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1.
STAR Protoc ; 3(2): 101328, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35496811

RESUMO

Retinal ganglion cell (RGC) transplantation has the potential to restore vision in optic neuropathy, but donor neuron survival and retinal integration remain challenging. Here, we present a protocol for ex vivo human RGC transplantation on flatmounted murine organotypic retinal explants, providing a robust platform for studying donor RGC survival, dendritic stratification, topographic distribution, donor-host interactions, and pro-engraftment strategies. The protocol includes microscopy-based analyses to evaluate donor cell engraftment and can be adapted to various donor cell types or culture systems. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021a, 2021b).


Assuntos
Doenças do Nervo Óptico , Células Ganglionares da Retina , Animais , Sobrevivência Celular , Humanos , Camundongos , Nervo Óptico , Retina
2.
An Acad Bras Cienc ; 94(2): e20210670, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507982

RESUMO

Fatty acid synthase (FASN) is the rate-limiting enzyme for the de novo synthesis of fatty acids in the cytoplasm of tumour cells. Many tumour cells express high levels of FASN, and its expression is associated with a poorer prognosis. Cervical cancer is a major public health problem, representing the fourth most common cancer affecting women worldwide. To date, only a few in silico studies have correlated FASN expression with cervical cancer. This study aimed to investigate in vitro FASN expression in premalignant lesions and cervical cancer samples and the effects of a FASN inhibitor on cervical cancer cells. FASN expression was observed in all cervical cancer samples with increased expression at more advanced cervical cancer stages. The FASN inhibitor (orlistat) reduced the in vitro cell viability of cervical cancer cells (C-33A, ME-180, HeLa and SiHa) in a time-dependent manner and triggered apoptosis. FASN inhibitor also led to cell cycle arrest and autophagy. FASN may be a potential therapeutic target for cervical cancer, and medicinal chemists, pharmaceutical researchers and formulators should consider this finding in the development of new treatment approaches for this cancer type.


Assuntos
Neoplasias do Colo do Útero , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Ácido Graxo Sintases/metabolismo , Ácido Graxo Sintases/farmacologia , Feminino , Humanos , Orlistate/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico
3.
Sci Rep ; 12(1): 7159, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35504928

RESUMO

Artificial cell fusion often serves as a valuable tool for studying different applications in biology and medicine, including natural development, immune response, cancer metastasis and production of therapeutic molecules. Plasmonic cell fusion, a technique that uses specific cell labeling by gold nanoparticles and resonant femtosecond pulse irradiation for fusing neighboring cells, has been demonstrated useful for such applications, allowing high cell specificity and an overall low toxicity. Despite these advantages, the numerous experimental factors contributing to plasmonic fusion have often led to subpar fusion efficiencies, requiring repeated experiments and extensive calibration protocols for achieving optimal results. In this work we present a study that aims to improve the overall performance of plasmonic cell fusion in terms of fusion efficiency and cell viability. By varying the pulse fluence, nanoparticle concentration, incubation times, and culture handling protocols, we demonstrate up to 100% fusion of malignant epithelial cells across the entire irradiated area of the culture. We also show that some of the smaller cells may stay viable for up to several days. The results would allow plasmonic fusion to play a key role in numerous studies and applications that require specific, high-efficiency cell-cell fusion.


Assuntos
Ouro , Nanopartículas Metálicas , Comunicação Celular , Fusão Celular , Sobrevivência Celular
4.
Methods Mol Biol ; 2451: 285-403, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35505024

