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1.
Int J Mol Med ; 51(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36382649

RESUMO

Excessive proliferation and migration of fibroblasts in the lumbar laminectomy area can lead to epidural fibrosis, eventually resulting in failed back surgery syndrome. It has been reported that laminin α1, a significant biofunctional glycoprotein in the extracellular matrix, is involved in several fibrosis­related diseases, such as pulmonary, liver and keloid fibrosis. However, the underlying mechanism of laminin α1 in epidural fibrosis remains unknown. The present study aimed to explore the effect and mechanism of laminin α1 in fibroblast proliferation, apoptosis and migration, and epidural fibrosis. Following the establishment of a laminectomy model, hematoxylin and eosin, Masson's trichrome and immunohistochemical staining were performed to determine the degree of epidural fibrosis, the number of fibroblasts, collagen content and the epidural expression levels of laminin α1, respectively. Furthermore, a stable small interfering RNA system was used to knock down the expression of laminin α1 in fibroblasts. The transfection efficiency was confirmed by reverse transcription­quantitative PCR and immunofluorescence staining. Western blot analysis, scratch wound assay, EdU incorporation assay, flow cytometric analysis and Cell Counting Kit 8 assay were performed to assess the proliferation, apoptosis, migration and viability of fibroblasts, as well as the expression levels of the AKT/mechanistic target of rapamycin (mTOR) signaling­related proteins. In vivo experiments revealed that laminin α1 was positively and time­dependently associated with epidural fibrosis. In addition, laminin α1 knockdown attenuated cell proliferation, viability and migration, and promoted apoptosis. Furthermore, the results revealed that the activation of the AKT/mTOR signaling pathway was involved in the aforementioned processes. Overall, the current study illustrated the positive association between laminin α1 and epidural fibrosis, and also verified the effect of laminin α1 on fibroblast proliferation, apoptosis and migration. Furthermore, the results suggested that the AKT/mTOR signaling pathway may serve a significant role in regulating the behavior of laminin α1­induced fibroblasts.


Assuntos
Espaço Epidural , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose , Espaço Epidural/patologia , Serina-Treonina Quinases TOR/metabolismo , Fibroblastos/metabolismo , Proliferação de Células , Sirolimo/farmacologia
2.
Behav Brain Res ; 437: 114129, 2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36179804

RESUMO

Evidence has demonstrated the hippocampal cholinergic system and the mammalian target of rapamycin (mTOR) participation during the memory formation of aversive events. This study assessed the role of these systems in the hippocampus for the extinction memory process by submitting male Wistar rats to fear-motivated step-down inhibitory avoidance (IA). The post-extinction session administration of the nicotinic and muscarinic cholinergic receptor antagonists, mecamylamine and scopolamine, respectively, both at doses of 2 µg/µl/side, and rapamycin, an mTOR inhibitor (0.02 µg/µl/side), into the CA1 region of the dorsal hippocampus, impaired the IA extinction memory. Furthermore, the nicotinic and muscarinic cholinergic receptor agonists, nicotine and muscarine, respectively, had a dose-dependent effect on the IA extinction memory when administered intra-CA1, immediately after the extinction session. Nicotine (0.6 µg/µl/side) and muscarine (0.02 µg/µl/side), respectively, had no effect, while the higher doses (6 and 2 µg/µl/side, respectively) impaired the IA extinction memory. Interestingly, the co-administration of muscarine at the lower dose blocked the impairment that was induced by rapamycin. This effect was not observed when nicotine at the lower dose was co-administered. These results have demonstrated the participation of the cholinergic receptors and mTOR in the hippocampus for IA extinction, and that the cholinergic agonists had a dose-dependent effect on the IA extinction memory. This study provides insights related to the behavioural aspects and the neurobiological properties underlying the early stage of fear-motivated IA extinction memory consolidation and suggests that there is hippocampal muscarinic receptor participation independent of mTOR in this memory process.


Assuntos
Aprendizagem da Esquiva , Extinção Psicológica , Medo , Hipocampo , Memória , Receptores Colinérgicos , Serina-Treonina Quinases TOR , Animais , Masculino , Ratos , Aprendizagem da Esquiva/fisiologia , Medo/fisiologia , Hipocampo/metabolismo , Muscarina/farmacologia , Antagonistas Muscarínicos/farmacologia , Nicotina/farmacologia , Ratos Wistar , Receptores Colinérgicos/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Extinção Psicológica/fisiologia , Memória/fisiologia
4.
Yakugaku Zasshi ; 142(11): 1191-1199, 2022.
Artigo em Japonês | MEDLINE | ID: mdl-36328449

RESUMO

The role of ß-estradiol (E2) in lipoprotein metabolism in mammary tumors remains unknown. Therefore the effect of E2 on secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells was examined. The E2-treated FM3A cells increased active LPL secretion in a time- and dose-dependent manner. The activity of mitogen-activated protein kinase (MAPK) was elevated in the tumor cells treated with E2, and E2-stimulated secretion of LPL was suppressed by the MAPK kinase 1/2 inhibitor PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor FR180204, p38 MAPK inhibitor SB202190, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In addition, the effect of E2 on active LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but not by the mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed the secretion of LPL by E2. Stimulatory secretion of LPL by E2 from the tumor cells is closely associated with activation of mTORC2 rather than mTORC1, possibly via the MAPK cascade.


