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1.
Life Sci ; 249: 117536, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32165211

RESUMO

AIMS: The malignancy of the Glioblastomas (GBM), the most frequent and aggressive brain tumors, have been associated with the presence of glioma stem cells (GSCs) which can form gliomaspheres (GS) in vitro. Progesterone (P) increases the proliferation, migration, and invasion of GBM cell lines through the interaction with its intracellular receptor (PR). However, it is unknown if the PR is expressed and the possible effects of P in the formation/differentiation of GS. MAIN METHODS: GS were grown from U251 and U87 cell lines by selective culture with serum-free neural stem cell medium. GSCs were identified by the detection of Sox2, Ki67, Nestin, CD133, and CD15 by immunofluorescence. Additionally, the relative expression of PROM1, NES, SOX2, OLIG2, EZH2, BMI1 and PR genes was evaluated by RT-qPCR. The GS were treated with P, and the number of cells was quantified. By RT-PCR the ßIII-TUB and GFAP differentiation genes were evaluated. KEY FINDINGS: GS were maintained until passage four. The expression of all GSCs markers was significantly higher in GS as compared with the basal culture of U251 and U87 cells. We demonstrated for the first time that PR is expressed in GS and this expression was higher as compared with the U251 and U87 cells in basal conditions. Also, we observed that P increased the number of cells derived primary gliomaspheres (GS1) from the U251 line, as well as the expression of the neuronal differentiation marker ßIII-TUB. SIGNIFICANCE: These results suggest the participation of P in the growth of GSCs.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Progesterona/farmacologia , Esferoides Celulares/patologia , Antígeno AC133/genética , Neoplasias Encefálicas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Humanos , Células-Tronco Neoplásicas/patologia , Regiões Promotoras Genéticas
2.
Gynecol Oncol ; 156(1): 251-259, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31767187

RESUMO

The majority of endometrial cancers are detected early with a favourable prognosis. However, for patients with advanced disease, chemotherapy response rates and overall survival remains poor. The endometrial cancer population is typically elderly with multiple co-morbidities and aggressive cytotoxic therapy may be hazardous. Therefore, there is an urgent need to define optimal treatment strategies for advanced and recurrent disease and personalise therapy based on individual tumour and patient characteristics. Three-dimensional (3D) models that preserve the tumour microenvironment and tumour-stromal interactions are increasingly important for translational research with the advent of immunotherapy and molecularly targeted agents. 3D patient-relevant pre-clinical models in endometrial cancer include spheroids, patient-derived organoids, microfluidic systems, patient-derived xenografts and patient-derived explants. Here we present a review of available 3D modelling systems in endometrial cancers, highlighting their current use, advantages, disadvantages and applications to translational research with a focus on the power of the patient-derived explant platform.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias do Endométrio/patologia , Animais , Carcinoma Endometrioide/tratamento farmacológico , Carcinoma Endometrioide/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias do Endométrio/tratamento farmacológico , Feminino , Xenoenxertos , Humanos , Transplante de Neoplasias/métodos , Organoides/patologia , Esferoides Celulares/patologia , Pesquisa Médica Translacional/métodos
3.
Cancer Sci ; 111(1): 239-252, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31729096

RESUMO

Hypoxia-inducible factor 1 (HIF-1) is a critical heterodimeric transcription factor for tumor malignancy. Recently, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) has been reported to function as a deubiquitinating enzyme for the stabilization of its α subunit (HIF-1α). In the present study, we showed that UCHL1 inhibition can be an effective therapeutic strategy against HIF-1-dependent tumor malignancy. In 2D monolayer culture, a UCHL1 inhibitor suppressed HIF activity and decreased the transcription of HIF downstream genes by inhibiting the UCHL1-mediated accumulation of HIF-1α. Phenotypically, UCHL1 inhibition remarkably blocked cell migration. In 3D spheroid culture models, ectopic expression of UCHL1 significantly upregulated malignancy-related factors such as solidity, volume, as well as viable cell number in an HIF-1α-dependent manner. Conversely, inhibition of the UCHL1-HIF-1 pathway downregulated these malignancy-related factors and also abolished UCHL1-mediated cell proliferation and invasiveness. Finally, inhibition of UCHL1 promoted HIF-1α degradation and lowered the expression of HIF-1 target genes in the 3D model, as also observed in 2D monolayer culture. Our research indicates that the UCHL1-HIF-1 pathway plays a crucial role in tumor malignancy, making it a promising therapeutic target for cancer chemotherapy.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Esferoides Celulares/patologia , Ubiquitina Tiolesterase/genética , Ubiquitinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Regulação para Cima/genética
4.
Cancer Sci ; 111(2): 467-476, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31845453

