death role of autophagy. However, the occurrence and relevance of autosis in neurons has never been clearly investigated, whereas we previously provided evidence that autophagy mechanisms could be involved in neuronal death in different in vitro and in vivo rodent models of hypoxia-ischemia (HI) and that morphological features of autosis were observed in dying neurons following rat perinatal cerebral HI. In the present study, we demonstrated that neuronal autosis could occur in primary cortical neurons using two different stimulations enhancing autophagy flux and neuronal death: a neurotoxic concentration of Tat-BECN1 (an autophagy-inducing peptide) and a hypoxic/excitotoxic stimulus (mimicking neuronal death induced by cerebral HI). Both stimulations induce autophagic neuronal death (dependent on canonical autophagic genes and independent on apoptotic, necroptotic or ferroptotic pathways) with all morphological and biochemical (ATP1a-dependent) features of autosis. However, we demonstrated that autosis is not dependent on the ubiquitous subunit ATP1a1 in neurons, as in dividing cell types, but on the neuronal specific ATP1a3 subunit. We also provided evidence that, in different in vitro and in vivo models where autosis is induced, ATP1a3-BECN1 interaction is increased and prevented by cardiac glycosides treatment. Interestingly, an increase in ATP1a3-BECN1 interaction is also detected in dying neurons in the autoptic brains of human newborns with severe hypoxic-ischemic encephalopathy (HIE). Altogether, these results suggest that ATP1a3-BECN1-dependent autosis could play an important role in neuronal death in HI conditions, paving the way for the development of new neuroprotective strategies in hypoxic-ischemic conditions including in severe case of human HIE.

SCFFBXW5-mediated degradation of AQP3 suppresses autophagic cell death through the PDPK1-AKT-MTOR axis in hepatocellular carcinoma cells

ABSTRACT AQP3 (aquaporin 3 (Gill blood group)), a member of the AQP family, is an aquaglyceroporin which transports water, glycerol and small solutes across the plasma membrane. Beyond its role in fluid transport, AQP3 plays a significant role in regulating various aspects of tumor cell behavior, including cell proliferation, migration, and invasion. Nevertheless, the underlying regulatory mechanism of AQP3 in tumors remains unclear. Here, for the first time, we report that AQP3 is a direct target for ubiquitination by the SCFFBXW5 complex. In addition, we revealed that downregulation of FBXW5 significantly induced AQP3 expression to prompt macroautophagic/autophagic cell death in hepatocellular carcinoma (HCC) cells. Mechanistically, AQP3 accumulation induced by FBXW5 knockdown led to the degradation of PDPK1/PDK1 in a lysosomal-dependent manner, thus inactivating the AKT-MTOR pathway and inducing autophagic death in HCC. Taken together, our findings revealed a previously undiscovered regulatory mechanism through which FBXW5 degraded AQP3 to suppress autophagic cell death via the PDPK1-AKT-MTOR axis in HCC cells. Abbreviation: BafA1: bafilomycin A1; CQ: chloroquine; CRL: CUL-Ring E3 ubiquitin ligases; FBXW5: F-box and WD repeat domain containing 5; HCC: hepatocellular carcinoma; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; 3-MA: 3-methyladenine; PDPK1/PDK1: 3-phosphoinositide dependent protein kinase 1; RBX1/ROC1: ring-box 1; SKP1: S-phase kinase associated protein 1; SCF: SKP1-CUL1-F-box protein.