Assuntos
Complexos Multiproteicos , Fosfatidilinositol 3-Quinases , Animais , Camundongos , Estradiol/farmacologia , Lipoproteínas , Mamíferos/genética , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno , Sirolimo/farmacologia , Neoplasias da Mama/metabolismo
5.
Front Immunol ; 13: 1020165, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36389788

RESUMO

Background: Immunocompromised (IC) patients show diminished immune response to COVID-19 mRNA vaccines (Co-mV). To date, there is no 'empirical' evidence to link the perturbation of translation, a rate-limiting step for mRNA vaccine efficiency (VE), to the dampened response of Co-mV. Materials and methods: Impact of immunosuppressants (ISs), tacrolimus (T), mycophenolate (M), rapamycin/sirolimus (S), and their combinations on Pfizer Co-mV translation were determined by the Spike (Sp) protein expression following Co-mV transfection in HEK293 cells. In vivo impact of ISs on SARS-CoV-2 spike specific antigen (SpAg) and associated antibody levels (IgGSp) in serum were assessed in Balb/c mice after two doses (2D) of the Pfizer vaccine. Spike Ag and IgGSp levels were assessed in 259 IC patients and 50 healthy controls (HC) who received 2D of Pfizer or Moderna Co-mV as well as in 67 immunosuppressed solid organ transplant (SOT) patients and 843 non-transplanted (NT) subjects following three doses (3D) of Co-mV. Higher Co-mV concentrations and transient drug holidays were evaluated. Results: We observed significantly lower IgGSP response in IC patients (p<0.0001) compared to their matched controls in 2D and 3D Co-mV groups. IC patients on M or S showed a profound dampening of IgGSP response relative to those that were not on these drugs. M and S, when used individually or in combination, significantly attenuated the Co-mV-induced Sp expression, whereas T did not exert significant influence. Sirolimus combo pretreatment in vivo significantly attenuated the Co-mV induced IgMSp and IgGSp production, which correlated with a decreasing trend in the early levels (after day 1) of Co-mV induced Sp immunogen levels. Neither higher Co-mV concentrations (6µg) nor withholding S for 1-day could overcome the inhibition of Sp protein levels. Interestingly, 3-days S holiday or using T alone rescued Sp levels in vitro. Conclusions: This is the first study to demonstrate that ISs, sirolimus and mycophenolate inhibited Co-mV-induced Sp protein synthesis via translation repression. Selective use of tacrolimus or drug holiday of sirolimus can be a potential means to rescue translation-dependent Sp protein production. These findings lay a strong foundation for guiding future studies aimed at improving Co-mV responses in high-risk IC patients.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Camundongos , Animais , Humanos , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico , Células HEK293 , COVID-19/prevenção & controle , SARS-CoV-2 , Imunoglobulina G , Sirolimo/farmacologia , Sirolimo/uso terapêutico
6.
BMC Cancer ; 22(1): 1193, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402986

RESUMO

The incidence of primary liver tumors, hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma (ICC), and combined HCC/ICC (cHCC/CC) is increasing. For ICC, targeted therapy exists only for a small subpopulation of patients, while for HCC, Sorafenib and Lenvatinib are in use. Diagnosis of cHCC/CC is a great challenge and its incidence is underestimated, bearing the risk of unintended non-treatment of ICC. Here, we investigated effects of targeted inhibitors on human ICC cell lines (HUH28, RBE, SSP25), in comparison to extrahepatic (E)CC lines (EGI1, CCC5, TFK1), and HCC/hepatoblastoma cell lines (HEP3B, HUH7, HEPG2). Cells were challenged with: AKT inhibitor MK-2206; multikinase inhibitors Sorafenib, Lenvatinib and Dasatinib; PI3-kinase inhibitors BKM-120, Wortmannin, LY294002, and CAL-101; and mTOR inhibitor Rapamycin. Dosage of the substances was based on the large number of published data of recent years. Proliferation was analyzed daily for four days. All cell lines were highly responsive to MK-2206. Thereby, MK-2206 reduced expression of phospho(p)-AKT in all ICC, ECC, and HCC lines, which mostly corresponded to reduction of p-mTOR, whereas p-ERK1/2 was upregulated in many cases. Lenvatinib showed inhibitory effects on the two HCC cell lines, but not on HEPG2, ICCs and ECCs. Sorafenib inhibited proliferation of all cells, except the ECC line CCC5. However, at reduced dosage, we observed increased cell numbers in some ICC experiments. Dasatinib was highly effective especially in ICC cell lines. Inhibitory effects were observed with all four PI3-kinase inhibitors. However, cell type-specific differences were also evident here. Rapamycin was most effective in the two HCC cell lines. Our studies show that the nine inhibitors differentially target ICC, ECC, and HCC/hepatoblastoma lines. Caution should be taken with Lenvatinib and Sorafenib administration in patients with cHCC/CC as the drugs may have no effects on, or might even stimulate, ICC.