RESUMO

Collective invasion of cancer cells is the key process of circulating tumor cell (CTC) cluster formation, and greatly contributes to metastasis. Cancer stem-like cells (CSC) have a distinct advantage of motility for metastatic dissemination. To verify the role of CSC in the collective invasion, we performed 3D assays to investigate the collective invasion from cancer cell spheroids. The results demonstrated that CSC can significantly promote both collective and single-cell invasion. Further study showed that CSC prefer to move outside and lead the collective invasion. More interestingly, approximately 60% of the leader CSC in collective invasion co-expressed both epithelial and mesenchymal genes, while only 4% co-expressed in single invasive CSC, indicating that CSC with hybrid epithelial/mesenchymal phenotype play a key role in cancer cell collective invasion.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Invasividade Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
5.
Int J Nanomedicine ; 14: 8543-8560, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31802868

RESUMO

Background: Nanoparticles exhibit great promise for improving the solubility and tissue-specific distribution of chemotherapeutic agents; however, the passive and highly variable enhanced permeability and retention (EPR) effects observed in tumors frequently leads to insufficient delivery of nanodrugs into tumors. The tumor-penetrating peptide iRGD can actively enhance tumor-selective delivery of nanoparticles into tumors by binding to integrin and interacting with tissue-penetrating receptor neuropilin-1. Materials and methods: To improve colorectal cancer treatment, in this study, we prepared a paclitaxel (PTX)-loaded PLGA nanoparticle (PLGA-PTX) and evaluated its tumor-targeting and antitumor activity by co-administration with iRGD. Results: Compared to free PTX, encapsulated PTX retained preferential cytotoxicity toward various colorectal cancer cells while effectively sparing healthy cells. PLGA-PTX treatment resulted in cell cycle arrest at the G2/M phase and apoptosis, leading to inhibition of cancer cell migration and invasion. PLGA-PTX combined with iRGD displayed little enhancement of cytotoxicity in vitro. Despite this, iRGD receptors integrin and neuropilin-1 were found to be primarily overexpressed on abundant tumor vessels in mice bearing colorectal tumors. Consequently, co-administration of nanoparticles with iRGD promoted the selective delivery of nanoparticles into tumor tissues in vivo. Additionally, the combined regimen enhanced the antitumor effects compared to those of each individual reagent. Conclusion: Our findings suggest that PLGA nanoparticles combined with the iRGD peptide provide a promising drug delivery strategy for facilitating active drug accumulation into tumors, given that iRGD receptors are overexpressed on tumor vessels. This co-administration system lacking covalent conjugation provides a more convenient means to combine various therapeutic agents with iRGD to achieve personalized nanotherapy.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Oligopeptídeos/administração & dosagem , Paclitaxel/uso terapêutico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/patologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/administração & dosagem , Invasividade Neoplásica , Oligopeptídeos/química , Paclitaxel/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Distribuição Tecidual
6.
Int J Nanomedicine ; 14: 9483-9496, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31819445