The Class I Histone Deacetylase Inhibitor as a Potent Therapeutic Target for Treating Glioblastoma and Mocetinostat as a Novel Inhibitor in the Induction Death of Cancer Cells

Epigenetic abnormality is one of the hallmarks of glioblastoma cancer cells. Histone deacetylase (HDAC) modification has a crucial role in epigenetic abnormality, which results in the initiation and progression of glioblastoma cancer cells. The selective HDAC inhibitors are well-known epigenetic regulators and promising anti-cancer agents that target specific HDAC enzymes and inhibit the proliferation of many cancer cells. Selective HDAC inhibitors isoform provides a high efficacy as chemotherapy in inhibiting cancer confirmation compared to non-selective HDAC inhibitors. Additionally, selective HDAC inhibitors suppress Class -I HDAC1, HDAC2, HDAC3, and HDAC11. HDAC class I inhibitors induce apoptosis, differentiation, autophagic death cells, and reactive oxygen species (ROS)- induced cell death, inhibit cell migration, invasion, and angiogenesis in cancer cells, while the normal cells showed more resistance to HDAC class I inhibitors. Mocetinostat (MGCD0103), a benzamide histone deacetylase, is a potent anti-cancer therapy for the treatment of several cancer cell lines and induction of autophagy. It has been approved by The Food and Drug Administration (FDA) for the treatment of Hodgkin lymphoma (HL) cell lines. MGCD0103 is a synthesized and selective HDAC inhibitor that has vigorous inhibitory activity against Class-I and IV HDAC. MGCD0103 is well tolerated and has favorable pharmacokinetic properties, pharmacodynamic profile, and fast absorption within 1 hour after oral administration, long elimination half-life, and sustained HDAC inhibition. Therefore, MGCD0103 is expected to be a promising anti-cancer drug for treating several types of human cancer cells. Keywords: MGCD0103; Apoptosis; Differentiation; Gliblastoma; HDAC; inhibitors.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt

Ethnopharmacological relevanceLicorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. Aim of the studyThis study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. Materials and methodsSW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. ResultsWighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a “closed” conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. ConclusionWighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Gut bacteria-driven homovanillic acid alleviates depression by modulating synaptic integrity

The gut-brain axis is implicated in depression development, yet its underlying mechanism remains unclear. We observed depleted gut bacterial species, including Bifidobacterium longum and Roseburia intestinalis, and the neurotransmitter homovanillic acid (HVA) in individuals with depression and mouse depression models. Although R.intestinalis does not directly produce HVA, it enhances B.longum abundance, leading to HVA generation. This highlights a synergistic interaction among gut microbiota in regulating intestinal neurotransmitter production. Administering HVA, B.longum, or R.intestinalis to mouse models with chronic unpredictable mild stress (CUMS) and corticosterone (CORT)-induced depression significantly improved depressive symptoms. Mechanistically, HVA inhibited synaptic autophagic death by preventing excessive degradation of microtubule-associated protein 1 light chain 3 (LC3) and SQSTM1/p62 proteins, protecting hippocampal neurons' presynaptic membrane. These findings underscore the role of the gut microbial metabolism in modulating synaptic integrity and provide insights into potential novel treatment strategies for depression.