Assuntos
Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Colangiocarcinoma , Hepatoblastoma , Neoplasias Hepáticas , Humanos , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/patologia , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Dasatinibe/uso terapêutico , Colangiocarcinoma/patologia , Fosfatidilinositol 3-Quinases , Sirolimo/farmacologia , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia
7.
Sci Rep ; 12(1): 19948, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402829

RESUMO

Severe malnutrition accounts for half-a-million deaths annually in children under the age of five. Despite improved WHO guidelines, inpatient mortality remains high and is associated with metabolic dysfunction. Previous studies suggest a correlation between hepatic metabolic dysfunction and impaired autophagy. We aimed to determine the role of mTORC1 inhibition in a murine model of malnutrition-induced hepatic dysfunction. Wild type weanling C57/B6 mice were fed a 18 or 1% protein diet for two weeks. A third low-protein group received daily rapamycin injections, an mTORC1 inhibitor. Hepatic metabolic function was assessed by histology, immunofluorescence, gene expression, metabolomics and protein levels. Low protein-fed mice manifested characteristics of severe malnutrition, including weight loss, hypoalbuminemia, hypoglycemia, hepatic steatosis and cholestasis. Low protein-fed mice had fewer mitochondria and showed signs of impaired mitochondrial function. Rapamycin prevented hepatic steatosis, restored ATP levels and fasted plasma glucose levels compared to untreated mice. This correlated with increased content of LC3-II, and decreased content mitochondrial damage marker, PINK1. We demonstrate that hepatic steatosis and disturbed mitochondrial function in a murine model of severe malnutrition can be partially prevented through inhibition of mTORC1. These findings suggest that stimulation of autophagy could be a novel approach to improve metabolic function in severely malnourished children.


Assuntos
Fígado Gorduroso , Desnutrição , Camundongos , Animais , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Serina-Treonina Quinases TOR , Desnutrição/complicações , Sirolimo/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina
8.
Int J Nanomedicine ; 17: 5049-5061, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36325149

RESUMO

Background: Transgenic C57BL/6-APC(Min/+) spontaneous cancer mouse model and the Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS) chemically induced orthotopic colorectal cancer mouse model represented distinct pathogenesis of colorectal cancers. Our previous study revealed that the combination of Rapamycin liposomes (Rapa/Lps) and 5-Fluorouracil (5-FU) has anti-colorectal cancer effects. However, the therapeutic efficacy of Rapa/Lps and 5-FU in other colorectal cancer mice models is yet to be thoroughly explored. The purpose of this study was to investigate the anti-tumor effect of Rapa/Lps combined with 5-FU in vivo and in vitro. Methods: In this study, we evaluated the effect of Rapa/Lps and 5-FU on APC (Min/+) mice and AOM/DSS-induced colorectal cancer mice. The small intestine, colorectum, serum, and plasma of mice in each group were collected following sacrifice to record the number of tumors. HE staining was utilized for observing pathological damage to intestine tissues. Tube formation assay, Transwell assay, wound healing assay, Western Blot were used to explore the anti-angiogenesis effect of drugs in HUVECs. Results: As expected, Rapa/Lps and 5-FU significantly suppressed tumor formation, decreased the number of tumors, and tumor load both in two mouse models, and had no influence on mouse weight. Mechanically, the anti-tumor effect of the drug also was associated in inhibiting angiogenesis and proliferation. Furthermore, we found that Rapa/Lps obviously inhibited HUVECs tube formation and migration. Conclusion: Altogether, we revealed the Rapa/Lps synergism with 5-FU decreased colon and small intestinal tumorigenesis in AOM/DSS-treated and APC (Min/+) mice, respectively, and correlated with anti-angiogenesis.


