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1.
Molecules ; 26(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34684822

RESUMO

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.


Assuntos
Membrana Corioalantoide/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Pró-Fármacos/metabolismo , Células A549 , Animais , Transporte Biológico Ativo , Embrião de Galinha , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Técnicas In Vitro , Células MCF-7 , Modelos Biológicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacocinética , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacocinética , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638879

RESUMO

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.


Assuntos
Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo , Dioxanos/farmacologia , Glicoesfingolipídeos , Pirrolidinas/farmacologia , Esferoides Celulares , Animais , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/genética , Células HCT116 , Humanos , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
3.
Nat Commun ; 12(1): 5551, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548489

RESUMO

While dysregulation of RNA splicing has been recognized as an emerging target for cancer therapy, the functional significance of RNA splicing and individual splicing factors in brain tumors is poorly understood. Here, we identify SON as a master regulator that activates PTBP1-mediated oncogenic splicing while suppressing RBFOX2-mediated non-oncogenic neuronal splicing in glioblastoma multiforme (GBM). SON is overexpressed in GBM patients and SON knockdown causes failure in intron removal from the PTBP1 transcript, resulting in PTBP1 downregulation and inhibition of its downstream oncogenic splicing. Furthermore, SON forms a complex with hnRNP A2B1 and antagonizes RBFOX2, which leads to skipping of RBFOX2-targeted cassette exons, including the PTBP2 neuronal exon. SON knockdown inhibits proliferation and clonogenicity of GBM cells in vitro and significantly suppresses tumor growth in orthotopic xenografts in vivo. Collectively, our study reveals that SON-mediated RNA splicing is a GBM vulnerability, implicating SON as a potential therapeutic target in brain tumors.


Assuntos
Neoplasias Encefálicas/genética , Proteínas de Ligação a DNA/genética , Glioblastoma/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Antígenos de Histocompatibilidade Menor/genética , Proteínas do Tecido Nervoso/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Splicing de RNA , Proteínas Repressoras/genética , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Éxons , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Xenoenxertos , Humanos , Íntrons , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise de Sobrevida
4.
Sci Rep ; 11(1): 18571, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535719

RESUMO

The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , L-Lactato Desidrogenase/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Humanos , Camundongos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas
5.
Nat Commun ; 12(1): 5608, 2021 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-34556639

RESUMO

The formation of a hollow lumen in a formerly solid mass of cells is a key developmental process whose dysregulation leads to diseases of the kidney and other organs. Hydrostatic pressure has been proposed to drive lumen expansion, a view that is supported by experiments in the mouse blastocyst. However, lumens formed in other tissues adopt irregular shapes with cell apical faces that are bowed inward, suggesting that pressure may not be the dominant contributor to lumen shape in all cases. Here we use live-cell imaging to study the physical mechanism of lumen formation in Madin-Darby Canine Kidney cell spheroids, a canonical cell-culture model for lumenogenesis. We find that in this system, lumen shape reflects basic geometrical considerations tied to the establishment of apico-basal polarity. A physical model incorporating both cell geometry and intraluminal pressure can account for our observations as well as cases in which pressure plays a dominant role.


Assuntos
Algoritmos , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Modelos Teóricos , Esferoides Celulares/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Desamino Arginina Vasopressina/farmacologia , Cães , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Células Madin Darby de Rim Canino , Microscopia Confocal/métodos , Nocodazol/farmacologia , Ouabaína/farmacologia , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Moduladores de Tubulina/farmacologia
6.
Cells ; 10(9)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34572147

RESUMO

The current process of meat production using livestock has significant effects on the global environment, including high emissions of greenhouse gases. In recent years, cultured meat has attracted attention as a way to acquire animal proteins. However, the lack of markers that isolate proliferating cells from bovine tissues and the complex structure of the meat make it difficult to culture meat in a dish. In this study, we screened 246 cell-surface antibodies by fluorescence-activated cell sorting for their capacity to form colonies and their suitability to construct spheroid "meat buds". CD29+ cells (Ha2/5 clone) have a high potency to form colonies and efficiently proliferate on fibronectin-coated dishes. Furthermore, the meat buds created from CD29+ cells could differentiate into muscle and adipose cells in a three-dimensional structure. The meat buds embedded in the collagen gel proliferated in the matrix and formed large aggregates. Approximately 10 trillion cells can theoretically be obtained from 100 g of bovine tissue by culturing and amplifying them using these methods. The CD29+ cell characteristics of bovine tissue provide insights into the production of meat alternatives in vitro.


