RESUMO
As the pioneering study from Pakistan, our research distinctly focuses on validating the roles of autophagy-associated genes and MicroRNAs (miRs) in the unique context of our population for glioma prognosis. The study delves into the nuanced interplay of autophagy within a miR-modulated environment, prompting an exploration of its potential impact on glioma development and survival. Employing real-time PCR (qPCR), we meticulously assessed the expression profiles of autophagy genes and miRs in glioma tissues, complemented by immunohistochemistry on Formalin-fixed paraffin-embedded tissues from the same patients. Our comprehensive statistical analyses, including the data normality hypothesis Shapiro-Wilk test, the Mann-Whitney U-test, Spearman correlation test, and Kaplan-Meier survival analysis, were tailored to unravel the intricate associations specific to low- and high-grade glioma within our population. Clinicopathological analysis revealed a predominance of male patients (66%) with a median age of 35 years. Glioblastoma (32%) and Astrocytoma (36%) were the most prevalent histopathological subtypes. Molecular analysis showed significant correlations between prognostic markers (Ki-67, IDH-1, p53) and clinicopathological factors, including age, histological type, radiotherapy, and chemotherapy. In high-grade glioma, increased expression of AKT and miR-21, coupled with reduced ULK2 and LC3 expression was distinctly observed. While correlation analysis identified a strong positive correlation between ULK2 and UVRAG, PTEN, miR-7, and miR-100 in low-grade glioma, unveiling distinctive molecular signatures unique to our study. Furthermore, a moderate positive correlation emerged between ULK2 and mTOR, miR-7, miR-30, miR-100, miR-204, and miR-374, also between miR-21 and miR-126. Similarly, a positive correlation appeared between ULK2 and AKT, LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7 and miR-374. AKT positively correlated with LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7, miR-30, miR-204, miR-374, miR-126 and miR-21 weakly correlated with AKT and miR-30 in high-grade glioma, providing further insights into the autophagy pathway within our population. The enrichment analysis for miR-21, miR-126, and miR-374 showed MAPK pathway as a common pathway along with Ras, PI3K, and mTOR pathway. The low ULK2, UVRAG, and miR-374 expression group exhibited significantly poor overall survival in glioma, while miR-21 over-expression indicated a poor prognosis in glioma patients, validating it in our population. This study provides comprehensive insights into the molecular landscape of gliomas, highlighting the dysregulation of autophagy genes ULK2, and UVRAG and the associated miR-21, miR-126 and miR-374 as potential prognostic biomarkers and emphasizing their unique significance in shaping survival outcomes in gliomas within the specific context of the Pakistani population.
Assuntos
Autofagia , Biomarcadores Tumorais , Glioma , Peptídeos e Proteínas de Sinalização Intracelular , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Masculino , Glioma/genética , Glioma/patologia , Glioma/metabolismo , Feminino , Prognóstico , Adulto , Autofagia/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Pessoa de Meia-Idade , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Regulação Neoplásica da Expressão Gênica , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Adulto Jovem , Idoso , Adolescente , Proteínas Serina-Treonina QuinasesRESUMO
Adiponectin, a unique adipose-derived factor, is significantly downregulated in obesity, making it a crucial target for tumor-related metabolic research. AdipoRon is a novel adiponectin receptor agonist with the advantages of a small molecular weight, high stability and a long half-life. By screening the cervical adipose tissue of papillary thyroid carcinoma (PTC) patients with adipokine antibody array, we found that adiponectin was a potential correlation factor between obesity and PTC progression. AdipoRon has oral activity and is easily absorbed and delivered to target tissues. The effects of AdipoRon on thyroid cancer have not been reported. In this study, we identified adiponectin receptor 1 (AdipoR1) and AdipoR2 