Assuntos
Colite , Neoplasias Colorretais , Camundongos , Animais , Azoximetano/toxicidade , Azoximetano/uso terapêutico , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Lipossomos/uso terapêutico , Sulfato de Dextrana/toxicidade , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Lipopolissacarídeos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Colite/induzido quimicamente
9.
Drug Des Devel Ther ; 16: 3817-3828, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36388085

RESUMO

Purpose: Mammalian Target of rapamycin (mTOR) plays a central role in regulating cell growth, proliferation, and cell cycle. The key component of mTORC2 is highly expressed in docetaxel-resistant prostate cells. However, the underlying molecular effects on prostate cells remain unclear. Methods: A docetaxel-resistant human prostate cell line (PC-3/DTX) was constructed to investigate the role of mTORC2 in docetaxel resistance. The lentivirus was transfected into cells to knock down the expression of Rictor, and cell viability was measured by Cell Counting Kit 8 (CCK-8). Flow cytometry was used to analyze the cell cycle, and the changes in related signal cascades were assessed by immunohistochemistry (IHC) staining and Western blot. Results: Docetaxel showed the lowest IC50 (50% inhibitory concentration) in PC-3/DTX cells with sh-RNA. Decreased Rictor expression resulted in a larger proportion of arrested cells in the G0/G1 phase in PC-3/DTX cells. The IC50 values of the AZD8055 group were lower than in the Rapamycin group when treated with docetaxel again. Furthermore, a larger proportion of PC-3/DTX cells were arrested in the G0/G1 phase in the AZD8055 group compared to the Rapamycin group. The IHC results of the prostate cancer tissues from a CRPC patient revealed the over expression of Rictor only, while Raptor expression was unaffected. Conclusion: We investigated the role of mTORC2 signaling on the acquired docetaxel -resistant PC-3 cells to identify potential methods for clinical treatment. MTORC2 expression is essential for docetaxel drug resistance of PC-3 cells. The mTORC1/2 inhibitor AZD8055 caused more significant disruption of mTORC2 kinase activity than the mTORC1 inhibitor Rapamycin, which lead to decreased docetaxel-mediated resistance. Therefore, reversing docetaxel resistance, may become a therapeutic option in the treatment of mCRPC patients.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Docetaxel/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Linhagem Celular Tumoral , Alvo Mecanístico do Complexo 2 de Rapamicina , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Sirolimo/farmacologia
10.
Endocrinology ; 164(1)2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36320101

RESUMO

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors suppress adrenal cortical carcinoma cell proliferation and cortisol production; the relationship between mTOR and aldosterone production has not been examined. METHODS: HAC15 cells were incubated with an mTOR activator and several inhibitors including AZD8055 (AZD) in the presence and absence of angiotensin II (AngII). The expression of rapamycin-sensitive adapter protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), adaptor proteins of mTOR complex 1 and 2, respectively, were studied in the HAC15 cells and deleted by CRISPR/gRNA. RESULTS: The mTOR inhibitors decreased aldosterone induced by AngII. Inhibition of mTOR by AZD significantly suppressed AngII-induced aldosterone and cortisol formation in a dose-dependent manner, whereas the mTOR activator MHY had no effect. AZD did not alter forskolin-induced aldosterone production showing that it is specific to the AngII signaling pathway. AngII-mediated ERK and mTOR activation were suppressed by AZD, along with a concomitant dose-dependent reduction of AngII-induced steroidogenic enzymes including steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase-type 2, CYP17A1, and aldosterone synthase protein. Furthermore, mTOR components ribosomal protein S6 kinase (P70S6K) and protein kinase B phosphorylation levels were decreased by AZD. As mTOR exerts its main effects by forming complexes with adaptor proteins Raptor and Rictor, the roles of these individual complexes were studied. We found an increase in the phosphorylation of Raptor and Rictor by AngII and that their CRISPR/gRNA-mediated knockdown significantly attenuated AngII-induced aldosterone and cortisol production. CONCLUSION: mTOR signaling has a critical role in transducing the AngII signal initiating aldosterone and cortisol synthesis in HAC15 cells and that inhibition of mTOR could be a therapeutic option for conditions associated with excessive renin-angiotensin system-mediated steroid synthesis.


Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Humanos , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Aldosterona/metabolismo , Hidrocortisona/metabolismo , Sirolimo/farmacologia , RNA Guia , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Serina-Treonina Quinases TOR
11.
Aging (Albany NY) ; 14(20): 8140-8149, 2022 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-36332147

RESUMO

Making provocative headlines, three outstanding publications demonstrated that early-life treatment with rapamycin, including treatments during developmental growth, extends lifespan in animals, confirming predictions of hyperfunction theory, which views aging as a quasi-program (an unintended continuation of developmental growth) driven in part by mTOR. Despite their high theoretical importance, clinical applications of two of these studies in mice, Drosophila and Daphnia cannot be implemented in humans because that would require growth retardation started at birth. A third study demonstrated that a transient (around 20% of total lifespan in Drosophila) treatment with rapamycin early in Drosophila adult life is as effective as lifelong treatment, whereas a late-life treatment is not effective. However, previous studies in mice demonstrated that a transient late-life treatment is highly effective. Based on hyperfunction theory, this article attempts to reconcile conflicting results and suggests the optimal treatment strategy to extend human lifespan.