Assuntos
Técnicas de Cultura de Células/métodos , Tecnologia de Alimentos/métodos , Produtos da Carne/análise , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Tecido Adiposo/citologia , Animais , Bovinos , Diferenciação Celular/genética , Proliferação de Células/fisiologia , Células Cultivadas , Citometria de Fluxo/métodos , Gado/genética , Carne , Células-Tronco Mesenquimais/metabolismo , Esferoides Celulares/metabolismo , Células-Tronco/metabolismo
7.
J Biol Chem ; 297(4): 101139, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34461098

RESUMO

MS imaging (MSI) is a powerful tool in drug discovery because of its ability to interrogate a wide range of endogenous and exogenous molecules in a broad variety of samples. The impressive versatility of the approach, where almost any ionizable biomolecule can be analyzed, including peptides, proteins, lipids, carbohydrates, and nucleic acids, has been applied to numerous types of complex biological samples. While originally demonstrated with harvested organs from animal models and biopsies from humans, these models are time consuming and expensive, which makes it necessary to extend the approach to 3D cell culture systems. These systems, which include spheroid models, prepared from immortalized cell lines, and organoid cultures, grown from patient biopsies, can provide insight on the intersection of molecular information on a spatial scale. In particular, the investigation of drug compounds, their metabolism, and the subsequent distribution of their metabolites in 3D cell culture systems by MSI has been a promising area of study. This review summarizes the different ionization methods, sample preparation steps, and data analysis methods of MSI and focuses on several of the latest applications of MALDI-MSI for drug studies in spheroids and organoids. Finally, the application of this approach in patient-derived organoids to evaluate personalized medicine options is discussed.


Assuntos
Antineoplásicos/uso terapêutico , Descoberta de Drogas , Neoplasias , Medicina de Precisão , Esferoides Celulares/metabolismo , Animais , Técnicas de Cultura de Células , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Esferoides Celulares/patologia , Microambiente Tumoral/efeitos dos fármacos
8.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445479

RESUMO

A spaceflight to the International Space Station (ISS) is a dream of many researchers. We had the chance to investigate the effect of real microgravity (CellBox-2 Space mission) on the transcriptome and proteome of FTC-133 human follicular thyroid cancer cells (TCC). The cells had been sent to the ISS by a Falcon 9 rocket of SpaceX CRS-13 from Cape Canaveral (United States) and cultured in six automated hardware units on the ISS before they were fixed and returned to Earth. Multicellular spheroids (MCS) were detectable in all spaceflight hardware units. The VCL, PXN, ITGB1, RELA, ERK1 and ERK2 mRNA levels were significantly downregulated after 5 days in space in adherently growing cells (AD) and MCS compared with ground controls (1g), whereas the MIK67 and SRC mRNA levels were both suppressed in MCS. By contrast, the ICAM1, COL1A1 and IL6 mRNA levels were significantly upregulated in AD cells compared with 1g and MCS. The protein secretion measured by multianalyte profiling technology and enzyme-linked immunosorbent assay (AngiogenesisMAP®, extracellular matrix proteins) was not significantly altered, with the exception of elevated angiopoietin 2. TCC in space formed MCS, and the response to microgravity was mainly anti-proliferative. We identified ERK/RELA as a major microgravity regulatory pathway.