on the surface of thyroid cancer cell lines. AdipoRon inhibited the proliferation and migration of thyroid cancer cells, limited energy metabolism in thyroid cancer cells, promoted differentiation of thyroid cancer cells, and induced autophagy and apoptosis. Mechanistic studies revealed that AdipoRon inhibited p-mTOR Ser2448 and p-p70S6K Thr389, and activated ULK1 and p-ULK1. ULK1 knockdown suppressed the effect of AdipoRon on LC3BII/I protein and lysosomes. AdipoR2 knockdown reduced AdipoRon-induced autophagy in thyroid cancer cells. This study is the first to demonstrate the role of AdipoRon in PTC. Our findings illustrate a previously unknown function and mechanism of the AdipoRon-AdipoR2-ULK/p-ULK1 axis in PTC and lay the foundation for clinical translation of AdipoRon to PTC. Targeting the AdipoRon-AdipoR2-ULK/p-ULK1 axis may represent a new therapeutic strategy for PTC.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Proliferação de Células , Obesidade , Receptores de Adiponectina , Neoplasias da Glândula Tireoide , Humanos , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/agonistas , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Linhagem Celular Tumoral , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Movimento Celular/efeitos dos fármacos , Masculino , Apoptose/efeitos dos fármacos , Câncer Papilífero da Tireoide/patologia , Câncer Papilífero da Tireoide/tratamento farmacológico , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Pessoa de Meia-Idade , Serina-Treonina Quinases TOR/metabolismo , Adiponectina/metabolismo , Piperidinas , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. Genome-wide association studies identified TRIM47 at the 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found highly expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we predicted a highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription, and vacuole formation. Together, we demonstrate that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation.
Assuntos
Autofagia , Encéfalo , Células Endoteliais , Animais , Camundongos , Humanos , Células Endoteliais/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Substância Branca/metabolismo , Substância Branca/patologia , Fatores de Risco , Masculino , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and adolescents. Conventional chemotherapy remains unsatisfactory due to drug toxicity and resistance issues. Therefore, there is an urgent need to develop more effective treatments for advanced osteosarcoma. In the current study, we focused on evaluating the anticancer efficacy of avermectin B1, a novel avermectin analog, against osteosarcoma cells. METHODS: The half-inhibitory concentration of avermectin B1 was calculated in three osteosarcoma cell lines. Then, functional experiments were conducted to evaluate the effects of avermectin B1 on cell proliferation, the cell cycle, apoptosis and autophagy. Moreover, the AMPK/ULK1 signaling pathway was detected by Western blot assay. Finally, the in vivo effect of avermectin B1 on tumor growth and metastasis was investigated using the xenograft mouse model. To examine the role of the AMPK/ULK1 pathway, an AMPK-specific inhibitor (dorsomorphin) was used in combination with avermectin B1. RESULTS: Avermectin B1 inhibited the proliferation of osteosarcoma cells in a dose-dependent manner based on CCK8 and colony formation assays. Then, it was found to inhibit migration and invasion by wound healing assay and cell migration and invasion assay. In addition, avermectin B1 induced osteosarcoma cell apoptosis and autophagy. In vivo, avermectin B1 effectively inhibited osteosarcoma cell growth and pulmonary metastasis. Mechanistically, avermectin B1 activated the AMPK/ULK1 pathway to exert antitumor activity in vitro and in vivo. Dorsomorphin significantly attenuated the Avermectin B1-induced antitumor activities. CONCLUSION: Our study suggests that avermectin B1 is a potential agent to treat osteosarcoma cells through the AMPK/ULK1 signaling pathway.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Neoplasias Ósseas , Ivermectina , Osteossarcoma , Transdução de Sinais , Animais , Humanos , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases.