Assuntos
Envelhecimento , Sirolimo , Humanos , Camundongos , Animais , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Longevidade , Drosophila
12.
Eur J Pharmacol ; 934: 175301, 2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36191631

RESUMO

In this study we aimed to reduce tau pathology, a hallmark of Alzheimer's Disease (AD), by activating mTOR-dependent autophagy in a transgenic mouse model of tauopathy by long-term dosing of animals with mTOR-inhibitors. Rapamycin treatment reduced the burden of hyperphosphorylated and aggregated pathological tau in the cerebral cortex only when applied to young mice, prior to the emergence of pathology. Conversely, PQR530 which exhibits better brain exposure and superior pharmacokinetic properties, reduced tau pathology even when the treatment started after the onset of pathology. Our results show that dosing animals twice per week with PQR530 resulted in intermittent, rather than sustained target engagement. Nevertheless, this pulse-like mTOR inhibition followed by longer intervals of re-activation was sufficient to reduce tau pathology in the cerebral cortex in P301S tau transgenic mice. This suggests that balanced therapeutic dosing of blood-brain-barrier permeable mTOR-inhibitors can result in a disease-modifying effect in AD and at the same time prevents toxic side effects due to prolonged over activation of autophagy.


Assuntos
Doença de Alzheimer , Animais , Camundongos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Proteínas tau/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Camundongos Transgênicos , Encéfalo , Sirolimo/farmacologia , Modelos Animais de Doenças
13.
Biochem Pharmacol ; 205: 115280, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36198355

RESUMO

BACKGROUND: Marfan syndrome (MFS) is a genetic disorder leading to medial aortic degeneration and life-limiting dissections. To date, there is no causal prevention or therapy. Rapamycin is a potent and selective inhibitor of the mechanistic target of rapamycin (mTOR) protein kinase, regulating cell growth and metabolism. The mgR/mgR mice represent an accepted MFS model for studying aortic pathologies to understand the underlying molecular pathomechanisms. This study investigated whether rapamycin inhibits the development of thoracic aortic aneurysms and dissections in mgR/mgR mice. METHODS: Isolated primary aortic smooth muscle cells (mAoSMCs) from mgR/mgR mice were used for in vitro studies. Two mg kg/BW rapamycin was injected intraperitoneally daily for two weeks, beginning at 7-8 weeks of age. Mice were sacrificed 30 days post-treatment. Histopathological and immunofluorescence analyses were performed using adequate tissue specimens and techniques. Animal survival was evaluated accompanied by periodic echocardiographic examinations of the aorta. RESULTS: The protein level of the phosphorylated ribosomal protein S6 (p-RPS6), a downstream target of mTOR, was significantly increased in the aortic tissue of mgR/mgR mice. In mAoSMCs isolated from these animals, expression of mTOR, p-RPS6, tumour necrosis factor α, matrix metalloproteinase-2 and -9 was significantly suppressed by rapamycin, demonstrating its anti-inflammatory capacity. Short-term rapamycin treatment of Marfan mice was associated with delayed aneurysm formation, medial aortic elastolysis and improved survival. CONCLUSIONS: Short-term rapamycin-mediated mTOR inhibition significantly reduces aortic aneurysm formation and thus increases survival in mgR/mgR mice. Our results may offer the first causal treatment option to prevent aortic complications in MFS patients.


Assuntos
Aneurisma Aórtico , Síndrome de Marfan , Camundongos , Animais , Síndrome de Marfan/complicações , Síndrome de Marfan/tratamento farmacológico , Metaloproteinase 2 da Matriz/metabolismo , Fibrilina-1/genética , Fator de Necrose Tumoral alfa , Modelos Animais de Doenças , Longevidade , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Proteína S6 Ribossômica , Camundongos Endogâmicos C57BL , Aneurisma Aórtico/tratamento farmacológico , Aneurisma Aórtico/etiologia , Aneurisma Aórtico/prevenção & controle , Serina-Treonina Quinases TOR
14.
Zool Res ; 43(6): 989-1004, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36257830

RESUMO

Ketamine, a rapid-acting antidepressant drug, has been used to treat major depressive disorder and bipolar disorder (BD). Recent studies have shown that ketamine may increase the potential risk of treatment-induced mania in patients. Ketamine has also been applied to establish animal models of mania. At present, however, the underlying mechanism is still unclear. In the current study, we found that chronic lithium exposure attenuated ketamine-induced mania-like behavior and c-Fos expression in the medial prefrontal cortex (mPFC) of adult male mice. Transcriptome sequencing was performed to determine the effect of lithium administration on the transcriptome of the PFC in ketamine-treated mice, showing inactivation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Pharmacological inhibition of AKT signaling by MK2206 (40 mg/kg), a selective AKT inhibitor, reversed ketamine-induced mania. Furthermore, selective knockdown of AKT via AAV-AKT-shRNA-EGFP in the mPFC also reversed ketamine-induced mania-like behavior. Importantly, pharmacological activation of AKT signaling by SC79 (40 mg/kg), an AKT activator, contributed to mania in low-dose ketamine-treated mice. Inhibition of PI3K signaling by LY294002 (25 mg/kg), a specific PI3K inhibitor, reversed the mania-like behavior in ketamine-treated mice. However, pharmacological inhibition of mammalian target of rapamycin (mTOR) signaling with rapamycin (10 mg/kg), a specific mTOR inhibitor, had no effect on ketamine-induced mania-like behavior. These results suggest that chronic lithium treatment ameliorates ketamine-induced mania-like behavior via the PI3K-AKT signaling pathway, which may be a novel target for the development of BD treatment.


Assuntos
Transtorno Depressivo Maior , Ketamina , Doenças dos Roedores , Masculino , Camundongos , Animais , Ketamina/toxicidade , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , Lítio/farmacologia , Mania , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinase/farmacologia , RNA Interferente Pequeno , Serina-Treonina Quinases TOR/genética , Transdução de Sinais , Antidepressivos/uso terapêutico , Antidepressivos/farmacologia , Sirolimo/farmacologia , Compostos de Lítio/farmacologia , Mamíferos , Doenças dos Roedores/tratamento farmacológico
15.
Front Immunol ; 13: 975233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36189285

RESUMO

Gene-modified cellular therapies carry inherent risks of severe and potentially fatal adverse events, including the expansion of alloreactive cells or malignant transformation due to insertional mutagenesis. Strategies to mitigate uncontrolled proliferation of gene-modified cells include co-transfection of a suicide gene, such as the inducible caspase 9 safety switch (ΔiC9). However, the activation of the ΔiC9 fails to completely eliminate all gene-modified cells. Therefore, we tested a two suicide gene system used independently or together, with the goal of complete cell elimination. The first approach combined the ΔiC9 with an inducible caspase 8, ΔiC8, which lacks the endogenous prodomain. The rationale was to use a second caspase with an alternative and complementary mechanism of action. Jurkat cells co-transduced to co-express the ΔiC8, activatable by a BB homodimerizer, and the ΔiC9 activatable by the rapamycin analog sirolimus were used in a model to estimate the degree of inducible cell elimination. We found that both agents could activate each caspase independently, with enhanced elimination with superior reduction in cell regrowth of gene-modified cells when both systems were activated simultaneously. A second approach was employed in parallel, combining the ΔiC9 with the RQR8 compact suicide gene. RQR8 incorporates a CD20 mimotope, targeted by the anti-CD20 monoclonal antibody rituxan, and the QBend10, a ΔCD34 selectable marker. Likewise, enhanced cell elimination with superior reduction in cell regrowth was observed when both systems were activated together. A dose-titration effect was also noted utilizing the BB homodimerizer, whereas sirolimus remained very potent at minimal concentrations. Further in vivo studies are needed to validate these novel combination systems, which may play a role in future cancer therapies or regenerative medicine.


Assuntos
Genes Transgênicos Suicidas , Sirolimo , Caspase 8/genética , Caspase 9/genética , Caspase 9/metabolismo , Genes Transgênicos Suicidas/genética , Humanos , Rituximab , Sirolimo/farmacologia
16.
Iran J Immunol ; 19(3): 219-231, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36190377

RESUMO

BACKGROUND: Impaired renal function is considered as a significant risk factor for cardiovascular events in chronic kidney disease patients. Several immunosuppressive drugs are used in these patients, which necessitates to minimize the drug-related side effects by employing alternative strategies. OBJECTIVE: This study aimed to evaluate prospectively the influence of low dose ATG induction therapy with two different protocols (Sirolimus versus Mycophenolate mofetil) on the expression of functional markers (LAG-3, CD39, and intracellular CTLA-4) on conventional Tregs in renal recipients. METHODS: Thirty-eight renal transplant recipients were enrolled in this study. The patients were randomly assigned into two groups, including TMP: Tacrolimus (Tac), Mycophenolate mofetil (MMF), and Prednisolone (n=23); and TSP: Tac, Sirolimus (SRL), and Prednisolone (n=15). The frequency of LAG-3, CD39, and intracellular CTLA-4 on circulating Tregs was analyzed by flow cytometry before and after transplantation. RESULTS: Analysis of the flow cytometry data showed that the frequency of CD4+CD25+FOXP3+ Tregs increased 4 months post-transplantation compared to pre-transplantation in both groups, although this increase was only significant in TMP group. In TMP treated patients, the frequency of LAG-3+ Tregs and CD39+ Tregs increased, whereas the frequency of intracellular CTLA-4+ Tregs decreased 4 months post-transplantation. In TSP group, while the frequency of CD39+ Tregs increased, the frequency of CTLA-4+ Tregs decreased in post-transplantation compared to pre-transplantation. CONCLUSIONS: it seems that both treatment regimen protocols with a low dose ATG induction therapy may be clinically applicable in kidney transplant recipients.


Assuntos
Transplante de Rim , Ácido Micofenólico , Sirolimo , Linfócitos T Reguladores , Aloenxertos , Antígeno CTLA-4 , Protocolos Clínicos , Fatores de Transcrição Forkhead , Humanos , Imunossupressores/farmacologia , Rim/fisiologia , Ácido Micofenólico/farmacologia , Prednisolona/farmacologia , Sirolimo/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Tacrolimo/farmacologia
17.
Magy Onkol ; 66(3): 239-241, 2022 Oct 05.
Artigo em Húngaro | MEDLINE | ID: mdl-36200504

RESUMO

We investigated the activity and inhibition of mTOR and other metabolic pathways with their clinical significance in human breast tumors (using ten cell lines and nearly a hundred biopsy samples).Based on our results, the metabolic and mTOR inhibitor treatments showed a moderate tumor growth inhibitory effect in the cell lines subtype independently, which indicates tumor cell and tissue adaptation. Providing human tissue samples, we found a subtype independent correlation between high mTOR activity and protein expression characterizing alternative metabolic pathways with increased expression and the poor prognosis of breast tumors. Breast tumors are characterized by metabolic heterogeneity and significant metabolic plasticity, which can be targeted by combining anti-metabolic treatments and new therapies. Concerning these, an immunohistochemical evaluation (IHC panel) can be recommended, which is suitable for both metabolic plasticity evaluation and recognition of cases that may require stricter follow-up or metabolic targeted therapy due to the expected poor prognosis.


Assuntos
Neoplasias da Mama , Sirolimo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Transdução de Sinais , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologia
18.
Molecules ; 27(19)2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36234769

RESUMO

Autophagy is the multistep mechanism for the elimination of damaged organelles and misfolded proteins. This mechanism is preceded and may induce other program cell deaths such as apoptosis. This study unraveled the potential pharmacological effect of 24MD in inducing the autophagy of lung cancer cells. Results showed that 24MD was concomitant with autophagy induction, indicating by autophagosome staining and the induction of ATG5, ATG7 and ubiquitinated protein, p62 expression after 12-h treatment. LC3-I was strongly conversed to LC3-II, and p62 was downregulated after 24-h treatment. The apoptosis-inducing activity was found after 48-h treatment as indicated by annexin V-FITC/propidium iodide staining and the activation of caspase-3. From a mechanistic perspective, 24-h treatment of 24MD at 60 µM substantially downregulated p-mTOR. Meanwhile, p-PI3K and p-Akt were also suppressed by 24MD at concentrations of 80 and 100 µM, respectively. We further confirmed m-TOR-mediated autophagic activity by comparing the effect of 24MD with rapamycin, a potent standard mTOR1 inhibitor through Western blot and immunofluorescence assays. Although 24MD could not suppress p-mTOR as much as rapamycin, the combination of rapamycin and 24MD could increase the mTOR suppressive activity and LC3 activation. Changing the substituent groups (R groups) from dimethylphenol to ethylphenol in EMD or changing methylazanedyl to cyclohexylazanedyl in 24CD could only induce apoptosis activity but not autophagic inducing activity. We identified 24MD as a novel compound targeting autophagic cell death by affecting mTOR-mediated autophagy.


Assuntos
Morte Celular Autofágica , Neoplasias Pulmonares , Apoptose , Autofagia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Propídio/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Ubiquitinadas/farmacologia , Proteínas Ubiquitinadas/uso terapêutico , Xilenos
19.
Int J Mol Sci ; 23(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36232303

RESUMO

Combined cisplatin-gemcitabine treatment causes rapid resistance development in patients with advanced urothelial carcinoma. The present study investigated the potential of the natural isothiocyanates (ITCs) allyl-isothiocyanate (AITC), butyl-isothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC) to suppress growth and proliferation of gemcitabine- and cisplatin-resistant bladder cancer cells lines. Sensitive and gemcitabine- and cisplatin-resistant RT112, T24, and TCCSUP cells were treated with the ITCs, and tumor cell growth, proliferation, and clone formation were evaluated. Apoptosis induction and cell cycle progression were investigated as well. The molecular mode of action was investigated by evaluating cell cycle-regulating proteins (cyclin-dependent kinases (CDKs) and cyclins A and B) and the mechanistic target of the rapamycin (mTOR)-AKT signaling pathway. The ITCs significantly inhibited growth, proliferation and clone formation of all tumor cell lines (sensitive and resistant). Cells were arrested in the G2/M phase, independent of the type of resistance. Alterations of both the CDK-cyclin axis and the Akt-mTOR signaling pathway were observed in AITC-treated T24 cells with minor effects on apoptosis induction. In contrast, AITC de-activated Akt-mTOR signaling and induced apoptosis in RT112 cells, with only minor effects on CDK expression. It is concluded that AITC, BITC, and PEITC exert tumor-suppressive properties on cisplatin- and gemcitabine-resistant bladder cancer cells, whereby the molecular action may differ among the cell lines. The integration of these ITCs into the gemcitabine-/cisplatin-based treatment regimen might optimize bladder cancer therapy.


Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Apoptose , Linhagem Celular Tumoral , Cisplatino/farmacologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Desoxicitidina/análogos & derivados , Humanos , Isotiocianatos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/metabolismo
20.
Pain Physician ; 25(7): E1137-E1151, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36288601

RESUMO

BACKGROUND: In neuropathic pain following peripheral nerve injury, microglia are rapidly activated and accumulated in the spinal cord. Physical exercise can alleviate neuropathic pain. However, the exact mechanism underlying this analgesic effect is not fully understood. OBJECTIVES: We aimed to investigate the molecular mechanisms by which exercise alleviates neuropathic pain in relation to brain-derived neurotrophic factor (BDNF), microglia polarization, and autophagy. STUDY DESIGN: A randomized controlled animal study divided into 2 stages. The first stage comprised 4 groups each with 6 mice, and the second stage comprised 6 groups, 3 with 18 mice and 3 with 12 mice. SETTING: Department of Anesthesiology, Lanzhou University Second Hospital, Orthopaedics Key Laboratory of Gansu Province, Lanzhou University. METHODS: Von Frey filaments, Western blotting, immunofluorescence, and transmission electron microscopy analyses were conducted to detect relevant markers. RESULTS: After peripheral nerve injury, exercise training downregulated BDNF expression and reversed microglial activation, as indicated by the increased expression of the M2 marker CD206 and decreased expression of the M1 marker CD86 in the spinal dorsal horn of mice. Autophagy flux was enhanced after exercise training, as suggested by the increased expression of the autophagy markers LC3-II/LC3-I and Beclin1 and decreased expression of the autophagy adaptor protein p62. Furthermore, autophagy inhibition by 3-methyladenine aggravated M1 polarization and hyperalgesia, whereas autophagy induced by rapamycin promoted M2 polarization and reduced hyperalgesia. Intrathecal injection of BDNF significantly upregulated BDNF expression, inhibited autophagy, triggered M1 polarization of spinal microglia, and aggravated hyperalgesia. Furthermore, BDNF regulated autophagy through the AKT/mTOR pathway, thereby participating in exercise training-mediated polarization of microglia after nerve injury. LIMITATIONS: The effect of exercise on autophagy and pain cannot be assessed in an in vitro model. The influence of intrathecal injection of BDNF on the metabolic changes in other neuronal cells and the subsequent effects on pain should be investigated. Further studies on how exercise training modulates microglial autophagy to alleviate neuropathic pain are needed. CONCLUSIONS: Exercise training promoted the recovery of sciatic nerve injury in mice, possibly by regulating microglial polarization through BDNF/AKT/mTOR signaling-mediated autophagy flux. We confirmed the efficacy of exercise training in alleviating neuropathic pain and suggest a new therapeutic target for neuropathic pain.


Assuntos
Neuralgia , Traumatismos dos Nervos Periféricos , Ratos , Camundongos , Animais , Microglia/metabolismo , Fator Neurotrófico Derivado do Encéfalo , Hiperalgesia/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , Traumatismos dos Nervos Periféricos/metabolismo , Proteína Beclina-1/metabolismo , Proteína Beclina-1/farmacologia , Ratos Sprague-Dawley , Neuralgia/tratamento farmacológico , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologia , Serina-Treonina Quinases TOR/uso terapêutico , Autofagia , Corno Dorsal da Medula Espinal/metabolismo , Sirolimo/metabolismo , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Analgésicos/uso terapêutico
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