Abstract CT218: Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer

Abstract Background: Triptolide and its water-soluble prodrug, Minnelide™, have shown promising activity against pancreatic ductal adenocarcinoma (PDAC) in both in vitro and in vivo experiments. Triptolide induces apoptosis in pancreatic cancer cells by different pathways, including caspase-dependent apoptotic death and caspase -independent autophagic death. It inhibits the activity of the TFIIH general transcription factor complex and modulates the transcriptional landscape of both the stroma and cancer cell compartments. In the epithelial tumour compartment, triptolide down-regulates key transcription factors such as c-MYC. Preclinical investigations indicate significant activity against super enhancer targets. In the phase I clinical trial of Minnelide™ activity in highly refractory metastatic pancreatic carcinoma was observed. Hence, we undertook this study in patients with chemotherapy refractory metastatic PDAC. Our aims were to determine clinical anticancer effects, and to define effects of the drug on super enhancer expression and downstream effects. Methods: Eligibility criteria included stage IV PDAC progressing on standard chemotherapy, Karnofsky performance status ≥ 70%, and measurable disease by RECIST 1.1. Planned sample size was up to 35 patients with interim analysis after the first 18 evaluable patients, aiming for at least a 20% difference in DCR (10% v 30%). Minnelide™ 0.67 mg/m2 was administered intravenously over 30 minutes on days 1-21, followed by a 7-day rest period in a 28-day cycle. Exploratory objectives included: analyses of paired tumour biopsies for changes in immune cell infiltration, stromal activation, chromatin accessibility, proteomics and metabolism. Results: 17 patients were treated. There were no RECIST responses or stable disease. However, evidence of activity was observed in 4 of 15 evaluable patients. This activity included progression after 117 days treatment in 1 patient and reductions in CT tumour size or FDG-PET uptake and/or significant decreases in circulating CA19.9 levels in 3 others. Minnelide was generally well tolerated. Treatment related toxicities > 3 were: neutropenia 4 patients and cerebellar ataxia 1 patient.Exploratory analyses included ATAC-Seq on paired biopsies where gene ontology analyses showed downregulation of genes negatively regulating cell cycle and upregulation of genes controlling cell cycle checkpoints. Other exploratory analyses will be presented. Conclusions: The study identified a tolerable drug dose and evidence of activity in a highly chemotherapy-refractory population which provides encouraging proof for the principle of targeting TFIIH in human PDAC. Supported by SU2C, Cancer Research UK, Lustgarten Foundation Citation Format: David Propper, Erkut Borazanci, Peter J. O'Dwyer, Daniel Von Hoff, Joseph Hartlebury, Corina E. Antal, Gerard I. Evan, HaiYong Han, Michael Downes, Ronald Evans, Joshua Rabinowitz, Emmanuel Petricoin, Ashok Saluja, Mohana Velagapudi, Hitendra Patel. Phase II open label trial of Minnelide™ in patients with chemotherapy refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT218.

Cycloplatinated (II) Complex Based on Isoquinoline Alkaloid Elicits Ferritinophagy-Dependent Ferroptosis in Triple-Negative Breast Cancer Cells.

The development and optimization of metal-based anticancer drugs with novel cytotoxic mechanisms have emerged as key strategies to overcome chemotherapeutic resistance and side effects. Agents that simultaneously induce ferroptosis and autophagic death have received extensive attention as potential modalities for cancer therapy. However, only a limited set of drugs or treatment modalities can synergistically induce ferroptosis and autophagic tumor cell death. In this work, we designed and synthesized four new cycloplatinated (II) complexes harboring an isoquinoline alkaloid C∧N ligand. On screening the in vitro activity of these agents, we found that Pt-3 exhibited greater selectivity of cytotoxicity, decreased resistance factors, and improved anticancer activity compared to cisplatin. Furthermore, Pt-3, which we demonstrate can initiate potent ferritinophagy-dependent ferroptosis, exhibits less toxic and better therapeutic activity than cisplatin in vivo. Our results identify Pt-3 as a promising candidate or paradigm for further drug development in cancer treatment.

Cafestol inhibits colon cancer cell proliferation and tumor growth in xenograft mice by activating LKB1/AMPK/ULK1-dependent autophagy

Chemotherapy failure in colorectal cancer patients is the major cause of recurrence and poor prognosis. As a result, there is an urgent need to develop drugs that have a good chemotherapy effect while also being extremely safe. In this study, we found cafestol inhibited colon cancer growth and HCT116 proliferation in vivo and in vitro, and improved the composition of intestinal flora. Further metabolomic data showed that autophagy and AMPK pathways were involved in the process of cafestol's anti-colon cancer effects. The functional validation studies revealed that cafestol increased autophagy vesicles and LC3B-II levels. The autophagic flux induced by cafestol was prevented by using BafA1. The autophagy inhibitor 3-MA blocked the cafestol-induced increase in LC3B-II and cell proliferation inhibition. Then we found that cafestol induced the increased expressions of LKB1, AMPK, ULK1, p-LKB1, p-AMPK, and p-ULK1 proteins in vivo and in vitro. Using the siRNA targeted to the Lkb1 gene, the levels of AMPK, ULK1, and LC3B-II were suppressed under cafestol treatment. These results indicated that the effect of cafestol is through regulating LKB1/AMPK/ULK1 pathway-mediated autophagic death. Finally, a correlation matrix of the microbiome and autophagy-related proteins was conducted. We found that cafestol-induced autophagic protein expression was positively correlated with the beneficial intestinal bacteria (Muribaculaceae, Bacteroides, Prevotellacece, and Alloprevotella) and negatively correlated with the hazardous bacteria. Conclusions: This study found that cafestol inhibited colon cancer in vitro and in vivo by the mechanism that may be related to LKB1/AMPK/ULK1 pathway-mediated autophagic cell death and improved intestinal microenvironment.

