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1.
Life Sci ; 263: 118586, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33065148

RESUMO

AIMS: Mitochondrial dysfunction is receiving considerable attention due to irreplaceable biological function of mitochondria. Ionizing radiation and tigecycline (TIG) alone can cause mitochondrial dysfunction, playing important role in tumor therapy. However, prior studies fail to investigate combined mechanism of carbon ion irradiation (IR) and TIG on tumor proliferation inhibition. The study aimed to explore the combined effects of both on autophagy and apoptosis. MATERIALS AND METHODS: NSCLC cells A549 and H1299 were treated with carbon ion, TIG, or both. Cell survival rate, autophagy, apoptosis, expression of mitochondrial signaling proteins were determined by clone formation assay, immunofluorescence of LC3B, flow cytometry and western blotting, respectively; ATP content, mitochondrial membrane potential (MMP) and Ca2+ level in mitochondria were used to assessed mitochondrial function. KEY FINDINGS: Results showed IR combined TIG inhibited cells proliferation by increasing apoptosis in both cells and enhancing autophagy in H1299 cells. Additionally, combination treatment induced the most severe mitochondrial dysfunction by sharply reducing ATP, MMP and increasing Ca2+ level of mitochondria. Up-regulation and down-regulation of mitochondrial translation proteins (EF-Tu, GFM1 and MRPS12) expression affected apoptosis and autophagy, while the level of p-mTOR was consistent with their expression in both cell types. In A549 cells, p-AMPK level decreased while p-Akt and p-mTOR increased after combination treatment. SIGNIFICANCE: Overall, our results showed that p-Akt and p-AMPK antagonistically targeted p-mTOR to regulate mitochondrial translation proteins to affect autophagy and apoptosis. Furthermore, this study suggests that combination of carbon ion and TIG is a potential therapeutic option against tumors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Radioterapia com Íons Pesados/métodos , Neoplasias Pulmonares/terapia , Tigeciclina/administração & dosagem , Células A549 , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Humanos , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Serina-Treonina Quinases TOR/metabolismo , Tigeciclina/farmacologia
2.
Radiat Res ; 194(3): 236-245, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32942301

RESUMO

Autophagy has been reported to play a radioresistance role in high-dose-rate irradiation. However, its mechanisms and roles in continuous low-dose-rate (CLDR) irradiation have not been clearly understood. Iodine-125 (I-125) seed brachytherapy is a modality of CLDR irradiation and has been used in the treatment of various cancers. In this study, we investigated the mechanisms and roles of autophagy induced by I-125 seed radiation in human esophageal squamous cell carcinoma (ESCC) cell lines (Eca-109 and EC-109) and a xenograft mouse model. The results of this work showed that I-125 seed radiation induced a dose-dependent increase in autophagy in both cell lines. In Eca-109 cells, I-125 seed radiation-induced endoplasmic reticulum (ER) stress, manifesting as the increased levels of intracellular Ca2+ and Grp78/BiP, and activated PERK-eIF2α, IRE1, and ATF6 pathways of the unfolded protein response. Knockdown of PERK led to the decreased expression of autophagy marker, LC3B-II. Inhibition of autophagy by chloroquine or knockdown of ATG5 enhanced I-125 seed radiation-induced cell proliferation inhibition and apoptosis. Interestingly, chloroquine did not aggravate ER stress but promoted apoptosis via the mitochondrial pathway. The animal experiment showed that inhibition of autophagy by chloroquine improved the efficacy of I-125 seed radiation. In summary, our data demonstrate that I-125 seed CLDR radiation induces ER stress-mediated autophagy in ESCC. Autophagy plays a pro-survival role in I-125 seed CLDR irradiation, and chloroquine is a potential candidate for use in combination therapy with I-125 seed radiation treatment to improve efficacy against ESCC.