Assuntos
Cálcio , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Homeostase , Lisossomos , Proteínas de Membrana , Molécula 1 de Interação Estromal , Proteína Supressora de Tumor p53 , Lisossomos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Humanos , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Animais , Camundongos , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologiaRESUMO
Pyroptosis, a pro-inflammatory form of programmed cell death, is crucial for host defense against pathogens and danger signals. Proteolytic cleavage of gasdermin proteins B-E (GSDMB-GSDME) is well established as a trigger for pyroptosis, but the intracellular activation mechanism of GSDMA remains elusive. Here, we demonstrate that severe starvation induces pyroptosis through phosphorylation-induced activation of GSDMA. Nutrient stresses stimulate GSDMA activation via phosphorylation mediated by Unc-51-like autophagy-activating kinase 1 (ULK1). Phosphorylation of Ser353 on human GSDMA by ULK1 or the phospho-mimetic Ser353Asp mutant of GSDMA liberates GSDMA from auto-inhibition, facilitating its membrane targeting and initiation of pyroptosis. To further validate the significance of GSDMA phosphorylation, we generated a constitutively active mutant Ser354Asp of mouse Gsdma, which induced skin inflammation and hyperplasia in mice, reminiscent of phenotypes with activated Gsdma. This study uncovers phosphorylation of GSDMA as a mechanism underlying pyroptosis initiation and cellular response to nutrient stress.
Assuntos
Gasderminas , Piroptose , Animais , Humanos , Camundongos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Gasderminas/metabolismo , Células HEK293 , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Fosforilação , Inanição/metabolismoRESUMO
BACKGROUND AND AIMS: CircRNAs and autophagy are closely involved in the physiological and pathological processes of ovarian cancer; however, their exact mechanisms are still undetermined. This investigation aimed to elucidate the function and associated pathways of circFAM188A, which modulates proliferation, autophagy, and invasion in ovarian cancer (EOC). METHODS: The expression of circFAM188A in the tissues of EOC patients was assessed via RT-PCR. To elucidate proliferation, invasion, and autophagy in the tumor cells, Transwell, 5-ethynyl-2'-deoxyuridine (EdU), and mRFP-GFP-LC3 reporter assays were conducted. The binding sites between circ-FAM188A and the miR-670-3p, miR-670-3p and YY1 were predicted using bioinformatics and verified by dual-luciferase reporter assays. Pulldown assays demonstrated binding between ULK1 and circ-FAM188A. ULK1 was found to be crucial in the initial stage of autophagy. Moreover, an in vivo xenograft model was established by subcutaneous injection of nude mice with EOC cells. RESULT: Expression of circ-FAM188A was increased in EOC tissues relative to normal ovarian tissues and circ-FAM188A overexpression promoted proliferation, invasion, and autophagy; these effects were reversed by circ-FAM188A silencing. miR-670-3p and circ-FAM188A co-localized in the cytoplasm. circ-FAM188A enhanced YY1 expression by sponging miR-670-3p and was also shown to interact with ULK1. CONCLUSION: It is thus suggested that circ-FAM188A modulates autophagy by sponging miR-670-3p as well as interacting with ULK1.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Carcinoma Epitelial do Ovário , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos Nus , MicroRNAs , Neoplasias Ovarianas , RNA Circular , Humanos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Feminino , MicroRNAs/genética , Autofagia/genética , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/metabolismo , Animais , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Proliferação de Células/genética , RNA Circular/genética , RNA Circular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linhagem Celular Tumoral , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Movimento Celular/genética , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: To observe the effects of electroacupuncture (EA) on the morphological changes of intervertebral disc tissues, apoptosis of nucleus pulposus cells, and the protein expression of Unc-51 like autophagy-activated kinase 1 (ULK1), homologous series of yeast Atg6 (Beclin1), and light chain protease complication 3 type (LC3) in nucleus