Assuntos
Adenocarcinoma Folicular/patologia , Biomarcadores Tumorais/metabolismo , Proteoma/metabolismo , Esferoides Celulares/patologia , Neoplasias da Glândula Tireoide/patologia , Transcriptoma , Ausência de Peso , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/metabolismo , Biomarcadores Tumorais/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Proteoma/análise , Voo Espacial , Esferoides Celulares/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas
9.
Cancer Sci ; 112(10): 4220-4233, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34363722

RESUMO

The ascites that develops in advanced OC, both at diagnosis and upon recurrence, is a rich source of multicellular spheroids/aggregates (MCSs/MCAs), which are the major seeds of tumor cell dissemination within the abdominal cavity. However, the molecular mechanism by which specific ascites-derived tumor cells survive and metastasize remains largely unknown. In this study, we elucidated cancer stem cell (CSC) properties of ascites-derived MCSs, concomitant with enhanced malignancy, induced EMT, and low KLF9 (Krüppel-like factor 9) expression, compared with PTCs. KLF9 was also downregulated in OC cell line-derived spheroids and the CD117+ CD44+ subpopulation in MCSs. Functional experiments demonstrated that KLF9 negatively modulated stem-like properties in OC cells. Mechanistic studies revealed that KLF9 reduced the transcriptional expression of Notch1 by directly binding to the Notch1 promoter, thereby inhibiting the function of slug in a CSL-dependent manner. Clinically, expression of KLF9 was associated with histological grade and loss of KLF9 predicts poor prognosis in OC.


Assuntos
Ascite/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Receptor Notch1/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Esferoides Celulares/patologia , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Gradação de Tumores , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Prognóstico , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Esferoides Celulares/metabolismo
10.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445168

RESUMO

Oxytocin (OXT) is a neuropeptide involved in a plethora of behavioral and physiological processes. However, there is a prominent lack of 3D cell culture models that investigate the effects of OXT on a cellular/molecular level. In this study, we established a hypothalamic neuronal spheroid model to investigate the cellular response in a more realistic 3D setting. Our data indicate that the formation of spheroids itself does not alter the basic characteristics of the cell line and that markers of cellular morphology and connectivity are stably expressed. We found that both OXT and arginine vasopressin (AVP) treatment increase spheroid size (surface area and volume), as well as individual nucleus size, which serves as an indicator for cellular proliferation. The cellular response to both OXT and AVP seems mainly to be mediated by the AVP receptor 1a (V1aR); however, the OXT receptor (OXTR) contributes significantly to the observed proliferative effect. When we blocked the OXTR pharmacologically or knocked down the OXTR by siRNA, the OXT- or AVP-induced cellular proliferation decreased. In summary, we established a 3D cell culture model of the neuronal response to OXT and AVP and found that spheroids react to the treatment via their respective receptors but also via cross-talk between the two receptor types.


Assuntos
Hipotálamo/citologia , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Arginina Vasopressina/metabolismo , Linhagem Celular , Proliferação de Células , Hipotálamo/metabolismo , Ocitocina/metabolismo , Ratos , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
11.
Biomolecules ; 11(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34439818

RESUMO

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial-mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.


Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Neoplasias da Próstata/genética , Splicing de RNA , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
12.
Biomed Res Int ; 2021: 5540877, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337022

RESUMO

Mesenchymal stem cells (MSCs) are valuable candidates in tissue engineering and stem cell-based therapy. Traditionally, MSCs derived from various tissues have been successfully expanded in vitro using adherent culture plates commonly called as monolayer two-dimensional (2D) cultures. Recently, many studies demonstrated that stemness and multilineage differentiation potential could be enhanced to greater extent when MSCs are cultured as suspended aggregates by means of three-dimensional (3D) culturing techniques. However, there are limited reports on changed mitochondrial metabolism on 3D spheroid formation of MSCs. Therefore, the present study was aimed at investigating the stemness, differentiation potential, and mitochondrial metabolism capacity of 3D dental pulp-derived MSC (DPSC) spheroids in comparison to monolayer cultured DPSCs. We isolated dental pulp-derived MSCs (DPSCs) and successfully developed a 3D culture system which facilitated the formation of MSC spheroids. The cell aggregation was observed after 2 hours, and spheroids were formed after 24 hours and remained in shape for 72 hours. After spheroid formation, the levels of pluripotent markers increased along with enhancement in adipogenic and osteogenic potential compared to 2D cultured control cells. However, decreased proliferative capacity, cell cycle arrest, and elevated apoptosis rate were observed with the time course of the 3D culture except for the initial 24-hour aggregation. Furthermore, oxygen consumption rates of living cells decreased with the time course of the aggregation except for the initial 24 hours. Overall, our study indicated that the short-term 3D culture of MSCs could be a suitable alternative to culture the cells.