RESUMO

Photodynamic therapy (PDT) is a non-to-minimally invasive treatment modality that utilizes photoactivatable drugs called photosensitizers to disrupt tumors with locally photoproduced reactive oxygen species (ROS). Photosensitizer activation by light results in hyperoxidative stress and subsequent tumor cell death, vascular shutdown and hypoxia, and an antitumor immune response. However, sublethally afflicted tumor cells initiate several survival mechanisms that account for decreased PDT efficacy. The hypoxia inducible factor 1 (HIF-1) pathway is one of the most effective cell survival pathways that contributes to cell recovery from PDT-induced damage. Several hundred target genes of the HIF-1 heterodimeric complex collectively mediate processes that are involved in tumor cell survival directly and indirectly (e.g., vascularization, glucose metabolism, proliferation, and metastasis). The broad spectrum of biological ramifications culminating from the activation of HIF-1 target genes reflects the importance of HIF-1 in the context of therapeutic recalcitrance. This chapter elaborates on the involvement of HIF-1 in cancer biology, the hypoxic response mechanisms, and the role of HIF-1 in PDT. An overview of inhibitors that either directly or indirectly impede HIF-1-mediated survival signaling is provided. The inhibitors may be used as pharmacological adjuvants in combination with PDT to augment therapeutic efficacy.


Assuntos
Neoplasias , Fotoquimioterapia , Sobrevivência Celular , Humanos , Fator 1 Induzível por Hipóxia/genética , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo
5.
Bioengineered ; 13(5): 11417-11429, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35506308

RESUMO

Diabetic cardiomyopathy (DCM) is a diabetic mellitus-related complications and progression of DCM may eventually lead to heart failure, while mechanisms related to DCM pathophysiology remain unclear. The study was undertaken to identify possible hub genes associated with DCM progression through bioinformatics analysis and to validate the role of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) in DCM progression using a cellular model of high glucose (HG)-induced DCM. The common differentially expressed genes (DEGs) between GSE173884 and GSE161827 were used for PPI network analysis. Our results identified 17 common DEGs between GSE173384 and GSE161827. Further analysis of the protein-protein interaction network identified nine hub genes and HMGCS2. The in vitro functional assays showed that HG induced up-regulation of HMGCS2, suppressed cardiomyocyte viability, enhanced apoptosis, inflammation, and oxidative stress of cardiomyocytes. Gain-of-function assays showed that HMGCS2 overexpression reduced cell viability, increased apoptosis, caspase-3/-9 activity, up-regulated interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α (TNF-α) expression, decreased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase expression, increased malondialdehyde (MDA) content, and reactive oxygen species (ROS) level but inhibited total antioxidant activity, SOD activity, CAT activity, and glutathione content in cardiomyocytes. Rescue experiments demonstrated HMGCS2 silence attenuated HG-induced decrease in cardiomyocyte viability and increase in cardiomyocyte apoptosis, inflammation, and oxidative stress. All in all, our study identified HMGCS2 as a hub gene in DCM pathophysiology and further functional studies indicated that HMGCS2 may aggravate DCM progression by reducing cardiomyocyte viability, increasing cardiomyocyte apoptosis, and promoting inflammation and oxidative stress in cardiomyocytes.


Assuntos
Diabetes Mellitus , Cardiomiopatias Diabéticas , Antioxidantes , Apoptose/genética , Sobrevivência Celular/genética , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Glucose/toxicidade , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Inflamação/genética , Inflamação/patologia , Estresse Oxidativo , Superóxido Dismutase/metabolismo
6.
Bioengineered ; 13(5): 11489-11502, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35506311

RESUMO

Diabetic retinopathy (DR) is one of the most common retinal microvascular diseases in diabetic patients. Therefore, elucidating the underlying molecular mechanism of DR is of great significance for its clinical treatment. This study explores the effects of the upregulated circFTO in DR patients in terms of cell apoptosis and viability. Several molecular assays are employed to explore these molecular mechanistic aspects, such as luciferase reporter, RNA pull-down, RT-qPCR, Western blot, and ELISA assays. miR-148a-3p is downregulated in DR patients. The expression of circFTO promoted ARPE-19 cells apoptosis and inhibited proliferation, reflecting the regulatory effect of circFTO/miR-148a-3p on retinal epithelial cells injury. In addition, the absence of circFTO could reduce ARPE-19 cells injury caused by HG by inhibiting oxidative stress and inflammation. Further, the investigations at the molecular level showed that circFTO could regulate the level of miR-148a-3p and TGFA in vitro. As the molecular sponge of miR-148a-3p, circFTO regulated cell viability and apoptosis and promoted the progression of DR through regulating the expression of TGFA. Together, this study provides new targets and markers for early diagnosis and therapy of DR.