pulposus tissue of cervical spondylosis rabbits, so as to explore the role of cellular autophagy in EA treatment of cervical spondylosis. METHODS: A total of 24 New Zealand white rabbits were randomly divided into blank, model and EA groups, with 8 rabbits in each group. In the EA group, both sides of the cervical (C)3-C6 "Jiaji" (EX-B2) were stimulated by EA (2 Hz/100 Hz, 1 mA) for 25 min, once daily for 5 days in a course, with a 2-day interval between courses, totaling 4 treatment courses. X-ray was used to assess cervical spine radiographic changes and evaluate radiographic scoresï¼transmission electron microscopy was used to observe ultrastructural changes in nucleus pulposus cellsï¼HE staining was used to observe morphological changes of intervertebral disc tissues and conduct pathological scoringï¼TUNEL staining was used to observe apoptosis rate of nucleus pulposus cellsï¼Western blot was performed to detect protein expression levels of ULK1, Beclin1, and LC3 in nucleus pulposus tissue. RESULTS: Compared with the blank group, rabbits in the model group showed significantly higher cervical spine radiographic scores (P<0.01), higher pathological scores of intervertebral disc tissues (P<0.05), increased apoptosis rate of nucleus pulposus cells (P<0.01), and decreased expression levels of ULK1, Beclin1, and LC3â ¡ proteins in nucleus pulposus tissue (P<0.05). Compared with the model group, the EA group showed significantly lower pathological scores of intervertebral discs (P<0.05), lower apoptosis rate of nucleus pulposus cells (P<0.01), and higher protein expression levels of ULK1, Beclin1, and LC3â ¡ in nucleus pulposus tissue (P<0.01). Rabbits in the blank control group exhibited generally normal organelle structures in nucleus pulposus tissues with few autophagic vacuoles, indicative of early stages of autophagyï¼while those in the model group showed disrupted organelle structures with cytoplasmic condensation and those in the EA group exhibited autophagosomes with double-membrane structures in nucleus pulposus tissues. CONCLUSIONS: EA promotes the expression of ULK1, Beclin1, and LC3â ¡ proteins in nucleus pulposus tissues, reduces apoptosis of nucleus pulposus cells, and improves intervertebral disc degeneration.
Assuntos
Pontos de Acupuntura , Autofagia , Eletroacupuntura , Núcleo Pulposo , Espondilose , Animais , Coelhos , Núcleo Pulposo/metabolismo , Espondilose/terapia , Espondilose/metabolismo , Espondilose/genética , Humanos , Masculino , Apoptose , Proteína Beclina-1/metabolismo , Proteína Beclina-1/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Feminino , Vértebras Cervicais/metabolismo , Disco Intervertebral/metabolismoRESUMO
Distal hereditary motor neuropathy (dHMN) is a progressive neurological disease characterized by distal limb muscle weakness and amyotrophy. Sigma 1 receptor (σ1R), a gene product of SIGMAR1, mutations have been reported to induce dHMN, but its mechanism remains unknown. This study aims to explore the effect of C238T and 31_50del mutations in σ1R on neuronal SH-SY5Y cell functions. The SH-SY5Y cells that overexpressed σ1R, C238T mutant σ1R (σ1RC238T) or 31_50del mutant σ1R (σ1R31_50del) were constructed by pEGFPN1 vectors. We used Western blot (WB) and immunofluorescence (IF) staining to detect the expression of σ1R and green fluorescent proteins (GFP). Then, we evaluated the impact of σ1R mutation on apoptosis, autophagy, endoplasmic reticulum stress, and the involvement of the unfolded protein response (UPR) pathway in SH-SY5Y cells. We found that σ1RC238T and σ1R31_50del downregulated σ1R and promoted the apoptosis of SH-SY5Y cells. σ1RC238T and σ1R31_50del increased p-PERK, p-eIF2α, p-JNK, BIP, ATF4, CHOP, ATF6, XBP1, Caspase3, Caspase12 expressions and Ca2+ concentration, whereas decreased ATP content in SH-SY5Y cells. Besides, the expressions of LC3B, Lamp1, ATG7, Beclin-1 and phosphorylation of AMPK and ULK1 were increased, while the p62 level decreased after C238T or 31_50del mutation of σ1R. Additionally, AMPK knockdown abolished the apoptosis mediated by σ1RC238T or σ1R31_50del in SH-SY5Y cells. Our results indicated that C238T or 31_50del mutation in σ1R promoted motor neuron apoptosis through the AMPK/ULK1 pathway in dHMN. This study shed light on a better understanding of the neurons pathological mechanisms mediated by σ1R C238T and σ1R 31-50del in dHMN.