Assuntos
Diferenciação Celular , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/citologia , Esferoides Celulares/citologia , Adipogenia , Apoptose , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Ciclo Celular , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Consumo de Oxigênio , Células-Tronco Pluripotentes/metabolismo , Esferoides Celulares/metabolismo
13.
Exp Cell Res ; 406(2): 112765, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34358523

RESUMO

Nasopharyngeal carcinoma (NPC) originates in the nasopharynx epithelium. Although concurrent chemoradiation therapy followed by chemotherapy is considered as an effective treatment, there is substantial drug resistance in locally advanced NPC patients. One major contributor to the chemoresistance includes aberrant expression of cell adhesion molecules, such as integrin α and ß subunits, giving rise to cell adhesion-mediated drug resistance. Thus, the aim of this study was to investigate the effect of integrin α5 on the development of intrinsic cisplatin resistance in NPC and the associated underlying mechanisms using in vitro three-dimensional (3D) spheroid models, as well as induced cisplatin-resistant NPC (NPCcisR). We demonstrated that established 3D highly- (5-8F) and lowly- (6-10B) metastatic NPC spheroids overexpressed integrin α5 and aggravated their resistance to cisplatin. Besides, enhanced integrin α5 resulted in substantially reduced growth, corresponding to G0/G1 and G2/M cell cycle arrest. In addition, 5-8FcisR and 6-10BcisR cells in 3D forms synergistically strengthened endurance of their spheroids to cisplatin treatment as observed by increased resistance index (RI) and decreased apoptosis. Mechanistically, the aberrantly expressed integrin α5 decreased drug susceptibility in NPC spheroids by inactivating ERK and inhibition of caspase-3 inducing apoptosis. Furthermore, the effect of integrin α5 inducing intrinsic resistance was verified via treatment with ATN-161, a peptide inhibitor for integrin α5ß1. The results showed dramatic reduction in integrin α5 expression, reversal of ERK phosphorylation and caspase-3 cleavage, together with elevated cisplatin sensitivity, indicating regulation of innate drug resistance via integrin α5. Taken together, our findings suggest that integrin α5 could act as a promising target to enhance the chemotherapeutic sensitivity in NPC.


Assuntos
Apoptose , Caspase 3/química , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Integrina alfa5/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/química , Carcinoma Nasofaríngeo/patologia , Esferoides Celulares/patologia , Antineoplásicos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular , Humanos , Integrina alfa5/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/secundário , Fosforilação , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo
14.
Sci Rep ; 11(1): 17076, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426602

RESUMO

Multicellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.


Assuntos
Ciclo Celular , Esferoides Celulares/metabolismo , Carcinoma de Células Renais/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA/metabolismo , Humanos , Microscopia Confocal/métodos , Sonicação/métodos , Esferoides Celulares/citologia , Células Tumorais Cultivadas
15.
Cells ; 10(7)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34359874

RESUMO

AFM-based rheology methods enable the investigation of the viscoelastic properties of cancer cells. Such properties are known to be essential for cell functions, especially for malignant cells. Here, the relevance of the force modulation method was investigated to characterize the viscoelasticity of bladder cancer cells of various invasiveness on soft substrates, revealing that the rheology parameters are a signature of malignancy. Furthermore, the collagen microenvironment affects the viscoelastic moduli of cancer cell spheroids; thus, collagen serves as a powerful proxy, leading to an increase of the dynamic moduli vs. frequency, as predicted by a double power law model. Taken together, these results shed new light on how cancer cells and tissues adapt their viscoelastic properties depending on their malignancy and the microenvironment. This method could be an attractive way to control their properties in the future, based on the similarity of spheroids with in vivo tumor models.