Arabidopsis BECLIN1-induced autophagy mediates reprogramming in tapetal programmed cell death by altering the gross cellular homeostasis

In flowering plants, the tapetum degeneration in post-meiotic anther occurs through developmental programmed cell death (dPCD), which is one of the most critical and sensitive steps for the proper development of male gametophytes and fertility. Yet the pathways of dPCD, its regulation, and its interaction with autophagy remain elusive. Here, we report that high-level expression of Arabidopsis autophagy-related gene BECLIN1 (BECN1 or AtATG6) in the tobacco tapetum prior to their dPCD resulted in developmental defects. BECN1 induces severe autophagy and multiple cytoplasm-to-vacuole pathways, which alters tapetal cell reactive oxygen species (ROS)-homeostasis that represses the tapetal dPCD. The transcriptome analysis reveals that BECN1- expression caused major changes in the pathway, resulting in altered cellular homeostasis in the tapetal cell. Moreover, BECN1-mediated autophagy reprograms the execution of tapetal PCD by altering the expression of the key developmental PCD marker genes: SCPL48, CEP1, DMP4, BFN1, MC9, EXI1, and Bcl-2 member BAG5, and BAG6. This study demonstrates that BECN1-mediated autophagy is inhibitory to the dPCD of the tapetum, but the severity of autophagy leads to autophagic death in the later stages. The delayed and altered mode of tapetal degeneration resulted in male sterility.

DUSP4 maintains the survival and LSD1 protein stability in esophageal squamous cell carcinoma cells by inhibiting JNK signaling-dependent autophagy.

DUSP4 is a biomarker of esophageal squamous cell carcinoma (ESCC), which is responsible for the prognosis in ESCC. However, the underlying mechanism of DUSP4-regulated ESCC carcinogenesis is unknown. As a negative regulator of JNK, DUSP4 can inhibit autophagy, which contributes to tumorigenesis. This study aimed to explore the role of autophagy in DUSP4-regulated ESCC carcinogenesis. Our results showed that DUSP4 overexpression inhibited autophagy and promoted LSD1 protein expression in ESCC cells, while DUSP4 silencing showed the opposite effects. However, DUSP4 overexpression and silencing did not affect LSD1 mRNA expression. But the regulatory ability of DUSP4 overexpression on autophagy, death level, and LSD1 protein was reversed by rapamycin. In addition, DUSP4 overexpression inhibited JNK and Bcl2 phosphorylation and the dissociation of Bcl2-Beclin1 complex, while DUSP4 silencing promoted JNK and Bcl2 phosphorylation. Moreover, the regulatory ability of DUSP4 overexpression on autophagy, death, and LSD1 protein was reversed by JNK activator anisomycin. The xenograft assays also showed that DUSP4 overexpression-promoted ESCC tumor growth in vivo and LC3II and LSD1 protein expression in tumor tissues were reversed by rapamycin or anisomycin. Overall, DUSP4 inhibits Bcl2-Beclin1-autophagy signal transduction through the negative regulation of JNK, thus suppressing autophagic death and the autophagic degradation of LSD1 in ESCC, by which DUSP4 promotes ESCC carcinogenesis.