Assuntos
MicroRNAs , Apoptose/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Glucose/toxicidade , Humanos , MicroRNAs/metabolismo
7.
Nan Fang Yi Ke Da Xue Xue Bao ; 42(4): 584-590, 2022 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-35527495

RESUMO

OBJECTIVE: To explore the effect of inhibiting polyribonucleotide nucleotidyl-transferase 1 (PNPT1) on oxygen-glucose deprivation (OGD)-induced apoptosis of mouse atrial myocytes. METHODS: Cultured mouse atrial myocytes (HL-1 cells) with or without OGD were transfected with PNPT1-siRNA or a negative control siRNA (NC-siRNA group), and the cell survival rate was detected using CCK-8 assay. The expression levels of ACTB and TUBA mRNA were detected with qPCR, and the protein expression of PNPT1 was detected with Western blotting. The apoptosis rate of the treated cells was determined with flow cytometry, the mitochondrial membrane potential was detected using JC-1 kit, and the mitochondrial morphology was observed using transmission electron microscope. RESULTS: With the extension of OGD time, the protein expression levels of PNPT1 increased progressively in the cytoplasm of HL-1 cells (P < 0.05). Transfection with PNPT1-siRNA significantly reduced PNPT1 expression in HL-1 cells (P < 0.05). Exposure to OGD significantly enhanced degradation of ACTB and TUBA mRNA (P < 0.05) and markedly increased the apoptosis rate of HL-1 cells (P < 0.05), and these changes were significantly inhibited by transfection with PNPT1-siRNA (P < 0.05), which obviously increased mitochondrial membrane potential and improved mitochondrial morphology of HL-1 cells exposed to OGD. CONCLUSION: Inhibition of PNPT1 improves mitochondrial damage and reduces degradation of apoptotic-associated mRNAs to alleviate OGD-induced apoptosis of mouse atrial myocyte.


Assuntos
Glucose , Oxigênio , Animais , Apoptose , Sobrevivência Celular , Glucose/farmacologia , Camundongos , Miócitos Cardíacos , Oxigênio/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
8.
Cancer Biol Ther ; 23(1): 369-377, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-35491899

RESUMO

Glioma-associated oncogene (Gli) antagonist-61 (GANT61) not only suppresses the malignant behavior of several cancers but also presents synergistic effects with other anticancer agents on suppressing the progression of cancers, while relevant information is rare in anaplastic thyroid carcinoma (ATC). This study aimed to explore the therapeutic effect of GANT61 in ATC and its molecular mechanism. ATC cells (8505C and CAL-62) were treated with GANT61, followed by detection of cell proliferation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) markers. Subsequently, RNA sequencing was performed to explore the potential downstream pathway. Following that, rescue experiments were conducted by SC79 (AKT activator) or colivelin (STAT3 activator) monotreatment or combined with GANT61 in ATC cells. GANT61 reduced Gli1 expression, suppressed proliferation at several time settings, promoted apoptosis, inhibited invasion and increased E-cadherin while decreased Vimentin and Snail expressions (EMT markers) in ATC cells. The subsequent RNA sequence identified 85 upregulated differentially expressed genes (DEGs) and 71 downregulated DEGs in GANT61-treated ATC cells, which were mainly enriched in PI3K/AKT, JAK/STAT, Hedgehog and mTOR pathways. Next, the inactivation of AKT/mTOR and JAK/STAT3 pathways by GANT61 treatment was verified by western blot. The following rescue experiments showed that SC79 or colivelin treatment promoted the malignant behaviors of ATC cells. More importantly, SC79 or colivelin treatment compensated the effect of GANT61 treatment on cell proliferation at several time settings and apoptosis, invasion, and part of that on EMT in ATC cells. GANT61 suppresses cell survival, invasion and EMT through inactivating AKT/mTOR or JAK/STAT3 pathways in ATC.


Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Transição Epitelial-Mesenquimal/genética , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas , Pirimidinas , Fator de Transcrição STAT3/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/patologia
9.
Sci Rep ; 12(1): 7398, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513484

RESUMO

The endocannabinoid system has been postulated to help restrict cancer progression and maintain osteoblastic function during bone metastasis. Herein, the effects of cannabinoid receptor (CB) type 1 and 2 activation on breast cancer cell and osteoblast interaction were investigated by using ACEA and GW405833 as CB1 and CB2 agonists, respectively. Our results showed that breast cancer cell (MDA-MB-231)-derived conditioned media markedly decreased osteoblast-like UMR-106 cell viability. In contrast, media from MDA-MB-231 cells pre-treated with GW405833 improved UMR-106 cell viability. MDA-MB-231 cells were apparently more susceptible to both CB agonists than UMR-106 cells. Thereafter, we sought to answer the question as to how CB agonists reduced MDA-MB-231 cell virulence. Present data showed that co-activation of CB1 and CB2 exerted cytotoxic effects on MDA-MB-231 cells by increasing apoptotic cell death through suppression of the NF-κB signaling pathway in an ROS-independent mechanism. ACEA or GW405833 alone or in combination also inhibited MDA-MB-231 cell migration. Thus, it can be concluded that the endocannabinoid system is able to provide protection during breast cancer bone metastasis by interfering cancer and bone cell interaction as well as by the direct suppression of cancer cell growth and migration.


Assuntos
Neoplasias Ósseas , Neoplasias da Mama , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Endocanabinoides/farmacologia , Feminino , Humanos , Osteoblastos/metabolismo , Receptores de Canabinoides
10.
Bioengineered ; 13(5): 11767-11781, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35543385

RESUMO

Globally, age-related macular degeneration (AMD) is a common irreversible ophthalmopathy. Oxidative stress of retinal pigment epithelial cells is involved in AMD occurrence and development. Klotho is an anti-aging protein with antioxidant properties. We investigated the protective properties of Klotho on hydrogen peroxide (H2O2)-induced injury of retinal pigment epithelial cells (ARPE-19 cells) and its associated pathomechanisms. We found that Klotho pretreatment for 24 h could up-regulate Bcl-2 levels, decrease the cleaved-caspase-3 and Bax levels, inhibit H2O2-induced ARPE-19 cell apoptosis, and promote cell proliferation. Klotho pretreatment inhibited the H2O2-mediated elevations of reactive oxygen species (ROS) in ARPE-19 cells. It enhanced antioxidant activities of the cells and restored the glutathione peroxidase (GPX), superoxide dismutase (SOD2), catalase (CAT), as well as malondialdehyde (MDA) levels to close to the normal level. N-acetylcysteine (NAC), a reactive oxygen scavenger, could reverse the harmful effects of H2O2 on proliferation, apoptosis, and oxidative stress of ARPE-19 cells. Further, Klotho pretreatment enhanced Akt phosphorylation and expression as well as nuclear translocation of Nrf2 in H2O2-treated ARPE-19 cells. This indicates that Klotho protects cells from oxidative stress by activating phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)-nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathway. Klotho is, therefore, a potential preventive or treatment option for AMD.