Assuntos
Apoptose , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Estresse do Retículo Endoplasmático , Receptores sigma , Receptor Sigma-1 , Humanos , Receptores sigma/metabolismo , Receptores sigma/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Linhagem Celular Tumoral , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Resposta a Proteínas não Dobradas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , MutaçãoRESUMO
Corilagin (CLG) has antitumor activities in certain human malignant cancers. Herein, the effects and mechanisms of CLG on osteosarcoma (OS) were investigated. OS cell viability and proliferation were detected by MTT and colony formation assay. Cell cycle and apoptosis were examined using flow cytometry. The interaction between TRAF6 and FLT3 was investigated using a co-immunoprecipitation assay. Results demonstrated that CLG treatment inhibited OS cell viability and proliferation but promoted OS cell autophagy and apoptosis in a concentration-dependent manner. Mechanically, CLG inhibited TRAF6-mediated FLT3 ubiquitination degradation. TRAF6 overexpression abolished the effects of CLG on OS cell proliferation, autophagy, and apoptosis. Finally, CLG administration inhibited OS tumor growth in mice by inducing autophagy-dependent apoptosis. Taken together, CLG inhibited OS progression by facilitating mTOR/ULK1 pathway-mediated autophagy through inhibiting TRAF6-mediated FLT3 ubiquitination, which indicated that CLG was a promising candidate for the treatment of OS.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Proliferação de Células , Osteossarcoma , Fator 6 Associado a Receptor de TNF , Serina-Treonina Quinases TOR , Ubiquitinação , Tirosina Quinase 3 Semelhante a fms , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Humanos , Animais , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Sobrevivência Celular/efeitos dos fármacosRESUMO
One of the key events in autophagy is the formation of a double-membrane phagophore, and many regulatory mechanisms underpinning this remain under investigation. WIPI2b is among the first proteins to be recruited to the phagophore and is essential for stimulating autophagy flux by recruiting the ATG12-ATG5-ATG16L1 complex, driving LC3 and GABARAP lipidation. Here, we set out to investigate how WIPI2b function is regulated by phosphorylation. We studied two phosphorylation sites on WIPI2b, S68 and S284. Phosphorylation at these sites plays distinct roles, regulating WIPI2b's association with ATG16L1 and the phagophore, respectively. We confirm WIPI2b is a novel ULK1 substrate, validated by the detection of endogenous phosphorylation at S284. Notably, S284 is situated within an 18-amino acid stretch, which, when in contact with liposomes, forms an amphipathic helix. Phosphorylation at S284 disrupts the formation of the amphipathic helix, hindering the association of WIPI2b with membranes and autophagosome formation. Understanding these intricacies in the regulatory mechanisms governing WIPI2b's association with its interacting partners and membranes, holds the potential to shed light on these complex processes, integral to phagophore biogenesis.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Autofagia , Proteínas de Membrana , Humanos , Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Ligação ProteicaRESUMO
Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.