RESUMO

Background: The efficacy of radiotherapy for glioma is often limited by the radioresistance of glioma cells. The radiosensitizing effects of silver nanoparticles (AgNPs) on glioma were found in the previous studies of our group. In order to enhance the radiosensitivity of tumor cells and selectively kill them while reducing the side effects of irradiation therapy, targeted modification of AgNPs is urgently needed. Materials and methods: In the present study, AgNPs functionalized with polyethylene glycol (PEG) and aptamer As1411 (AsNPs) were synthesized and subsequently characterized by transmission electron microscopy, ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Then the targeting property of AsNPs was evaluated by dark-field imaging, confocal microscopy and in vivo imaging. Both colony formation assay and glioma-bearing mouse model were employed to study the radiosensitizing effect of AsNPs. Results: The characterization results revealed a spherical shape of AgNPs with an average diameter of 18 nm and the successful construction of AsNPs. AsNPs were confirmed to specifically target C6 glioma cells, but not normal human microvascular endothelial cells. Moreover, AsNPs could not only internalize into tumor cells, but also penetrate into the core of tumor spheroids. In vitro experiments showed that AsNPs exhibited a better radiosensitizing effect than AgNPs and PEGylated AgNPs (PNPs), inducing a higher rate of apoptotic cell death. In vivo imaging demonstrated that Cy5-AsNPs preferentially accumulated at the tumor site, and the ratio of fluorescence intensity of Cy5-AsNPs to that of Cy5-PNPs reached the maximum at 6 h post-systemic administration. Furthermore, the combination of AsNPs with irradiation significantly prolonged the median survival time of C6 glioma-bearing mice. Conclusion: Our results indicated that AsNPs could be an effective nano-radiosensitizer for glioma targeting treatment.


Assuntos
Aptâmeros de Nucleotídeos/química , Glioma/radioterapia , Nanopartículas Metálicas/química , Oligodesoxirribonucleotídeos/química , Polietilenoglicóis/química , Radiossensibilizantes/farmacologia , Prata/química , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Feminino , Glioma/tratamento farmacológico , Humanos , Hidrodinâmica , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos BALB C , Camundongos Nus , Tamanho da Partícula , Ratos , Esferoides Celulares/patologia , Eletricidade Estática , Distribuição Tecidual
7.
Int J Nanomedicine ; 14: 6831-6842, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695364

RESUMO

Background: Cancer relapse and metastasis is an obstacle to the treatment of breast cancer. Breast cancer stem cells (BCSCs), which can evade the killing effect of traditional chemotherapies, such as doxorubicin (DOX), may contribute to cancer development. Therefore, it is necessary to develop novel drugs that can target and eliminate BCSCs. While multiple strategies have been conceived, they are normally limited by the low drug loading capacity. Purpose: An aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX, which consists of a CD44 aptamer TA6, DNA building blocks M1 and M2 conjugated with an AKT inhibitor peptide AKTin individually and DOX, was designed. Methods: This DNA nanotrain was prepared through hybridization chain reactionand this highly ordered DNA duplex has plenty of sites where DOX and AKTin can be intercalated or anchored. By performing on MCF-7 BCSCs and tumors by xenografting BCSCs into nude mice, efficacy of the newly prepared drug was evaluated and compared with that of free DOX and various DNA nanotrains. Results: TA6NT-AKTin-DOX showed better efficacy both in vitro and in vivo. To some extent, the enhanced efficacy could be attributed to the targeting effect of TA6 and the high drug loading capacity of the nanotrain (~20 DOX molecules). Besides, a synergistic response was demonstrated by combining DOX with AKTin, probably due to that the anchored AKTin can reverse the drug resistance of BCSCs including apoptosis resistance and ABC transporters overexpression via the AKT signaling pathway. Conclusion: The aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX demonstrated its targeting capability to BCSCs.


Assuntos
Aptâmeros de Nucleotídeos/química , Neoplasias da Mama/patologia , DNA/química , Doxorrubicina/uso terapêutico , Nanopartículas/química , Células-Tronco Neoplásicas/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Dano ao DNA , Doxorrubicina/farmacologia , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos
8.
Biomed Pharmacother ; 118: 109371, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31545281