Assuntos
Autofagia/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos da radiação , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/radioterapia , Radioisótopos do Iodo/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação
3.
Radiat Res ; 194(2): 103-115, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32845995

RESUMO

Radiation is a critical pillar in cancer therapeutics, exerting its anti-tumor DNA-damaging effects through various direct and indirect mechanisms. Radiation has served as an effective mode of treatment for a number of cancer types, providing both curative and palliative treatment; however, resistance to therapy persists as a fundamental limitation. While cancer cell death is the ideal outcome of any anti-tumor treatment, radiation induces several responses, including apoptotic cell death, mitotic catastrophe, autophagy and senescence, where autophagy and senescence may promote cell survival. In most cases, autophagy, a conventionally cytoprotective mechanism, is a "first" responder to damage incurred from chemotherapy and radiation treatment. The paradigm developed on the premise that autophagy is cytoprotective in nature has provided the rationale for current clinical trials designed with the goal of radiosensitizing cancer cells through the use of autophagy inhibitors; however, these have failed to produce consistent results. Delving further into pre-clinical studies, autophagy has actually been shown to take diverse, sometimes opposing, forms, such as acting in a cytotoxic or nonprotective fashion, which may be partially responsible for the inconsistency of clinical outcomes. Furthermore, autophagy can have both pro- and anti-tumorigenic effects, while also having an important immune modulatory function. Senescence often occurs in tandem with autophagy, which is also the case with radiation. Radiation-induced senescence is frequently followed by a phase of proliferative recovery in a subset of cells and has been proposed as a tumor dormancy model, which can contribute to resistance to therapy and possibly also disease recurrence. Senescence induction is often accompanied by a unique secretory phenotype that can either promote or suppress immune functions, depending on the expression profile of cytokines and chemokines. Novel therapeutics selectively cytotoxic to senescent cells (senolytics) may prove to prolong remission by delaying disease recurrence in patients. Accurate assessment of primary responses to radiation may provide potential targets that can be manipulated for therapeutic benefit to sensitize cancer cells to radiotherapy, while sparing normal tissue.


Assuntos
Autofagia/efeitos da radiação , Senescência Celular/efeitos da radiação , Neoplasias/patologia , Neoplasias/radioterapia , Animais , Apoptose/efeitos da radiação , Ensaios Clínicos como Assunto , Humanos
4.
Adv Exp Med Biol ; 1207: 301-313, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32671756

RESUMO

Autophagy is critical for the survival and stemness maintenance of cancer stem cells (CSCs) and is an enhancer of CSC tumorigenesis. At the same time, autophagy contributes to conditions optimal for facilitating the invasion and metastasis of CSCs. Moreover, autophagy induces the dormant state of CSCs to help them resist the cytotoxic effects of chemotherapy and radiotherapy, thereby improving the likelihood of their survival. The combination of autophagy inhibitors with specific drugs targeting specific CSC subpopulations is expected to act specifically on CSCs and produce fewer toxic side effects on normal tissues. This in-depth study is very timely and important for further identifying the potential role of autophagy in different states of CSCs and places a particular emphasis on exploring molecular mechanisms in the regulation of autophagy via advanced techniques based on molecular biology.


Assuntos
Autofagia , Células-Tronco Neoplásicas , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/radioterapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação
5.
Anticancer Res ; 40(5): 2537-2548, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366398

RESUMO

BACKGROUND/AIM: Radiotherapy-induced autophagy affects radiation-sensitivity and radiotherapy efficacy. Histone modifications also occur during radiotherapy. This study assessed radiotherapy effects on histone modification and autophagy in non-small cell lung cancer (NSCLC) cells. MATERIALS AND METHODS: NSCLC cells were subjected to γ-irradiation. Autophagy was detected using western blotting and acridine orange staining. Radiation effect on cell growth was evaluated by clonogenic assay. Histone modifications were assessed by western blotting. Next generation sequencings (NGSs) were conducted to identify histone modification target genes. RESULTS: Radio-protective autophagy and histone H4 lysine 20 trimethylation (H4K20me3) were up-regulated after irradiation. By NGSs, genes that are differentially expressed upon irradiation were identified, including the candidate H4K20me3 target gene GABARAPL1. Furthermore, we showed that GABARAPL1 is essential for the radiation-induced autophagy. CONCLUSION: Our findings revealed the regulatory axis of radiation-induced H4K20me3-GABARAPL1 in radio-protective autophagy. Modulation of this axis may be a new strategy to enhance radiotherapy efficacy in NSCLC.