Assuntos
Heme Oxigenase-1 , Fator 2 Relacionado a NF-E2 , Antioxidantes/farmacologia , Apoptose , Sobrevivência Celular , Células Epiteliais/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , Transdução de Sinais
11.
Proc Natl Acad Sci U S A ; 119(21): e2202016119, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35537042

RESUMO

SignificanceAutophagy defects are a risk factor for inflammatory bowel diseases (IBDs), but the mechanism remains unknown. We show here that conditional whole-body deletion of Atg5 or Fip200, but not Atg7, is lethal due to loss of ileum stem cells and barrier function likely caused by different kinetics of autophagy loss, which was rescued by slow deletion. Specific autophagy loss in PDGFRα+ mesenchymal cells (PMCs) resulted in loss of Wnt signaling responsible for failed stem cell renewal. We also observed depletion of aspartate and nucleotides throughout the ileum. Our results illustrate that autophagy is required for PMC metabolism and survival necessary to sustain intestinal stem cells and mouse survival, and failure to maintain PMCs through autophagy contributes to IBD.


Assuntos
Autofagia , Intestinos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Células-Tronco , Animais , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Sobrevivência Celular , Camundongos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células-Tronco/metabolismo
12.
Cell Death Dis ; 13(5): 448, 2022 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-35538058

RESUMO

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.


Assuntos
Hexoquinase , Leucemia Mieloide Aguda , Sobrevivência Celular/genética , Glicólise/genética , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células Mieloides/metabolismo
13.
J Biomed Sci ; 29(1): 30, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35538574

RESUMO

BACKGROUND: Autophagy plays important roles in cell homeostasis and protein quality control. Long non-coding RNAs (lncRNAs) have been revealed as an emerging class of autophagy regulators, but the majority of them function in regulating the expression of autophagy-related genes. LncRNAs that directly act on the core autophagic proteins remain to be explored. METHODS: Immunofluorescence staining and Western blotting were used to evaluate the function of BCRP3 in autophagy and aggrephagy. RNA immunoprecipitation and in vitro RNA-protein binding assay were used to evaluate the interaction of BCRP3 with its target proteins. Phosphatidylinositol 3-phosphate ELISA assay was used to quantify the enzymatic activity of VPS34 complex. qRT-PCR analysis was used to determine BCRP3 expression under stresses, whereas mass spectrometry and Gene Ontology analyses were employed to evaluate the effect of BCRP3 deficiency on proteome changes. RESULTS: We identified lncRNA BCRP3 as a positive regulator of autophagy. BCRP3 was mainly localized in the cytoplasm and bound VPS34 complex to increase its enzymatic activity. In response to proteotoxicity induced by proteasome inhibition or oxidative stress, BCRP3 was upregulated to promote aggrephagy, thereby facilitating the clearance of ubiquitinated protein aggregates. Proteomics analysis revealed that BCRP3 deficiency under proteotoxicity resulted in a preferential accumulation of proteins acting in growth inhibition, cell death, apoptosis, and Smad signaling. Accordingly, BCRP3 deficiency in proteotoxic cells compromised cell proliferation and survival, which was mediated in part through the upregulation of TGF-ß/Smad2 pathway. CONCLUSIONS: Our study identifies BCRP3 as an RNA activator of the VPS34 complex and a key role of BCRP3-mediated aggrephagy in protein quality control and selective degradation of growth and survival inhibitors to maintain cell fitness.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases , RNA Longo não Codificante , Autofagia , Sobrevivência Celular/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Proteostase , RNA Longo não Codificante/metabolismo
14.
World J Surg Oncol ; 20(1): 125, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35439960

RESUMO

BACKGROUND: Cervical cancer is a kind of malignant gynecological tumor. The first choice for treating cervical cancer is still a combination of surgery and chemoradiotherapy, but the 5-year survival rate remains poor. Therefore, researchers are trying to find new ways to diagnose and treat cervical cancer early. METHODS: The expression level of KIF14 in cells and tissues was determined via qRT-PCR. The ability of the cells to proliferate, migrate, and invade was examined using CCK-8 assay kits, colony formation assays, and Transwell chambers. The expression levels of Cyclin D1, Cyclin B1, p21, and p27 were also detected using western blot assays. RESULTS: The results suggested that p27 is a key regulatory factor in the KIF14-mediated regulation of the cell cycle. In addition, KIF14 knockdown promotes malignancy in cervical cancer cells by inhibiting p27 degradation, resulting in cell cycle arrest. CONCLUSIONS: KIF14 is an oncogene in cervical cancer, and knocking down KIF14 causes cell cycle arrest by inhibiting p27 degradation, thus affecting cell viability, proliferation, and migration. These results provide a potential therapeutic target for cervical cancer.