Assuntos
Aciltransferases , Autofagossomos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular , Lipoilação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/fisiologia , Humanos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Aciltransferases/metabolismo , Aciltransferases/genética , Autofagossomos/metabolismo , Células HEK293 , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Transporte Proteico , Proteínas de Transporte VesicularRESUMO
Autophagy initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células HEK293 , Fosforilação , Proteínas Relacionadas à Autofagia/metabolismo , Ligação Proteica , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células HeLa , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Chaperonas MolecularesRESUMO
OBJECTIVE: Reduced adiponectin (ADPN) levels have been implicated in the pathogenesis of prostate cancer (PCa). The role of glycolysis in cancer development and treatment has attracted increasing attention. The present study aimed to elucidate its impact on PCa and to explore the mechanistic involvement of glycolysis. METHODS: An RM-1 cell xenograft model of Adpn-knockout mice was used to corroborate the effects of glycolysis, AMP-activated protein kinase (AMPK) signaling, and autophagy on tumor xenograft progression. The effect of ADPN on PCa cells was evaluated using the Cell Counting Kit-8 (CCK-8), lactate levels, and flow cytometry. The expression of glycolysis-related genes was detected using real-time RT-PCR in LNCaP and PC-3 cells after incubation with ADPN. Autophagic flux after ADPN treatment was quantified by chloroquine intervention and confocal analysis of mRFP-GFP-LC3. Alterations in the levels of adiponectin receptor 1 (AdipoR1), AMPK, Unc-51-like kinase 1 (ULK1), autophagy-related protein 7 (ATG7), p62, and microtubule-associated protein 1 light chain 3 beta (LC3B) were assessed after incubation of LNCaP cells with ADPN. RESULTS: Proteomic analysis of xenograft tumors demonstrated significant upregulation of glycolysis in Adpn-/- mice. Lower levels of ADPN accelerated tumor xenograft growth, diminished p-AMPKα/AMPKα ratio and LC3B II/I ratio, and elevated levels of proliferating cell nuclear antigen (PCNA) within the tumor microenvironment. ADPN inhibited proliferation and glycolysis and potentiated apoptosis in both cell lines. Expression of glycolysis-related genes decreased after ADPN treatment. Autophagic flux was elevated, as evidenced by changes in autophagy-related proteins and confocal microscopy analysis of mRFP-GFP-LC3. It led to the suppression of p62 while inducing phosphorylation of AMPKα and upregulating AdipoR1, ULK1, ATG7, and LC3B II/I ratio. CONCLUSION: ADPN inhibited the proliferation and progression of PCa cell-derived tumor xenografts by inhibiting glycolysis. Specifically, ADPN effectively inhibits glycolysis and activates the downstream AMPK/ULK1 signaling pathway to suppress proliferation of PCa cells.
Assuntos
Proteínas Quinases Ativadas por AMP , Adiponectina , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Glicólise , Camundongos Knockout , Neoplasias da Próstata , Transdução de Sinais , Masculino , Animais , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Glicólise/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Humanos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Adiponectina/metabolismo , Adiponectina/genética , Linhagem Celular Tumoral , Autofagia/efeitos dos fármacos , Camundongos , Proliferação de Células/efeitos dos fármacos , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/genéticaRESUMO
Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent U.S. Food and Drug Administration approval of covalent inhibitors of KRASG12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRASG12C-driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients who do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRASG12C, efforts are underway to develop effective combination therapies. Here, we report that the inhibition of KRASG12C signaling increases autophagy in KRASG12C-expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C-driven lung cancer cell proliferation in vitro and superior tumor control in vivo. Additionally, in genetically engineered mouse models of KRASG12C-driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRASG12C in lung cancer.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Animais , Feminino , Humanos , Masculino , Camundongos , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Piperazinas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Piridinas , Pirimidinas/farmacologiaRESUMO