Assuntos
Autofagia/efeitos da radiação , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Histonas/metabolismo , Neoplasias Pulmonares/metabolismo , Lisina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral , Epigênese Genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Metilação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética
6.
Oncol Rep ; 43(6): 2028-2044, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32323828

RESUMO

Iodine­125 (125I) seed brachytherapy has been proven to be a safe and effective treatment for advanced esophageal cancer; however, the mechanisms underlying its actions are not completely understood. In the present study, the anti­cancer mechanisms of 125I seed radiation in human esophageal squamous cell carcinoma (ESCC) cells (Eca­109 and KYSE­150) were determined, with a particular focus on the mode of cell death. The results showed that 125I seed radiation significantly inhibited cell proliferation, and induced DNA damage and G2/M cell cycle arrest in both ESCC cell lines. 125I seed radiation induced cell death through both apoptosis and paraptosis. Eca­109 cells were primarily killed by inducing caspase­dependent apoptosis, with 6 Gy radiation resulting in the largest response. KYSE­150 cells were primarily killed by inducing paraptosis, which is characterized by extensive cytoplasmic vacuolation. 125I seed radiation induced autophagic flux in both ESCC cell lines, and autophagy inhibition by 3­methyladenine enhanced radiosensitivity. Furthermore 125I seed radiation induced increased production of reactive oxygen species (ROS) in both ESCC cell lines. Treatment with an ROS scavenger significantly attenuated the effects of 125I seed radiation on endoplasmic reticulum stress, autophagy, apoptosis, paraptotic vacuoles and reduced cell viability. In vivo experiments showed that 125I seed brachytherapy induced ROS generation, initiated cell apoptosis and potential paraptosis, and inhibited cell proliferation and tumor growth. In summary, the results demonstrate that in ESCC cells, 125I seed radiation induces cell death through both apoptosis and paraptosis; and at the same time initiates protective autophagy. Additionally, 125I seed radiation­induced apoptosis, paraptosis and autophagy was considerably mediated by ROS.


Assuntos
Neoplasias Esofágicas/radioterapia , Carcinoma de Células Escamosas do Esôfago/radioterapia , Radioisótopos do Iodo/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Distribuição Aleatória , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Int J Radiat Biol ; 96(6): 823-835, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32149569

RESUMO

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown.Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing ß-galactosidase (SA-ß-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19Kip1/Cip1, p21Waf1/Cip1 and p27Kip1/Cip1, senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair.Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells.Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures.


Assuntos
Células Epiteliais/patologia , Mesoderma/patologia , Neoplasias da Próstata/patologia , Tolerância a Radiação , Rabdomiossarcoma/patologia , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Linhagem Celular Tumoral , Senescência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo
8.
Oxid Med Cell Longev ; 2020: 5135893, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32190174

RESUMO

Objective: The mechanism of enhanced radiosensitivity induced by mitochondrial uncoupling protein UCP2 was investigated in HeLa cells to provide a theoretical basis as a novel target for cervical cancer treatment. Methods: HeLa cells were irradiated with 4 Gy X-radiation at 1.0 Gy/min. The expression of UCP2 mRNA and protein was assayed by real-time quantitative polymerase chain reaction and western blotting. UCP2 siRNA and negative control siRNA fragments were constructed and transfected into HeLa cells 24 h after irradiation. The effect of UCP2 silencing and irradiation on HeLa cells was determined by colony formation, CCK-8 cell viability, γH2AX immunofluorescence assay of DNA damage, Annexin V-FITC/PI apoptosis assay, and propidium iodide cell cycle assay. The effects on mitochondrial structure and function were investigated with fluorescent probes including dichlorodihydrofluorescein diacetate (DCFH-DA) assay of reactive oxygen species (ROS), rhodamine 123, and MitoTracker Green assay of mitochondrial structure and function. Results: Irradiation upregulated UCP2 expression, and UCP2 knockdown decreased the survival of irradiated HeLa cells. UCP2 silencing sensitized HeLa cells to irradiation-induced DNA damage and led to increased apoptosis, cell cycle arrest in G2/M, and increased mitochondrial ROS. Increased radiosensitivity was associated with an activation of P53, decreased Bcl-2, Bcl-xl, cyclin B, CDC2, Ku70, and Rad51 expression, and increased Apaf-1, cytochrome c, caspase-3, and caspase-9 expression. Conclusions: UCP2 inhibition augmented the radiosensitivity of cervical cancer cells, and it may be a potential target of radiotherapy of advanced cervical cancer.


Assuntos
Tolerância a Radiação , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2/antagonistas & inibidores , Neoplasias do Colo do Útero/metabolismo , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Feminino , Inativação Gênica/efeitos da radiação , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Modelos Biológicos , Tolerância a Radiação/efeitos da radiação , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo , Neoplasias do Colo do Útero/patologia
9.
Cancer Lett ; 483: 114-126, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32014456

RESUMO

Radioresistance reduces the success of therapy for patients with ESCC. Enhancing our understanding of the cardinal principles of radioresistance may improve the response of patients to irradiation. MicroRNAs perform a key role in posttranscriptional regulation, which is linked with the response of tumors to irradiation. Here, we successfully constructed a radioresistant cell line model, ECA109R, from parental esophageal cancer cell line ECA109. We used RNA-Seq analysis and qRT-PCR to compare the miRNA expression profiles of the ECA109 and ECA109R cell lines. The results revealed that miR-450a-5p was downregulated in the radioresistant cells. Functional analysis indicated that miR-450a-5p increases cellular radiosensitivity and suppresses autophagy in ESCC cells. We utilized a luciferase reporter assay to identify the target gene, DUSP10, as an indispensable regulator of the p38 and SAPK/JNK signaling pathways. Upregulation or downregulation of DUSP10 expression could reverse the effects of miR-450a-5p overexpression or inhibition. Tumor xenograft experiments verified that miR-450a-5p overexpression could increase sensitivity to radiation therapy in vivo. In general, our findings indicate that miR-450a-5p is a latent radiosensitizer and may represent a potential novel therapeutic target for radioresistance in ESCC.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Neoplasias Esofágicas/radioterapia , Carcinoma de Células Escamosas do Esôfago/radioterapia , MicroRNAs/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Tolerância a Radiação , Animais , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Linhagem Celular Tumoral , Fosfatases de Especificidade Dupla/genética , Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/enzimologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Tolerância a Radiação/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Gene ; 738: 144485, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32087272

RESUMO

Impairment of neurogenesis is thought to be one of the important mechanisms underlying radiation-induced cognitive decline. Self-renewal and differentiation of neural stem cells (NSCs) are important components of neurogenesis. It has been well established that autophagy plays an important role in neurodegenerative conditions, however, its role in radiation-induced cognitive decline remains unclear. Our previous studies have found that ionizing radiation (IR) induces autophagy in mouse neurons, and minocycline, an antibiotic that can cross the blood-brain barrier, protects neurons from radiation-induced apoptosis through promoting autophagy, thus may contribute to the improvement of mouse cognitive performance after whole-brain irradiation. In the present study, we investigated whether autophagy is involved in radiation-induced damage in self-renewal and differentiation of NSCs. We found that NSCs were extremely sensitive to IR. Irradiation induced autophagy in NSCs in a dose-dependent manner. Atg7 knockdown significantly decreased autophagy, thus increased the apoptosis levels in irradiated NSCs, suggesting that autophagy protected NSCs from radiation-induced apoptosis. Moreover, compared with the negative control NSCs, the neurosphere size was significantly reduced and the neuronal differentiation was notably inhibited in Atg7-deficient NSCs after irradiation, indicating that autophagy defect could exacerbate radiation-induced reduction in NSC self-renewal and differentiation potential. In conclusion, down-regulating autophagy by selective Atg7 knockdown in NSCs enhanced radiation-induced NSC damage, suggesting an important protective role of autophagy in maintaining neurogenesis. Along with the protective effect of autophagy on irradiated neurons, our results on NSCs not only shed the light on the involvement of autophagy in the development of radiation-induced cognitive decline, but also provided a potential target for preventing cognitive impairment after cranial radiation exposure.


Assuntos
Proteína 7 Relacionada à Autofagia/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Neurogênese/efeitos da radiação , Neurônios/efeitos da radiação , Gravidez , Radiação Ionizante
11.
Mol Plant ; 13(3): 515-531, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32087368

RESUMO

Light is arguably one of the most important environmental factors that determines virtually all aspects of plant growth and development, but the molecular link between light signaling and the autophagy pathway has not been elucidated in plants. In this study, we demonstrate that autophagy is activated during light-to-dark conversion though transcriptional upregulation of autophagy-related genes (ATGs). We showed that depletion of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to enhanced autophagy activity and resistance to extended darkness and nitrogen starvation treatments, contributing to higher expression of ATGs. HY5 interacts with and recruits HISTONE DEACETYLASE 9 (HDA9) to ATG5 and ATG8e loci to repress their expression by deacetylation of the Lys9 and Lys27 of histone 3. Furthermore, we found that both darkness and nitrogen depletion induce the degradation of HY5 via 26S proteasome and the concomitant disassociation of HDA9 from ATG5 and ATG8e loci, leading to their depression and thereby activated autophagy. Genetic analysis further confirmed that HY5 and HDA9 act synergistically and function upstream of the autophagy pathway. Collectively, our study unveils a previously unknown transcriptional and epigenetic network that regulates autophagy in response to light-to-dark conversion and nitrogen starvation in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Autofagia/efeitos da radiação , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Escuridão , Histona Desacetilases/metabolismo , Nitrogênio/deficiência , Transcrição Genética/efeitos da radiação , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Loci Gênicos/genética
12.
Sci Rep ; 10(1): 2015, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029749

RESUMO

Although metallothionein-3 (MT3), a brain-enriched form of metallothioneins, has been linked to Alzheimer's disease, little is known regarding the role of MT3 in glioma. As MT3 plays a role in autophagy in astrocytes, here, we investigated its role in irradiated glioma cells. Irradiation increased autophagy flux in GL261 glioma cells as evidenced by increased levels of LC3-II but decreased levels of p62 (SQSTM1). Indicating that autophagy plays a cytoprotective role in glioma cell survival following irradiation, measures inhibiting autophagy flux at various steps decreased their clonogenic survival of irradiated GL261 as well as SF295 and U251 glioma cells. Knockdown of MT3 with siRNA in irradiated glioma cells induced arrested autophagy, and decreased cell survival. At the same time, the accumulation of labile zinc in lysosomes was markedly attenuated by MT3 knockdown. Indicating that such zinc accumulation was important in autophagy flux, chelation of zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), induced arrested autophagy in and reduced survival of GL261 cells following irradiation. Suggesting a possible mechanism for arrested autophagy, MT3 knockdown and zinc chelation were found to impair lysosomal acidification. Since autophagy flux plays a cytoprotective role in irradiated glioma cells, present results suggest that MT3 and zinc may be regarded as possible therapeutic targets to sensitize glioma cells to ionizing radiation therapy.


Assuntos
Autofagia/efeitos da radiação , Neoplasias Encefálicas/radioterapia , Glioma/radioterapia , Metalotioneína/metabolismo , Fótons/uso terapêutico , Animais , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quelantes/farmacologia , Etilenodiaminas/farmacologia , Técnicas de Silenciamento de Genes , Glioma/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/efeitos da radiação , Metalotioneína/genética , Camundongos , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação , Zinco/metabolismo
13.
ACS Appl Mater Interfaces ; 12(4): 4265-4275, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31903741

RESUMO

Conventional therapeutic approaches to treat malignant tumors such as surgery, chemotherapy, or radiotherapy often lead to poor therapeutic results, great pain, economic burden, and risk of recurrence and may even increase the difficulty in treating the patient. Long-term drug administration and systemic drug delivery for cancer chemotherapy would be accompanied by drug resistance or unpredictable side effects. Thus, the use of photothermal therapy, a relatively rapid tumor elimination technique that regulates autophagy and exerts an antitumor effect, represents a novel solution to these problems. Heat shock protein 90 (HSP90), a protein that reduces photothermal or hypothermic efficacy, is closely related to AKT (protein kinase B) and autophagy. Therefore, it was hypothesized that autophagy could be controlled to eliminate tumors by combining exogenous light with a selective HSP90 inhibitor, for example, SNX-2112. In this study, an efficient tumor-killing strategy using graphene oxide loaded with SNX-2112 and folic acid for ultrafast low-temperature photothermal therapy (LTPTT) is reported. A unique mechanism that achieves remarkable therapeutic performance was discovered, where overactivated autophagy induced by ultrafast LTPTT led to direct apoptosis of tumors and enabled functional recovery of T cells to promote natural immunity for actively participating in the attack against tumors. This LTPTT approach resulted in residual tumor cells being rendered in an "injured" state, opening up the possibility of concurrent antitumor and antirecurrence treatment.


Assuntos
Antineoplásicos/administração & dosagem , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Neoplasias/tratamento farmacológico , Fotoquimioterapia/métodos , Animais , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Fotoquimioterapia/instrumentação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Temperatura
14.
Lab Invest ; 100(3): 387-399, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31558773

RESUMO

Electron beam (EB) irradiation is useful to reduce the recurrence of keloids; however, the underlying mechanism remains unknown. MicroRNA-21 (miR-21), which regulates autophagy during cancer radiation therapy, was identified as a potential therapeutic target for keloids. Here, we investigate the regulatory mechanism(s) of miR-21-5p on keloid fibroblast autophagy and migration after EB irradiation. The microRNA expression profile of the keloid dermis was examined by performing a microRNA microarray. Levels of LC3B and Beclin-1 were detected by immunohistochemical and western blot analysis in the keloid dermis and fibroblasts. Autophagy and apoptosis were tested in keloid fibroblasts after EB irradiation or transfection with an miR-21-5p inhibitor using electron microscopy, a Cyto-ID Green Autophagy Detection Kit, and an Annexin V PE Apoptosis Detection Kit. Migration was analyzed by an in vitro scratch-wound healing assay. Mechanistic tests were performed using small interfering RNAs to phosphatase and tensin homolog (siPTEN). Levels of miR-21-5p, PTEN, programmed cell death 4 (PDCD4), p-AKT, and apoptosis- and autophagy-associated genes were examined by qRT-PCR and western blotting. LC3B expression and migration ability were enhanced in fibroblasts and the keloid margin dermis compared with those in the adjacent normal skin. Both EB irradiation and an miR-21-5p inhibitor reduced keloid fibroblast autophagy, which was accompanied by decreased expression of miR-21-5p, p-AKT, and LC3B-II and increased expression of PTEN, PDCD4, and apoptosis-related genes. MiR-21-5p downregulation inhibited migration and suppressed LC3B expression and this was reversed by PTEN reduction. In conclusion, with increasing apoptosis, EB irradiation inhibits autophagy in keloid fibroblasts by reducing miR-21-5p, which regulates migration and LC3B expression via PTEN/AKT signaling. These data suggest a potential mechanism wherein miR-21-5p inhibition regulates autophagy and migration in EB-irradiated keloid fibroblasts, effectively preventing local invasion and recurrence. Therefore, miR-21-5p could be a new therapeutic target, to replace EB irradiation, and control keloid relapse.


Assuntos
Autofagia/efeitos da radiação , Fibroblastos , Queloide/metabolismo , MicroRNAs , PTEN Fosfo-Hidrolase/metabolismo , Adulto , Apoptose/efeitos da radiação , Movimento Celular/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Elétrons , Feminino , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Transcriptoma/efeitos da radiação , Adulto Jovem
15.
Photodermatol Photoimmunol Photomed ; 36(2): 111-117, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31553079

RESUMO

BACKGROUND: Autophagy is known as an intracellular cleanup system necessary to maintain homeostasis of the skin. Many studies have pointed out the relationship between aging and the inactivation of autophagy function, which suggests that the inactivation of autophagy occurs in aged skin. However, the aging process of the skin is complicated compared with other organs, because the skin is localized at the border between the inside of the body and the environment. Thus, skin aging is strongly affected by environmental factors, and it is well recognized that ultraviolet (UV) radiation is an important environmental factor that promotes skin aging. Therefore, characterizing the autophagic phenotypes induced by environmental factors is important to understand the process of skin aging. METHODS: In order to demonstrate the status of autophagy during environment-induced aging of the skin, we investigated the autophagy profiles of normal human dermal fibroblasts (NHDFs) treated with repetitive UVA irradiation as model fibroblasts in photoaged skin. RESULTS: Repetitively UVA-irradiated NHDFs showed increased numbers of autophagosomes, which coincided with the accumulation of p62 and increased levels of LAMP-1 and lysosomes. The behavior of repetitively UVA-irradiated NHDFs on autophagy was similar to that of NHDFs treated with hydroxychloroquine (HCQ), which is an inhibitor of lysosomal proteinase. CONCLUSION: In summary, these results demonstrate that repetitively UVA-irradiated fibroblasts have reduced autophagy function due to the dysfunction of lysosomes.


Assuntos
Autofagia/efeitos da radiação , Fibroblastos/metabolismo , Envelhecimento da Pele/efeitos da radiação , Pele/metabolismo , Raios Ultravioleta/efeitos adversos , Fibroblastos/patologia , Humanos , Pele/patologia
16.
Nucleic Acids Res ; 48(2): 736-747, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31740976

RESUMO

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are predominantly repaired by non-homologous end joining (NHEJ). IR-induced DNA damage activates autophagy, an intracellular degradation process that delivers cytoplasmic components to the lysosome. We identified the deubiquitinase USP14 as a novel autophagy substrate and a regulator of IR-induced DNA damage response (DDR) signaling. Inhibition of autophagy increased levels and DSB recruitment of USP14. USP14 antagonized RNF168-dependent ubiquitin signaling and downstream 53BP1 chromatin recruitment. Here we show that autophagy-deficient cells are defective in NHEJ, as indicated by decreased IR-induced foci (IRIF) formation by pS2056-, pT2609-DNA-PKcs, pS1778-53BP1, RIF1 and a reporter assay activation. Moreover, chromatin recruitment of key NHEJ proteins, including, Ku70, Ku80, DNA-PKcs and XLF was diminished in autophagy-deficient cells. USP14 inhibition rescued the activity of NHEJ-DDR proteins in autophagy-deficient cells. Mass spectrometric analysis identified USP14 interaction with core NHEJ proteins, including Ku70, which was validated by co-immunoprecipitation. An in vitro assay revealed that USP14 targeted Ku70 for deubiquitination. AKT, which mediates Ser432-USP14 phosphorylation, was required for IRIF formation by USP14. Similar to USP14 block, AKT inhibition rescued the activity of NHEJ-DDR proteins in autophagy- and PTEN-deficient cells. These findings reveal a novel negative PTEN/Akt-dependent regulation of NHEJ by USP14.


Assuntos
Reparo do DNA por Junção de Extremidades/efeitos da radiação , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ubiquitina Tiolesterase/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Autofagia/efeitos da radiação , Cromatina/genética , Cromatina/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Células HEK293 , Humanos , Autoantígeno Ku/genética , PTEN Fosfo-Hidrolase/deficiência , Radiação Ionizante , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
17.
Biochem Biophys Res Commun ; 522(3): 612-617, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-31785812

RESUMO

Endoplasmic reticulum (ER) stress is a conserved cellular process for cells to clear unfolded or misfolded proteins and maintain cell homeostasis under stress conditions. Autophagy may act as a pro-survival strategy to cope with multiple stress conditions in tumor progression and distant metastasis. Although many studies have demonstrated that there is a close correlation between radiation-induced ER stress and autophagy, the molecular mechanisms currently remain unclear. In the present study, we performed an in vivo study concerning the effect of autophagy induced by ER stress on the radiosensitivity of mouse sarcoma using X-rays. Our results documented that X-rays could induce ER stress in sarcoma and then autophagy was activated by unfolded protein response (UPR) through the IRE1-JNK-pBcl2-Beclin1 signaling axis. The induction of autophagy caused a decline in cell apoptosis while inhibiting the autophagy resulted in increased apoptosis and inhibition of tumor progression. Combined treatment of X-ray exposure and chloroquine increased ER stress-related apoptosis and enhanced the radiosensitivity of mouse sarcoma that was not sensitive to X-ray irradiation alone. Thus, our study indicates that inhibition of ER stress-induced autophagy might be a novel strategy to improve the efficacy of radiotherapy against radioresistant sarcoma.


Assuntos
Antimaláricos/uso terapêutico , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Cloroquina/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos da radiação , Sarcoma/radioterapia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Tolerância a Radiação/efeitos dos fármacos
18.
Biochem Biophys Res Commun ; 522(4): 910-916, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31806377

RESUMO

As a cancer treatment strategy, irradiation therapy is widely used that can cause DNA breakage and increase free radicals, which leads to different types of cell death. Among them, apoptosis and autophagy are the most important and the most studied cell death processes. Although the exploration of the relationship between apoptosis and autophagy has been a major area of focus, still the molecular mechanisms of autophagy on apoptosis remain unclear. Here, we have revealed that apoptosis was enhanced by the death receptor 5 (DR5) pathway, and the effect of autophagy on apoptosis was promoted by DR5 interacting with LC3B as well as Caspase8 in gliomas after irradiation. Interestingly, we observed that the addition of four different autophagy inducers, rapamycin (RAP), CCI779, ABT737 and temozolomide (TMZ), induced the differences of DR5 expression and cell apoptosis after irradiation. Unlike RAP and CCI779, ABT737 and TMZ were able to increase DR5 expression and further induce cell death. Therefore, we have concluded that DR5 plays a novel and indispensable role in promoting cell apoptosis under irradiation and suggest a potential therapeutic approach for glioblastoma treatment.


Assuntos
Apoptose , Autofagia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Glioma/patologia , Glioma/radioterapia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose/genética , Apoptose/efeitos da radiação , Autofagia/genética , Autofagia/efeitos da radiação , Neoplasias Encefálicas/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Histonas/metabolismo , Humanos , Metilação , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Transcrição Genética
19.
J Photochem Photobiol B ; 201: 111653, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31710929

RESUMO

Autophagy is an important process for maintaining intracellular homeostasis. Our previous study demonstrated that autophagy was down-regulated in ultraviolet B (UVB)-irradiated keratinocytes. Raffinose is a natural oligosaccharide that serves as a novel activator of autophagy and as a balancing agent to regulate the diversity of environmental stress. However, whether raffinose balances ultraviolet stress through the autophagy activation pathway has yet to be established. In this study, we found that raffinose treatment inhibited the LDH release and trypan blue staining in UVB-challenged human keratinocytes cell line HaCaT but did not affect the cleavage of apoptotic markers Caspase-3 and PARP, as well as translocation into nucleus of other cell death markers Endonuclease G and AIF. Moreover, we confirmed that raffinose treatment enhanced autophagy flux in an MTOR-independent manner in HaCaT cells. Importantly, decrease of LC3-II turnover in UVB-irradiated keratinocytes could be rescued by raffinose treatment, indicating that raffinose treatment increased autophagy in UVB-irradiated HaCaT cells. Furthermore, the effect on cell death by raffinose was inhibited when autophagy was suppressed with either a small interfering RNA targeting ATG5 (siATG5) or autophagic inhibitor wortmannin. In conclusion, we demonstrated that raffinose increases MTOR-independent autophagy and reduces cell death in UVB-irradiated keratinocytes. Our study indicated that the natural agent raffinose presents the potential value in opposing photodamage.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Rafinose/farmacologia , Raios Ultravioleta , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
20.
ACS Appl Mater Interfaces ; 11(50): 46408-46418, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31729218

RESUMO

Recent studies suggest that cancer cell death accompanied by organelle dysfunction might be a promising approach for cancer therapy. The Golgi apparatus has a key role in cell function and may initiate signaling pathways to mitigate stress and, if irreparable, start apoptosis. It has been shown that Golgi disassembly and fragmentation under oxidative stress act as indicators for stress-mediated cell death pathways through cell cycle arrest in the G2/M phase. The present study shows that UV-induced reactive oxygen species (ROS) generation by Ag@ZnO nanoparticles (NPs) transform the Golgi structures from compressed perinuclear ribbons into detached vesicle-like structures distributed in the entire cytoplasm of melanoma cells. This study also demonstrates that Ag@ZnO NP-induced Golgi fragmentation cooccurs with G2 block of cell cycle progression, preventing cells from entering the mitosis phase. Additionally, the increased intracellular ROS production triggered by Ag@ZnO NPs upon UV exposure promoted autophagy. Taken together, Ag@ZnO NPs induce stress-related Golgi fragmentation and autophagy, finally leading to melanoma cell apoptosis. Intracellular oxidative stress generated by Ag@ZnO NPs upon UV irradiation may thus represent a targeted approach to induce cancer cell death through organelle destruction in melanoma cells, while fibroblast cells remained largely unaffected.


Assuntos
Proliferação de Células/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Melanoma/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Complexo de Golgi/genética , Humanos , Melanoma/genética , Melanoma/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Espécies Reativas de Oxigênio/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Prata/química , Prata/farmacologia , Raios Ultravioleta , Óxido de Zinco/química , Óxido de Zinco/farmacologia
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