RESUMO

BACKGROUND: Disulfiram (DSF) is a drug used for treatment of alcoholism that has also displayed promising anti-cancer activity. It unfolds its effects by inhibiting the enzyme activity of aldehyde dehydrogenase (ALDH) isoforms. METHODS: MTT assay, spheroid formation, clonogenicity assay, qRT-PCR, and ALDH enzyme activity analysis were performed using ovarian cancer cell lines IGROV1, SKOV3 and SKOV3IP1. Cell cycle analyses and measurement of intracellular reactive oxygen species (ROS) were carried out by flow cytometry. ALDH+ and ALDH- cells were isolated by FACS sorting. RESULTS: ALDH activity was inhibited in ovarian cancer stem cells (the proportion of ALDH+ cells was reduced from 21.7% to 0.391%, 8.4% to 0%, 6.88% to 0.05% in cell lines IGROV1, SKOV3, and SKOV3IP1, respectively). DSF with or without the cofactor copper (Cu2+) exhibited cytotoxicity dose- and time-dependent and enhanced cisplatin-induced apoptosis. DSF + Cu2+ increased intracellular ROS levels triggering apoptosis of ovarian cancer stem cells (CSC). Significantly more colony and spheroid formation was observed in ALDH+ compared with ALDH- cells (P < 0.01). Moreover, ALDH+ cells were more resistant to cisplatin treatment compared with ALDH-cells (P < 0.05) and also exhibited a lower basal level of ROS. However, no significant difference in ROS accumulation nor in cellular viability was observed in ALDH + cells in comparison to ALDH- cells after pre-treatment with DSF (0.08 µM). CONCLUSION: Our findings provide evidence that DSF might be employed as a novel adjuvant chemotherapeutic agent in combination with cisplatin for treatment of ovarian cancer.


Assuntos
Aldeído Desidrogenase/metabolismo , Cobre/farmacologia , Dissulfiram/farmacologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Humanos , Concentração Inibidora 50 , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
9.
Tissue Cell ; 59: 39-43, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383287

RESUMO

Major Glioblastoma's hallmarks include proliferation, invasion and heterogeneity. Biological 3D tumor spheroid models can serve as intermediate systems between traditional 2D cell culture and complex in vivo models. Tumor spheroids have been shown to more accurately reproduce the spatial organization and microenvironmental factors of in vivo micro-tumors, such as relevant gradients of nutrients and other molecular agents, while they maintain cell-to-cell and cell-to-matrix interactions. In vitro 3D assays are useful to monitor these properties. Here, we test the suitability of the well-known T98 G Glioblastoma cell line in such a 3D assay. The doubling time and death rate parameters of T98 G are estimated, as well as their spheroidal growth-expansion curves with and without the presence of basement membrane substrate. The T98 G invasive profile is characterized by collective morphology and proliferation-associated invasion. We show that the T98 G secondary GB cell line exhibits both invasive and proliferative capabilities in 3D and thus, can serve as control cell line for the 3D in vitro study of primary GB cell cultures.


Assuntos
Glioblastoma , Modelos Biológicos , Esferoides Celulares , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
10.
Biomed Pharmacother ; 118: 109281, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377469