Perioperative neurocognitive disorders (PND) are intractable, indistinct, and considerably diminish the postoperative quality of life of patients. It has been proved that Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was involved in neurodegenerative diseases by regulating mitochondrial biogenesis. The underlying mechanisms of PGC-1α and Nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome in PND are not well understood. In this study, we constructed a model of laparotomy in aged mice, and then examined the cognition changes with novel object recognition tests and fear condition tests. The protein levels of PGC-1α and NLRP3 in the hippocampus were detect after surgery. Our results showed that NLRP3 and downstream PI3K/AKT pathway expressions were augmented in the hippocampus after surgery, whereas, the expressions of PGC-1α/estrogen-related receptor α (ERRα)/Unc-51-like autophagy activating kinase 1 (ULK1) pathway were diminished after surgery. In addition, we found that NLRP3 was mainly co-localized with neurons in the hippocampus, and synaptic-related proteins were reduced after surgery. At the same time, transmission electron microscopy (TEM) showed that mitochondria were impaired after surgery. Pharmacological treatment of MCC950, a selective NLRP3 inhibitor, effectively alleviated PND. Activation of PGC-1α with ZLN005 significantly ameliorated PND by enhancing the PGC-1α/ERRα/ULK1 signaling pathway, and further suppressing NLRP3 activation. As a result, we conclude that suppression of the PGC-1α/ERRα/ULK1 signaling pathway is the primary mechanism of PND which caused mitochondrial dysfunction, and activated NLRP3 inflammasome and downstream PI3K/AKT pathway, eventually improved cognitive dysfunction.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Hipocampo , Inflamassomos , Camundongos Endogâmicos C57BL , Mitocôndrias , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transtornos Neurocognitivos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores de Estrogênio , Transdução de Sinais , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Inflamassomos/metabolismo , Camundongos , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Hipocampo/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Mitocôndrias/metabolismo , Masculino , Receptores de Estrogênio/metabolismo , Transtornos Neurocognitivos/metabolismo , Transtornos Neurocognitivos/etiologia , Envelhecimento/metabolismo , Laparotomia/efeitos adversos , Sulfonamidas/farmacologia , Furanos , IndenosRESUMO
BACKGROUND: Left ventricular diastolic dysfunction (LVDD) is a manifestation of heart failure, with both its incidence and prevalence increasing annually. Currently, no pharmacological treatments are available for LVDD, highlighting the urgent need for new therapeutic discoveries. Ginsenosides are commonly used in cardiovascular therapy. Previous research has synthesized the ginsenoside precursor molecule, 20S-O-Glc-DM (C20DM), through biosynthesis. C20DM shows greater bioavailability, eco-friendliness, and cost-effectiveness compared to traditional ginsenosides, positioning it as a promising option for treating LVDD. PURPOSE: This study firstly documents the therapeutic activity of C20DM against LVDD and unveils its potential mechanisms of action. It provides a pharmacological basis for C20DM as a new cardiovascular therapeutic agent. METHODS: In this study, models of LVDD in mice and ISO-induced H9C2 cell damage were developed. Cell viability, ROS and Ca2+ levels, mitochondrial membrane potential, and proteins associated with mitochondrial biogenesis and autophagy were evaluated in the in vitro experiments. Animal experiments involved administering medication for 3 weeks to validate the therapeutic effects of C20DM and its impact on mitochondria and autophagy. RESULTS: Research has shown that C20DM is more effective than Metoprolol in treating LVDD, significantly lowering the E/A ratio, e'/a' ratio, and IVRT, and ameliorating myocardial inflammation and fibrosis. C20DM influences the activity of PGC-1α, downregulates PINK1 and Parkin, thereby enhancing mitochondrial quality control, and restoring mitochondrial oxidative respiration and membrane potential. Furthermore, C20DM reduces excessive autophagy in cardiomyocytes via the AMPK-mTOR-ULK1 pathway, diminishing cardiomyocyte hypertrophy and damage. CONCLUSIONS: Overall, our research indicates that C20DM has the potential to enhance LVDD through the regulation of mitochondrial quality control and cellular autophagy, making it a promising option for heart failure therapy.