Assuntos
Colágeno/farmacologia , Células Epiteliais/patologia , Esferoides Celulares/patologia , Neoplasias da Bexiga Urinária/patologia , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colágeno/química , Elasticidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Força Atômica , Modelos Biológicos , Reologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Microambiente Tumoral , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Viscosidade
16.
Cells ; 10(7)2021 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-34359860

RESUMO

A major problem in psychiatric research is a deficit of relevant cell material of neuronal origin, especially in large quantities from living individuals. One of the promising options is cells from the olfactory neuroepithelium, which contains neuronal progenitors that ensure the regeneration of olfactory receptors. These cells are easy to obtain with nasal biopsies and it is possible to grow and cultivate them in vitro. In this work, we used RNAseq expression profiling and immunofluorescence microscopy to characterise neurospheres-derived cells (NDC), that simply and reliably grow from neurospheres (NS) obtained from nasal biopsies. We utilized differential expression analysis to explore the molecular changes that occur during transition from NS to NDC. We found that processes associated with neuronal and vascular cells are downregulated in NDC. A comparison with public transcriptomes revealed a depletion of neuronal and glial components in NDC. We also discovered that NDC have several metabolic features specific to neuronal progenitors treated with the fungicide maneb. Thus, while NDC retain some neuronal/glial identity, additional protocol alterations are needed to use NDC for mass sample collection in psychiatric research.


Assuntos
Mucosa Olfatória/citologia , Esferoides Celulares/citologia , Adulto , Biomarcadores/metabolismo , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Análise de Componente Principal , Esferoides Celulares/metabolismo , Transcriptoma/genética
17.
Cells ; 10(7)2021 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-34359918

RESUMO

The regulator of G-protein signaling 5 (RGS5) acts as an inhibitor of Gαq/11 and Gαi/o activity in vascular smooth muscle cells (VSMCs), which regulate arterial tone and blood pressure. While RGS5 has been described as a crucial determinant regulating the VSMC responses during various vascular remodeling processes, its regulatory features in resting VSMCs and its impact on their phenotype are still under debate and were subject of this study. While Rgs5 shows a variable expression in mouse arteries, neither global nor SMC-specific genetic ablation of Rgs5 affected the baseline blood pressure yet elevated the phosphorylation level of the MAP kinase ERK1/2. Comparable results were obtained with 3D cultured resting VSMCs. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promoted their resting state as evidenced by microarray-based expression profiling and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, RGS5 overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation, and migration, which was mimicked by selectively inhibiting Gαi/o but not Gαq/11 activity. Collectively, the heterogeneous expression of Rgs5 suggests arterial blood vessel type-specific functions in mouse VSMCs. This comprises inhibition of acute agonist-induced Gαq/11/calcium release as well as the support of a resting VSMC phenotype with low ERK1/2 activity by suppressing the activity of Gαi/o.


Assuntos
Pontos de Checagem do Ciclo Celular , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Proteínas RGS/metabolismo , Animais , Pressão Sanguínea , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Diástole , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Fosforilação , Esferoides Celulares/metabolismo , Sístole , Proteína rhoA de Ligação ao GTP/metabolismo
18.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361027

RESUMO

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias Bucais/patologia , Neovascularização Patológica/patologia , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Escamosas/metabolismo , Exossomos/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Bucais/metabolismo , Neovascularização Patológica/metabolismo , Cultura Primária de Células/métodos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/normas
19.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360970

RESUMO

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.


Assuntos
Ligamento Cruzado Anterior/fisiologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Ligamento Cruzado Anterior/citologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Conexinas/genética , Conexinas/metabolismo , Decorina/genética , Decorina/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibroblastos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Masculino , Poliésteres/química , Coelhos , Regeneração , Esferoides Celulares/metabolismo , Estresse Mecânico
20.
Biochem Biophys Res Commun ; 570: 131-136, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34280616

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs.


Assuntos
Neoplasias da Mama/genética , Proteínas Cdc20/metabolismo , Regulação para Baixo , Metilcolantreno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Esferoides Celulares/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Transcriptoma/genética
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