Total Flavonoids of Aurantii Fructus Immaturus Regulate miR-5100 to Improve Constipation by Targeting Fzd2 to Alleviate Calcium Balance and Autophagy in Interstitial Cells of Cajal.

Aurantii Fructus Immaturus total flavonoids (AFIF) is the main effective fraction extracted from AFI, which has a good effect on promoting gastrointestinal motility. This study aimed to investigate AFIF which regulates miR-5100 to improve constipation symptoms in mice by targeting Frizzled-2 (Fzd2) to alleviate interstitial cells of Cajal (ICCs) calcium ion balance and autophagy apoptosis. The constipated mouse model was induced by an antibiotic suspension, and then treated with AFIF. RNA-seq sequencing, luciferase assay, immunofluorescence staining, transmission electron microscopy, ELISA, flow cytometry, quantitative polymerase chain reaction (PCR), and Western blot were applied in this study. The results showed that AFIF improved constipation symptoms in antibiotic-induced constipated mice, and decreased the autophagy-related protein Beclin1 levels and the LC3-II/I ratio in ICCs. miR-5100 and its target gene Fzd2 were screened as key miRNAs and regulator associated with autophagy. Downregulation of miR-5100 caused increased expression of Fzd2, decreased proliferation activity of ICCs, increased apoptotic cells, and enhanced calcium ion release and autophagy signals. After AFIF treatment, miR-5100 expression was upregulated and Fzd2 was downregulated, while autophagy-related protein levels and calcium ion concentration decreased. Furthermore, AFIF increased the levels of SP, 5-HT, and VIP, and increased the expression of PGP9.5, Sy, and Cx43, which alleviated constipation by improving the integrity of the enteric nervous system network. In conclusion, AFIF could attenuate constipation symptoms by upregulating the expression of miR-5100 and targeting inhibition of Fzd2, alleviating calcium overload and autophagic death of ICCs, regulating the content of neurotransmitters, and enhancing the integrity of the enteric nervous system network.

Capsaicin Attenuates LPS-Induced Acute Lung Injury by Inhibiting Inflammation and Autophagy Through Regulation of the TRPV1/AKT Pathway.

Acute lung injury (ALI) is a severe pulmonary disease characterized by damage to the alveoli and pulmonary blood vessels, leading to severe impairment of lung function. Studies on the effect of capsaicin (8-methyl-N-geranyl-6-nonamide, CAP) on lipopolysaccharide (LPS)-induced ALI in bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B) are still limited. This study aimed to investigate the effect and specific mechanism by which CAP improves LPS-induced ALI. The present study investigated the effect of CAP and the potential underlying mechanisms in LPS-induced ALI in vitro and vivo via RNA sequencing, Western blotting (WB), quantitative real-time reverse transcription PCR (qRT‒PCR), enzyme-linked immunosorbent assay (ELISA), and transmission electron microscopy (TEM). The TRPV1 inhibitor AMG9810 and the AKT agonist SC79 were used to confirm the protective effect of the TRPV1/AKT axis against ALI. The autophagy agonist rapamycin (Rapa) and the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (Baf-A1) were used to clarify the characteristics of LPS-induced autophagy. Our findings demonstrated that CAP effectively suppressed inflammation and autophagy in LPS-induced ALI, both in vivo and in vitro. This mechanism involves regulation by the TRPV1/AKT signaling pathway. By activating TRPV1, CAP reduces the expression of P-AKT, thereby exerting its anti-inflammatory and inhibitory effects on pro-death autophagy. Furthermore, prior administration of CAP provided substantial protection to mice against ALI induced by LPS, reduced the lung wet/dry ratio, decreased proinflammatory cytokine expression, and downregulated LC3 expression. Taken together, our results indicate that CAP protects against LPS-induced ALI by inhibiting inflammatory responses and autophagic death through the TRPV1/AKT signaling pathway, presenting a novel strategy for ALI therapy.