Assuntos
Neoplasias do Colo do Útero , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Sobrevivência Celular , Colo do Útero/patologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Humanos , Proteínas Oncogênicas , Neoplasias do Colo do Útero/patologia
15.
Acta Neurobiol Exp (Wars) ; 82(1): 77-87, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35451425

RESUMO

Alzheimer's disease (AD) is the most common neurodegenerative disease and is manifested by memory loss and spatial disorientation. There is currently no effective treatment for AD. Abnormalities of the chromosome 9 open reading frame 72 (C9ORF72) gene have been associated with various neurodegenerative diseases. However, its intrinsic roles in AD remain to be elucidated. Here we found that Aß25­35 increased the expression of C9orf72 in PC12 cells at both mRNA and protein levels. In Aß25­35­treated PC12 cells, C9orf72 overexpression induced an abnormally condensed and fragmented nucleus and apoptosis, as well as significantly enhanced reactive oxygen species (ROS) levels. Mechanistically, an Aß25­35­induced decrease of superoxide dismutase activity was augmented by C9orf72 overexpression, which in contrast increased malondialdehyde content. Consistently, further apoptotic analysis revealed significant downregulation of Bcl­2 and Bcl­xL expression and enhanced cleavage of caspase­3 with Aß25­35 treatment, all of which were exacerbated by C9orf72 overexpression. In addition, tau phosphorylation, another hallmark of AD pathology, was induced by Aß25­35 and was remarkably enhanced by C9orf72 overexpression. Our data indicate that C9orf72 plays important roles in intracellular ROS signaling and Aß25­35­induced neuronal apoptosis in AD. These findings provide insights into C9orf72 function in the pathogenesis of many related neurodegenerative diseases and provide a basis for potential therapeutic interventions.


Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Apoptose/genética , Proteína C9orf72/metabolismo , Proteína C9orf72/farmacologia , Sobrevivência Celular , Estresse Oxidativo , Células PC12 , Fragmentos de Peptídeos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Espécies Reativas de Oxigênio/uso terapêutico
16.
Int J Med Mushrooms ; 24(3): 35-50, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35467805

RESUMO

Neurological diseases are increasingly recognized as a health burden worldwide, mainly affecting the elderly population. Sanguinoderma rugosum (=Amauroderma rugosum) is a wild medicinal mushroom traditionally used to alleviate inflammation and prevent seizures. The present study aimed to investigate the neuroprotective and neurorescue effects as well as the possible mechanisms of S. rugosum extracts on glutamate-induced HT-22 mouse hippocampal neuronal cells. The mycelia of S. rugosum were subjected to submerged liquid fermentation followed by solvent extraction and fractionation. The neurotoxicity, neuroprotective, and neurorescue activities of S. rugosum extracts were assessed via the MTT viability assay at 24 and 48 h. The effects of S. rugosum extracts on glutamate-induced oxidative stress and cell death were investigated through flow cytometry. Gas chromatography/mass spectrometry (GC/MS) analysis was conducted to identify the bioactive compounds in the S. rugosum hexane fraction (SR-HF). All extracts were noncytotoxic toward HT-22 cells. Pretreatment with S. rugosum ethanolic extract (SR-EE; 12.5 µg/mL) or SR-HF (100 µg/mL) markedly (P < 0.05) improved the loss of cell viability and attenuated the accumulation of reactive oxygen species production. Pretreatment with SR-HF was also demonstrated to inhibit glutamate-induced cell death. The MTT assay showed that all extracts generally rescued glutamate-induced HT-22 cells at 24 and 48 h. The GC/MS analysis revealed the existence of 11 bioactive components in SR-HF, with linoleic acid, ergosterol, and ethyl linoleate being the main chemical constituents. The current findings suggest that SR-HF could be used as a potential therapeutic intervention to ameliorate oxidative stress and neuroinflammation.