Assuntos
Autofagia , Ginsenosídeos , Potencial da Membrana Mitocondrial , Miócitos Cardíacos , Disfunção Ventricular Esquerda , Animais , Autofagia/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Disfunção Ventricular Esquerda/tratamento farmacológico , Camundongos , Ginsenosídeos/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Linhagem Celular , Espécies Reativas de Oxigênio/metabolismo , Modelos Animais de Doenças , Ratos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Cálcio/metabolismoRESUMO
The five-year survival rate for patients with hepatocellular carcinoma (HCC) is only 20â¯%, highlighting the urgent need to identify new therapeutic targets and develop potential therapeutic options to improve patient prognosis. One promising approach is inhibiting autophagy as a strategy for HCC treatment. In this study, we established a virtual docking conformation of the autophagy promoter ULK1 binding XST-14 derivatives. Based on this conformation, we designed and synthesized four series of derivatives. By evaluating their affinity and anti-HCC effects, we confirmed that these compounds exert anti-HCC activity by inhibiting ULK1. The structure-activity relationship was summarized, with derivative A4 showing 10 times higher activity than XST-14 and superior efficacy to sorafenib against HCC. A4 has excellent effect on reducing tumor growth and enhancing sorafenib activity in HepG2 and HCCLM3 cells. Moreover, we verified the therapeutic effect of A4 in sorafenib-resistant HCC cells both in vivo and in vitro. These results suggest that inhibiting ULK1 to regulate autophagy may become a new treatment method for HCC and that A4 will be used as a lead drug for HCC in further research. Overall, A4 shows good drug safety and efficacy, offering hope for prolonging the survival of HCC patients.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Desenho de Fármacos , Indóis , Neoplasias Hepáticas , Inibidores de Proteínas Quinases , Sorafenibe , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Indóis/farmacologia , Indóis/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Sorafenibe/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Ischemia reperfusion (IR) causes impaired myocardial function, and autophagy activation ameliorates myocardial IR injury. Isoliquiritigenin (ISO) has been found to protect myocardial tissues via AMPK, with exerting anti-tumor property through autophagy activation. This study aims to investigate ISO capacity to attenuate myocardial IR through autophagy activation mediated by AMPK/mTOR/ULK1 signaling. METHODS: ISO effects were explored by SD rats and H9c2 cells. IR rats and IR-induced H9c2 cell models were established by ligating left anterior descending (LAD) coronary artery and hypoxia/re-oxygenation, respectively, followed by low, medium and high dosages of ISO intervention (Rats: 10, 20, and 40 mg/kg; H9c2 cells: 1, 10, and 100 µmol/L). Myocardial tissue injury in rats was assessed by myocardial function-related index, HE staining, Masson trichrome staining, TTC staining, and ELISA. Autophagy of H9c2 cells was detected by transmission electron microscopy (TEM) and immunofluorescence. Autophagy-related and AMPK/mTOR/ULK1 pathway-related protein expressions were detected with western blot. RESULTS: ISO treatment caused myocardial function improvement, and inhibition of myocardial inflammatory infiltration, fibrosis, infarct area, oxidative stress, CK-MB, cTnI, and cTnT expression in IR rats. In IR-modeled H9c2 cells, ISO treatment lowered apoptosis rate and activated autophagy and LC3 fluorescence expression. In vivo and in vitro, ISO intervention exhibited enhanced Beclin1, LC3II/LC3I, and p-AMPK/AMPK levels, whereas inhibited P62, p-mTOR/mTOR and p-ULK1(S757)/ULK1 protein expression, activating autophagy and protecting myocardial tissues from IR injury. CONCLUSION: ISO treatment may induce autophagy by regulating AMPK/mTOR/ULK1 signaling, thereby improving myocardial IR injury, as a potential candidate for treatment of myocardial IR injury.
Assuntos
Proteínas Quinases Ativadas por AMP , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Autofagia , Chalconas , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Masculino , Ratos , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Chalconas/farmacologia , Modelos Animais de Doenças , Fibrose , Infarto do Miocárdio/patologia , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
BACKGROUND: High expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets. METHODS: Kinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA. RESULTS: By using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly. CONCLUSION: Our research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies.