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1.
Anticancer Res ; 41(10): 4885-4894, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34593436

RESUMO

BACKGROUND/AIM: Advanced undifferentiated pleomorphic sarcoma (UPS) has a poor prognosis and there are few treatments that can improve overall survival. Recently, Rapalink-1, a third-generation mammalian target of rapamycin (mTOR) kinase inhibitor, has been developed and shown to be effective against other tumours. However, mTOR inhibitors have been shown to induce autophagy and resistance to anti-cancer drugs. This study aimed to investigate the antitumor effects of Rapalink-1 with an autophagy inhibitor. MATERIALS AND METHODS: The antitumor effect of Rapalink-1 and/or hydroxychloroquine in three UPS cell lines was examined via cell viability analysis, western blotting, flow cytometry and immunofluorescence. RESULTS: Rapalink-1 decreased cell proliferation and inhibited the PI3K/mTOR pathway. Combined treatment with Rapalink-1 and hydroxychloroquine enhanced the antitumor effect compared to treatment with Rapalink-1 alone by blocking the autophagy-inducing effect of mTOR inhibitors. CONCLUSION: Combined treatment with Rapalink-1 and hydroxychloroquine may be used as a potential therapeutic agent against UPS.


Assuntos
Apoptose , Autofagia , Hidroxicloroquina/farmacologia , Sarcoma/patologia , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proliferação de Células , Inibidores Enzimáticos/farmacologia , Humanos , Sarcoma/tratamento farmacológico , Sarcoma/metabolismo , Sirolimo/farmacologia , Células Tumorais Cultivadas
2.
BMC Cancer ; 21(1): 1061, 2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34565342

RESUMO

BACKGROUND: Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. METHODS: Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. RESULTS: Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. CONCLUSIONS: Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.


Assuntos
Acetanilidas/farmacologia , Azepinas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445268

RESUMO

Sporadic lymphangioleiomyomatosis (S-LAM) is a rare lung disease characterized by the proliferation of smooth muscle-like LAM cells and progressive cystic destruction. Sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has a proven efficacy in patients with LAM. However, the therapeutic mechanisms of sirolimus in LAM remain unclear. We aimed to evaluate sirolimus-related lung parenchymal changes and the potential effect in LAM cells and modulating pathological cystic destruction. Lung specimens were examined for histopathological changes by HMB45 staining and compared the LAM patients treated with and without sirolimus. We detected the overexpression of mTOR, HMB45, and phosphorylation of cofilin (p-cofilin) in LAM patients. Sirolimus showed efficacy in patients with LAM, who exhibited a reduced expression of mTOR and p-cofilin as well as reduced interstitial septal thickness. In addition, sirolimus suppresses mTOR and p-cofilin, thus suppressing the migration and proliferation of LAM cells isolated from the patient's lung tissue. This study demonstrates that interstitial septal thickness, as determined by histological structural analysis. Sirolimus effectively reduced the expression of p-cofilin and interstitial septal thickness, which may be a novel mechanism by sirolimus. Moreover, we develop a new method to isolate and culture the LAM cell, which can test the possibility of medication in vitro and impact this current study has on the LAM field. The development of approaches to interfere with mTOR-cofilin1-actin signaling may result in an option for S-LAM therapy.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , Proteínas de Neoplasias/metabolismo , Sirolimo/farmacologia , Adulto , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/tratamento farmacológico , Linfangioleiomiomatose/patologia , Fosforilação/efeitos dos fármacos
4.
Eur J Neurosci ; 54(6): 6104-6122, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34405459

RESUMO

Long-lasting cognitive impairment is one of the most central negative consequences related to the exposure to cannabis during adolescence and particularly of Δ-9-tetrahydrocannabinol (THC). The aim of this study was to compare the protracted effects of adolescent versus late-adolescent chronic exposure to THC on short-term memory and plasticity and to examine whether rapamycin, a blocker of the mammalian target of rapamycin (mTOR) pathway, can restore THC-induced deficits in memory and plasticity. Male rats were injected with ascending doses of THC [2.5, 5, 10 mg/kg; intraperitoneally (i.p.)] during adolescence and late-adolescence (post-natal days 30-41 and 45-56, respectively), followed by daily injections of rapamycin (1 mg/kg, i.p.) during the first 10 days of cessation from THC. Thirty days after the last injection, rats were tested for short-term and working memory, anxiety-like behaviour, and plasticity in the pathways projecting from the ventral subiculum (vSub) of the hippocampus to the prefrontal cortex (PFC) and nucleus accumbens (NAc). THC exposure in adolescence, but not late-adolescence, was found to induce long-term deficits in object recognition short-term memory and synaptic plasticity in the hippocampal-accumbens pathway. Importantly, rapamycin rescued these persistent effects of THC administered during adolescence. Our findings show that some forms of memory and plasticity are sensitive to chronic THC administration during adolescence and that rapamycin administered during THC cessation may restore cognitive function and plasticity, thus potentially protecting against the possible long-term harmful effects of THC.


Assuntos
Dronabinol , Alucinógenos , Animais , Dronabinol/farmacologia , Hipocampo , Masculino , Córtex Pré-Frontal , Ratos , Sirolimo/farmacologia
5.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360785

RESUMO

Metabolic alteration is characteristic during tumour growth and therapy; however, targeting metabolic rewiring could overcome therapy resistance. mTOR hyperactivity, autophagy and other metabolic processes, including mitochondrial functions, could be targeted in breast cancer progression. We investigated the growth inhibitory mechanism of rapamycin + doxycycline treatment in human breast cancer model systems. Cell cycle and cell viability, including apoptotic and necrotic cell death, were analysed using flow cytometry, caspase activity measurements and caspase-3 immunostainings. mTOR-, autophagy-, necroptosis-related proteins and treatment-induced morphological alterations were analysed by WesTM, Western blot, immunostainings and transmission electron microscopy. The rapamycin + doxycycline combination decreased tumour proliferation in about 2/3rd of the investigated cell lines. The continuous treatment reduced tumour growth significantly both in vivo and in vitro. The effect after short-term treatment was reversible; however, autophagic vacuoles and degrading mitochondria were detected simultaneously, and the presence of mitophagy was also observed after the long-term rapamycin + doxycycline combination treatment. The rapamycin + doxycycline combination did not cause apoptosis or necrosis/necroptosis, but the alterations in autophagy- and mitochondria-related protein levels (LC3-B-II/I, p62, MitoTracker, TOM20 and certain co-stainings) were correlated to autophagy induction and mitophagy, without mitochondria repopulation. Based on these results, we suggest considering inducing metabolic stress and targeting mTOR hyperactivity and mitochondrial functions in combined anti-cancer treatments.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Doxiciclina/farmacologia , Feminino , Células HT29 , Humanos , Células MCF-7 , Mitocôndrias/patologia , Sirolimo/farmacologia
6.
Nat Commun ; 12(1): 4814, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376668

RESUMO

Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Glutamina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Exp Cell Res ; 406(1): 112729, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34242625

RESUMO

Phosphatase and tensin homolog (PTEN) deleted on human chromosome 10 is a tumor suppressor with bispecific phosphatase activity, which is often involved in the study of energy metabolism and tumorigenesis. PTEN is recently reported to participate in the process of acute injury. However, the mechanism of PTEN in Ischemia-Reperfusion Injury (IRI) has not yet been clearly elucidated. In this study, mice with bilateral renal artery ischemia-reperfusion and HK-2 cells with hypoxia/reoxygenation (H/R) were used as acute kidney injury models. We demonstrated that PTEN was downregulated in IRI-induced kidney as well as in H/R-induced HK-2 cells. By silencing and overexpressing PTEN with si-PTEN RNA and PHBLV-CMV-PTEN-flag lentivirus before H/R, we found that PTEN protected HK-2 cells against H/R-induced injury reflected by the change in cell activity and the release of LDH. Furthermore, we inhibited HIF1-α with PX-478 and inactivated mTOR with Rapamycin before the silence of PTEN in H/R model. Our data indicated that the renoprotective effect of PTEN worked via PI3K/Akt/mTOR pathway and PI3K/Akt/HIF1-α pathway, hence alleviating apoptosis and improving autophagy respectively. Our findings provide valuable insights into the molecular mechanism underlying renoprotection of PTEN on autophagy and apoptosis induced by renal IRI, which offers a novel therapeutic target for the treatment of AKI.


Assuntos
Injúria Renal Aguda/prevenção & controle , Autofagia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Traumatismo por Reperfusão/prevenção & controle , Serina-Treonina Quinases TOR/genética , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Apoptose/genética , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/metabolismo , Rim/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos de Mostarda/farmacologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Fenilpropionatos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
8.
Methods Mol Biol ; 2312: 253-276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228295

RESUMO

Recent studies revealed the biological significance of dynamic multicomponent assemblies of biomolecules inside living cells. Protein and nucleic acid assemblies are biomolecular condensates or non-membrane-bound organelles that have attracted increasing attention. Synthetic tools that manipulate the dynamic assembly/disassembly process of the structures are useful in elucidating both biophysical mechanisms of their assembly/disassembly and physiological roles of the condensates. In this report, general protocols to form and observe synthetic polymer-based condensates in living cells are described using the tool iPOLYMER. Taking advantage of the modular design of the tool, both chemical and light stimuli can induce formation of synthetic condensates inside living cells, which are observed by laser-scanning confocal microscopy. The experimental design described herein should help those who conduct experiments on synthetic manipulation of biomolecular condensates using iPOLYMER and other tools for synthetic manipulation of condensates. Technical notes for using iPOLYMER, including basic protocols of chemical- or light-inducible dimerization techniques (CID/LID), choice of proper control experiments, and advantages/disadvantages are also presented.


Assuntos
Engenharia Celular , Grânulos Citoplasmáticos/genética , Regulação da Expressão Gênica , Mimetismo Molecular , Optogenética , Peptídeos/genética , RNA/genética , Biologia Sintética , Antígeno-1 Intracelular de Células T/genética , Animais , Células COS , Técnicas de Cultura de Células , Chlorocebus aethiops , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células HEK293 , Humanos , Hidrogéis , Luz , Microscopia Confocal , Microscopia de Fluorescência , Peptídeos/metabolismo , Domínios Proteicos , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sirolimo/farmacologia , Antígeno-1 Intracelular de Células T/metabolismo , Transfecção
9.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209274

RESUMO

Fetal alcohol spectrum disorders (FASDs) are one of the most common consequences of ethanol exposure during pregnancy. In adulthood, these disorders can be manifested by learning and memory deficits and depressive-like behavior. Ethanol-induced oxidative stress may be one of the factors that induces FASD development. The mammalian target of the Rapamycin (mTOR) signaling pathway that acts via two distinct multiprotein complexes, mTORC1 and mTORC2, can affect oxidative stress. We investigated whether mTOR-dependent or mTOR-independent mechanisms are engaged in this phenomenon. Thus, Rapamycin-a selective inhibitor of mTORC1, Torin-2-a non-selective mTORC1/mTORC2 inhibitor, and FK-506-a drug that impacts oxidative stress in an mTOR-independent manner were used. Behavioral tests were performed in adult (PND60-65) rats using a passive avoidance (PA) task (aversive learning and memory) and forced swimming test (FST) (depressive-like behaviors). In addition, the biochemical parameters of oxidative stress, such as lipid peroxidation (LPO), as well as apurinic/apyrimidinic (AP)-sites were determined in the hippocampus and prefrontal cortex in adult (PND65) rats. The rat FASD model was induced by intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4-9 (an equivalent to the third trimester of human pregnancy). All substances (3 mg/kg) were given 30 min before ethanol. Our results show that neonatal ethanol exposure leads to deficits in context-dependent fear learning and depressive-like behavior in adult rats that were associated with increased oxidative stress parameters in the hippocampus and prefrontal cortex. Because these effects were completely reversed by Rapamycin, an mTORC1 inhibitor, this outcome suggests its usefulness as a preventive therapy in disorders connected with prenatal ethanol exposure.


Assuntos
Comportamento Animal/efeitos dos fármacos , Depressão , Transtornos do Espectro Alcoólico Fetal , Deficiências da Aprendizagem , Estresse Oxidativo/efeitos dos fármacos , Sirolimo/farmacologia , Animais , Depressão/metabolismo , Depressão/fisiopatologia , Depressão/prevenção & controle , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Transtornos do Espectro Alcoólico Fetal/prevenção & controle , Deficiências da Aprendizagem/metabolismo , Deficiências da Aprendizagem/fisiopatologia , Deficiências da Aprendizagem/prevenção & controle , Ratos , Ratos Wistar
10.
Zool Res ; 42(4): 482-486, 2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34235896

RESUMO

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that begins with defective rod photoreceptor function, followed by impaired cone function, and complete blindness in its late stage. To date, however, there is no effective treatment for RP. By carrying a nonsense mutation in the Pde6b gene, rd1 mice display elevated cGMP in conjunction with higher intracellular Ca 2+ in their rod photoreceptors, resulting in fast retinal degeneration. Ca 2+ has been linked to activation of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway integrates extracellular and intracellular signals to sense the supply of nutrients and plays a central role in regulating protein and lipid synthesis as well as apoptosis and autophagy. In the present study, we showed that mTOR and phosphorylated mTOR (p-mTOR, activated form of mTOR) are up-regulated in rd1 photoreceptors at postnatal day 10 (P10), a pre-degenerative stage. Moreover, the downstream effectors of mTOR, such as pS6K and S6K, are also increased, suggesting activation of the mTOR signaling pathway. Intravitreal administration of rapamycin, a negative regulator of mTOR, inhibits the mTOR pathway in rd1 photoreceptors. Consequently, the progression of retinal degeneration is slower and retinal function is enhanced, possibly mediated by activation of autophagy in the photoreceptors. Taken together, these results highlight rapamycin as a potential therapeutic avenue for retinal degeneration.


Assuntos
Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/prevenção & controle , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/patologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Camundongos , Degeneração Retiniana/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sirolimo/uso terapêutico
11.
Nat Commun ; 12(1): 4407, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315870

RESUMO

Alcohol Use Disorder (AUD) affects a large portion of the population. Unfortunately, efficacious medications to treat the disease are limited. Studies in rodents suggest that mTORC1 plays a crucial role in mechanisms underlying phenotypes such as heavy alcohol intake, habit, and relapse. Thus, mTORC1 inhibitors, which are used in the clinic, are promising therapeutic agents to treat AUD. However, chronic inhibition of mTORC1 in the periphery produces undesirable side effects, which limit their potential use for the treatment of AUD. To overcome these limitations, we designed a binary drug strategy in which male mice were treated with the mTORC1 inhibitor RapaLink-1 together with a small molecule (RapaBlock) to protect mTORC1 activity in the periphery. We show that whereas RapaLink-1 administration blocked mTORC1 activation in the liver, RapaBlock abolished the inhibitory action of Rapalink-1. RapaBlock also prevented the adverse side effects produced by chronic inhibition of mTORC1. Importantly, co-administration of RapaLink-1 and RapaBlock inhibited alcohol-dependent mTORC1 activation in the nucleus accumbens and attenuated alcohol seeking and drinking.


Assuntos
Consumo de Bebidas Alcoólicas/patologia , Encéfalo/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Animais , Intolerância à Glucose/complicações , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Especificidade de Órgãos , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Perda de Peso/efeitos dos fármacos
12.
ACS Chem Neurosci ; 12(15): 2917-2928, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34264648

RESUMO

Neuropathic pain is a chronic condition with little specific treatment. Insulin-like growth factor 1 (IGF1), interacting with its receptor, IGF1R, serves a vital role in neuronal and brain functions such as autophagy and neuroinflammation. Yet, the function of spinal IGF1/IGF1R in neuropathic pain is unclear. Here, we examined whether and how spinal IGF1 signaling affects pain-like behaviors in mice with chronic constriction injury (CCI) of the sciatic nerve. To corroborate the role of IGF1, we injected intrathecally IGF1R inhibitor (nvp-aew541) or anti-IGF1 neutralizing antibodies. We found that IGF1 (derived from astrocytes) in the lumbar cord increased along with the neuropathic pain induced by CCI. IGF1R was predominantly expressed on neurons. IGF1R antagonism or IGF1 neutralization attenuated pain behaviors induced by CCI, relieved mTOR-related suppression of autophagy, and mitigated neuroinflammation in the spinal cord. These findings reveal that the abnormal IGF1/IGF1R signaling contributes to neuropathic pain by exacerbating autophagy dysfunction and neuroinflammation.


Assuntos
Fator de Crescimento Insulin-Like I , Neuralgia , Animais , Autofagia , Camundongos , Neuralgia/tratamento farmacológico , Transdução de Sinais , Sirolimo/farmacologia , Medula Espinal
13.
Ageing Res Rev ; 70: 101376, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34089901

RESUMO

Aging can not only shorten a healthy lifespan, but can also lead to multi-organ dysfunction and failure. Anti-aging is a complex and worldwide conundrum for eliminating the various pathologies of senility. The past decade has seen great progress in the understanding of the aging-associated signaling pathways and their application for developing anti-aging approaches. Currently, some drugs can improve quality of life. The activation of mammalian target of rapamycin (mTOR) signaling is one of the core and detrimental mechanisms related to aging; rapamycin can reduce the rate of aging, improve age-related diseases by inhibiting the mTOR pathway, and prolong lifespan and healthspan effectively. However, the current evidence for rapamycin in lifespan extension and organ aging is fragmented and scattered. In this review, we summarize the efficacy and safety of rapamycin in prolonging a healthy lifespan by systematically alleviating aging in multiple organ systems, i.e., the nervous, urinary, digestive, circulatory, motor, respiratory, endocrine, reproductive, integumentary and immune systems, to provide a theoretical basis for the future clinical application of rapamycin in anti-aging.


Assuntos
Envelhecimento , Sirolimo , Humanos , Longevidade , Qualidade de Vida , Transdução de Sinais , Sirolimo/farmacologia , Sirolimo/uso terapêutico
14.
Am J Physiol Cell Physiol ; 321(1): C176-C186, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34106788

RESUMO

Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, patients with MELAS treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in patients with MELAS are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared with control, healthy individuals. We observed that mitochondria from patients were fragmented, had a threefold decline in the average speed of motility, a twofold reduced mitochondrial membrane potential, and a 1.5- to 2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared with healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 h resulted in increased mitochondrial respiration compared with control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.


Assuntos
Fibroblastos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Síndrome MELAS/genética , Mitocôndrias/efeitos dos fármacos , Sirolimo/farmacologia , Estudos de Casos e Controles , Pré-Escolar , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Lactente , Lisossomos/metabolismo , Síndrome MELAS/tratamento farmacológico , Síndrome MELAS/metabolismo , Síndrome MELAS/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Adulto Jovem
15.
Front Immunol ; 12: 637469, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34113339

RESUMO

ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4+ and CD8+ T cells, and the emergence of a large population of CD4-CD8- (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment.


Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Fígado/imunologia , Sirolimo/farmacologia , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/biossíntese , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Células Estreladas do Fígado/metabolismo , Hepatite/imunologia , Hepatócitos/metabolismo , Antígenos de Histocompatibilidade Classe II/biossíntese , Tolerância Imunológica/efeitos dos fármacos , Macrófagos do Fígado/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Ovalbumina/imunologia , Poliésteres , Receptor de Morte Celular Programada 1/biossíntese
16.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065421

RESUMO

Dendritic cells (DCs) are unique immune cells that can link innate and adaptive immune responses and Immunometabolism greatly impacts their phenotype. Rapamycin is a macrolide compound that has immunosuppressant functions and is used to prevent graft loss in kidney transplantation. The current study evaluated the therapeutic potential of ex-vivo rapamycin treated DCs to protect kidneys in a mouse model of acute kidney injury (AKI). For the rapamycin single (S) treatment (Rapa-S-DC), Veh-DCs were treated with rapamycin (10 ng/mL) for 1 h before LPS. In contrast, rapamycin multiple (M) treatment (Rapa-M-DC) were exposed to 3 treatments over 7 days. Only multiple ex-vivo rapamycin treatments of DCs induced a persistent reprogramming of mitochondrial metabolism. These DCs had 18-fold more mitochondria, had almost 4-fold higher oxygen consumption rates, and produced more ATP compared to Veh-DCs (Veh treated control DCs). Pathway analysis showed IL10 signaling as a major contributing pathway to the altered immunophenotype after Rapamycin treatment compared to vehicle with significantly lower cytokines Tnfa, Il1b, and Il6, while regulators of mitochondrial content Pgc1a, Tfam, and Ho1 remained elevated. Critically, adoptive transfer of rapamycin-treated DCs to WT recipients 24 h before bilateral kidney ischemia significantly protected the kidneys from injury with a significant 3-fold improvement in kidney function. Last, the infusion of DCs containing higher mitochondria numbers (treated ex-vivo with healthy isolated mitochondria (10 µg/mL) one day before) also partially protected the kidneys from IRI. These studies demonstrate that pre-emptive infusion of ex-vivo reprogrammed DCs that have higher mitochondria content has therapeutic capacity to induce an anti-inflammatory regulatory phenotype to protect kidneys from injury.


Assuntos
Injúria Renal Aguda/tratamento farmacológico , Células Dendríticas/efeitos dos fármacos , Isquemia/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Sirolimo/farmacologia , Injúria Renal Aguda/metabolismo , Transferência Adotiva/métodos , Animais , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Isquemia/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Mol Immunol ; 137: 11-19, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34182227

RESUMO

OBJECTIVE: To investigate the influence of tolerance dendritic cells (tolDCs), generated from Bone marrow mesenchymal stem cells (BM-MSCs) treated with rapamycin (Rapa) on liver allograft survival in a rat acute liver transplantation model. METHODS: Different GM-CSF induction project was used to obtain immature DCs (imDCs), mature DCs (matDCs) or tolDCs from BM-MSCs. First, MLR was performed to analyze the activity of tolDCs on polyclonaly stimulated total T cells. Then, co-cultured imDCs, matDCs and tolDCs with CD8+T cells isolated by magnetic activated cell sorting to analyze the influence on its regulatory characteristic. Last, the established rat acute liver transplantation model were adoptive transfused with imDCs, matDCs or tolDCs isolated by anti-CD11c immunomagnetic beads. The phenotype of DC cells and level of CD8+Treg in the culture system and in vivo, the expression of CD8 and CD45RC in the tissues were analyzed by flow cytometry and immunohistochemistry, respectively. RESULTS: The loGM-CSF plus IL-4 decreased the costimulatory molecules of CD80/86 and MHC class II of DCs comparison with hiGM-CSF from BM-MSCs no matter whether stimulation by LPS (P<0.05). Rapa treated not only reduced the expression of CD80/86 and MHC class II but also down-regulated the expression of CD11c after LPS stimulation which was more obviously in tolDCs by loGM-CSF project (P<0.05). Moreover, tolDCs displayed a rather higher level of IL-10 and low level of IL-12p70 than others (P<0.01), which shown a rather lower stimulative effect on the proliferation of T cells comparison with matDCs and imDCs. Co-cultured with CD8+Treg showed an improvement on induction of CD8+TCR+CD45RC-T cells (CD8+Treg) in ex vivo. The rats transfused with tolDCs has a delayed survival benefits with high level of CD8+Tregs (P<0.01) and high expression of CD45RC in liver tissue (P<0.01) and spleen when comparison with other groups. The infused tolDCs improved a mean survival time (MST) of 32 days comparison with a MTS of 9.5 days and 15.75 days displayed by rat that per-infused with matDCs and imDCs, respectively. CONCLUSION: Rapa modified tolDCs derived from BM-MSCs reversed graft rejection by improve tolerance characteristics of CD8+CD45RC-Treg in acute liver rat transplantation.


Assuntos
Medula Óssea/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Antígenos Comuns de Leucócito/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Sirolimo/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Medula Óssea/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Transplante de Fígado/métodos , Células-Tronco Mesenquimais/metabolismo , Ratos , Linfócitos T Reguladores/metabolismo , Transplante Homólogo/métodos
18.
Methods Mol Biol ; 2277: 143-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080150

RESUMO

Mice missing the Complex I subunit NADH:Ubiquinone Oxidoreductase Fe-S Protein 4 (NDUFS4) of the electron transport chain are a leading model of the severe mitochondrial disease Leigh syndrome. These mice have enabled a better understanding of mitochondrial dysfunction in human disease, as well as in the discovery of interventions that can potentially suppress mitochondrial disease manifestations. In addition, increasing evidence suggests significant overlap between interventions that increase survival in NDUFS4 knockout mice and that extend life span during normative aging. This chapter discusses the practical aspects of handling and studying these mice, which can be challenging due to their severe disease phenotype. Common procedures such as breeding, genotyping, weaning, or treating these transgenic mice are also discussed.


Assuntos
Envelhecimento/genética , Ração Animal , Complexo I de Transporte de Elétrons/genética , Camundongos Knockout , Envelhecimento/fisiologia , Animais , Feminino , Técnicas de Genotipagem , Humanos , Doença de Leigh/genética , Masculino , Doenças Mitocondriais/genética , Sirolimo/farmacologia
19.
Nat Commun ; 12(1): 3660, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135321

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagAGTP mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state.


Assuntos
Jejum/metabolismo , Fígado/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Animais , Modelos Animais de Doenças , Glucose/metabolismo , Homeostase , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Nutrientes/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Fenótipo , Proteômica , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Transcrição Genética/efeitos dos fármacos , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo
20.
Front Cell Infect Microbiol ; 11: 676183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34123875

RESUMO

Deficiency in memory formation and increased immunosenescence are pivotal features of Trypanosoma cruzi infection proposed to play a role in parasite persistence and disease development. The vaccination protocol that consists in a prime with plasmid DNA followed by the boost with a deficient recombinant human adenovirus type 5, both carrying the ASP2 gene of T. cruzi, is a powerful strategy to elicit effector memory CD8+ T-cells against this parasite. In virus infections, the inhibition of mTOR, a kinase involved in several biological processes, improves the response of memory CD8+ T-cells. Therefore, our aim was to assess the role of rapamycin, the pharmacological inhibitor of mTOR, in CD8+ T response against T. cruzi induced by heterologous prime-boost vaccine. For this purpose, C57BL/6 or A/Sn mice were immunized and daily treated with rapamycin for 34 days. CD8+ T-cells response was evaluated by immunophenotyping, intracellular staining, ELISpot assay and in vivo cytotoxicity. In comparison with vehicle-injection, rapamycin administration during immunization enhanced the frequency of ASP2-specific CD8+ T-cells and the percentage of the polyfunctional population, which degranulated (CD107a+) and secreted both interferon gamma (IFNγ) and tumor necrosis factor (TNF). The beneficial effects were long-lasting and could be detected 95 days after priming. Moreover, the effects were detected in mice immunized with ten-fold lower doses of plasmid/adenovirus. Additionally, the highly susceptible to T. cruzi infection A/Sn mice, when immunized with low vaccine doses, treated with rapamycin, and challenged with trypomastigote forms of the Y strain showed a survival rate of 100%, compared with 42% in vehicle-injected group. Trying to shed light on the biological mechanisms involved in these beneficial effects on CD8+ T-cells by mTOR inhibition after immunization, we showed that in vivo proliferation was higher after rapamycin treatment compared with vehicle-injected group. Taken together, our data provide a new approach to vaccine development against intracellular parasites, placing the mTOR inhibitor rapamycin as an adjuvant to improve effective CD8+ T-cell response.


Assuntos
Vacinas Protozoárias , Trypanosoma cruzi , Animais , Linfócitos T CD8-Positivos , Camundongos , Camundongos Endogâmicos C57BL , Sirolimo/farmacologia , Vacinação
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