Assuntos
Agaricales , Fármacos Neuroprotetores , Agaricales/química , Idoso , Animais , Sobrevivência Celular , Ácido Glutâmico/toxicidade , Hipocampo , Humanos , Camundongos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Extratos Vegetais/química , Extratos Vegetais/farmacologia
17.
Int J Nanomedicine ; 17: 1495-1509, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35388270

RESUMO

Purpose: Nanoparticles are resources of advanced nanotechnology being present in several products. Titanium dioxide nanoparticles are among the five most widely used NP currently expanding their benefits from the oil industry to the areas of diagnostic medicine due to their properties and small size. However, its impact on human health is still controversial in the literature. We aimed to evaluate the cytotoxicity of a new titanium NP functionalized with sodium carboxylic ligand (COOH-Na+) in human keratinocytes (HaCaT) and human fibroblasts (HDFn). Methods: The physical-chemical characterization was performed by the transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential techniques, respectively. MTT and LDH assays were used to assess cytotoxicity and cell membrane damage respectively, ELISA to identify the inflammatory profile and, reactive oxygen species assay and cytometry to detect reactive oxygen species and their relationship with apoptosis/necrosis mechanisms. Results: The results demonstrated a decrease in cell viability at the highest concentrations tested for both cell lines, but no change in LDH release was detected for the HaCaT. The cell membrane damage was found only at 100.0 µg/mL for the HDFn. It was demonstrated that cytotoxicity in the highest concentrations evaluated for both cell lines for the 72 h period. The HDFn showed damage to the cell membrane at a concentration of 100 µg/mL followed by a significant increase in reactive oxygen species production. No inflammatory profile was detected. The HaCaT showed apoptosis when exposed to the highest concentration evaluated and HDFn showed both apoptosis and necrosis for the same concentration. Conclusion: Thus, it is possible to conclude that the cytotoxicity mechanism differs according to the cell type evaluated, with HDFn being the most sensitive line in this case, and this mechanism can be defined in a dose and time dependent manner, since the highest concentrations also triggered death cell.


Assuntos
Nanopartículas Metálicas , Nanopartículas , Apoptose , Sobrevivência Celular , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Nanopartículas/química , Nanopartículas/toxicidade , Necrose/induzido quimicamente , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Titânio/química , Titânio/toxicidade
18.
Int J Mol Sci ; 23(7)2022 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-35408947

RESUMO

Melanoma is a drug-resistant cancer, representing a serious challenge in cancer treatment. Dacarbazine (DTIC) is the standard drug in metastatic melanoma treatment, despite the poor results. Hyperthermia has been proven to potentiate chemotherapy. Hence, this work analyzed the combined action of hyperthermia and DTIC on A375 and MNT-1 cell lines. First, temperatures between 40 °C and 45 °C were tested. The effect of DTIC on cell viability was also investigated after exposures of 24, 48, and 72 h. Then, cells were exposed to 43 °C and to the respective DTIC IC10 or IC20 of each time exposure. Overall, hyperthermia reduced cell viability, however, 45 °C caused an excessive cell death (>90%). Combinational treatment revealed that hyperthermia potentiates DTIC's effect, but it is dependent on the concentration and temperature used. Also, it has different mechanisms from the treatments alone, delaying A375 cells at the G2/M phase and MNT-1 cells at the S and G2/M phases. Intracellular reactive oxygen species (ROS) levels increased after treatment with hyperthermia, but the combined treatment showed no additional differences. Also, hyperthermia highly increased the number of A375 early apoptotic cells. These results suggest that combining hyperthermia and DTIC should be more explored to improve melanoma treatment.


Assuntos
Hipertermia Induzida , Melanoma , Linhagem Celular Tumoral , Sobrevivência Celular , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Taiwan J Obstet Gynecol ; 61(2): 255-264, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35361385

RESUMO

OBJECTIVE: Abnormal expression of long non-coding RNAs (lncRNAs) is critical in preeclampsia (PE) pathogenesis. The current study explored the function of non-coding RNA activated by DNA damage (NORAD) in the progression of PE. MATERIALS AND METHODS: Quantitative real-time PCR was utilized to determine the expression of NORAD, microRNA (miR)-202-5p, and fragile X-related gene 1 (FXR1) in PE and normal placenta tissues. Cell viability was determined using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide assay, and cell migration and invasion were assessed using the Transwell assay. Target relationships were confirmed with the dual-luciferase reporter assay. Western blotting was performed to determine the protein level of FXR1. RESULTS: NORAD expression was markedly reduced in PE placenta. NORAD over-expression enhanced the viability, migration, and invasion of HTR-8/SVneo cells. miR-202-5p was a target and was negatively regulated by NORAD. Down-regulation of miR-202-5p promoted the viability, migration, and invasion of HTR-8/SVneo cells. miR-202-5p inversely regulated FXR1 expression by targeting the 3'UTR of FXR1. Both miR-202-5p up-regulation and FXR1 knockdown reversed the NORAD over-expression-induced enhancement in the viability, migration, and invasion of HTR-8/SVneo cells. CONCLUSION: Collectively, the results revealed that NORAD over-expression promoted trophoblast viability, invasion, and migration by regulating the miR-202-5p/FXR1 axis. These findings clarify PE pathogenesis and will inform the discovery of new targets for PE treatment.


Assuntos
MicroRNAs , Pré-Eclâmpsia , RNA Longo não Codificante , Proteínas de Ligação a RNA , Sobrevivência Celular/genética , Feminino , Humanos , MicroRNAs/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Gravidez , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Trofoblastos/metabolismo
20.
J Orthop Surg Res ; 17(1): 197, 2022 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-35366936

RESUMO

Functionalized self-assembling peptides, which display functional growth-factor bioactivity, can be designed by connecting the C-terminus of a pure self-assembling peptide with a short functional motif. In this study, we designed a novel functionalized peptide (RADA16-SNVI) in which an SNVI motif with hBMP-7 activity was conjugated onto the C-terminus of the RADA16 peptide via solid-phase synthesis. A mix of RADA16-SNVI and RADA16 solutions was used to create a functionalized peptide nanofiber scaffold (SNVI-RADA16). The hydrogels were analyzed by atomic force microscopy, circular dichroism, and scanning electron microscopy. The results showed that the SNVI-RADA16 solution effectively formed hydrogel. Next, we seeded the SNVI-RADA16 scaffold with adipose-derived stem cells (ADSCs) and investigated whether it displayed biological properties of nucleus pulposus tissue. SNVI-RADA16 displayed good biocompatibility with the ADSCs and induced their expression. Cells in SNVI-RADA16 gel had a greater secretion of the extracellular matrix marker collagen type II and aggrecan compared to ADSCs grown in monolayer and control gel (p < 0.05). The ratio of the aggrecan to collagen in cells in SNVI-RADA16 gel is approximately 29:1 after culture for 21 days. ADSCs in SNVI-RADA16 gels expressed the hypoxia-inducible factor 1α(HIF-1α) mRNA by real-time PCR. However, HIF-1 mRNA is absence in control gel and monolayer. The results suggested that the functionalized self-assembled peptide promotes the differentiation of ADSCs into nucleus pulposus-like cells. Thus, the designed SNVI-RADA16 self-assembling peptide hydrogel scaffolds may be suitable for application in nucleus pulposus tissue regeneration.


Assuntos
Núcleo Pulposo , Sobrevivência Celular , Células Cultivadas , Núcleo Pulposo/metabolismo , Peptídeos/farmacologia , Células-Tronco , Tecidos Suporte/química
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