RESUMO

In 2018 there were over 1.8 million new cases worldwide of colorectal cancer and relapses after clinical treatments. Many studies ascribe the risk of the appearance of this cancer to the Western life style : a sedentary life, obesity, and low -fiber, high -fat diets can promote the onset of disease. Several studies have shown supplement phytochemicals to have an inhibiting effect on the growth of various cancers through the activation of apoptosis. Our goal was to prove the effectiveness of a natural compound in the combined therapy of colorectal cancer. Trigno M supplement was an optimal candidate as anticancer product for its high concentrations of phenolic acids, flavonoids and anthocyanins. Our work showed the antitumor activity of Trigno M, extract of Prunus spinosa drupes combined with the nutraceutical activator complex (NAC), in 2D, 3D and in vivo colorectal cancer models. The cellular model we used both in vitro and in vivo was the HCT116 cell line, particularly suitable for engraftment after inoculation in mice. Trigno M inhibited the growth and colony formation of HCT116 cells (35%) as compared to the chemotherapy treatment with 5-fluorouracil (80%) used in clinical therapy. The reduction of the morphological dimensions in the spheroid cells after Trigno M, was compared with 5-fluorouracil demonstrating the efficacy of the Trigno M compound also in 3D models. Flow cytometric analysis on 3D cells showed a significant increase in the apoptotic cell fraction after Trigno M treatment (44.8%) and a low level of necrotic fraction (6.7%) as compared with control cells. Trigno M and 5-fluorouracil induced the apoptosis in a comparable percentage. Monotherapy with Trigno M in severely immunodeficient mice, carrying colon rectal cancer xenografts, significantly reduced tumor growth. The histopatological analysis of the ectopic tumors showed a lower level of necrosis after Trigno M treatment compared with the control. We conclude that Trigno M is well tolerated by mice, delays colorectal cancer growth in these animals and should be weighed up for integration of the current multi-drug protocols in the treatment of colon carcinoma.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Modelos Biológicos , Extratos Vegetais/uso terapêutico , Prunus/química , Acetilcisteína/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Neoplasias do Colo/ultraestrutura , Feminino , Fluoruracila/farmacologia , Células HCT116 , Humanos , Camundongos SCID , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Eur Phys J E Soft Matter ; 42(8): 112, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31456065

RESUMO

Computational models aiming at the spatio-temporal description of cancer evolution are a suitable framework for testing biological hypotheses from experimental data, and generating new ones. Building on our recent work (J. Theor. Biol. 389, 146 (2016)) we develop a 3D agent-based model, capable of tracking hundreds of thousands of interacting cells, over time scales ranging from seconds to years. Cell dynamics is driven by a Monte Carlo solver, incorporating partial differential equations to describe chemical pathways and the activation/repression of "genes", leading to the up- or down-regulation of specific cell markers. Each cell-agent of different kind (stem, cancer, stromal etc.) runs through its cycle, undergoes division, can exit to a dormant, senescent, necrotic state, or apoptosis, according to the inputs from its systemic network. The basic network at this stage describes glucose/oxygen/ATP cycling, and can be readily extended to cancer-cell specific markers. Eventual accumulation of chemical/radiation damage to each cell's DNA is described by a Markov chain of internal states, and by a damage-repair network, whose evolution is linked to the cell systemic network. Aimed at a direct comparison with experiments of tumorsphere growth from stem cells, the present model will allow to quantitatively study the role of transcription factors involved in the reprogramming and variable radio-resistance of simulated cancer-stem cells, evolving in a realistic computer simulation of a growing multicellular tumorsphere.


Assuntos
Carcinogênese/metabolismo , Evolução Clonal , Modelos Teóricos , Esferoides Celulares/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Dano ao DNA , Glucose/metabolismo , Humanos , Cadeias de Markov , Oxigênio/metabolismo , Esferoides Celulares/patologia , Células Tumorais Cultivadas
12.
BMC Cancer ; 19(1): 760, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370822

RESUMO

BACKGROUNDS: The role of sphere-forming culture in enriching subpopulations with stem-cell properties in hepatocellular carcinoma (HCC) is unclear. The present study investigates its value in enriching cancer stem cells (CSCs) subpopulations and the mechanism by which HCC CSCs are maintained. METHODS: HCC cell lines and fresh primary tumor cells were cultured in serum-free and ultra-low attachment conditions to allow formation of HCC spheres. In vitro and in vivo experiments were performed to evaluate CSC characteristics. Expression levels of CSC-related genes were assessed by qRT-PCR and the correlation between sphere formation and clinical characteristics was investigated. Finally, gene expression profiling was performed to explore the molecular mechanism underlying HCC CSC maintenance. RESULTS: We found that both cell lines and primary tumor cells formed spheres. HCC spheres possessed the capacity for self-renewal, proliferation, drug resistance, and contained different subpopulations of CSCs. Of interest, 500 sphere-forming Huh7 cells or 200 primary tumor cells could generate tumors in immunodeficient animals. Sphere formation correlated with size, multiple tumors, satellite lesions, and advanced stage. Further investigation identified that the PPARα-SCD1 axis plays an important role in maintenance of the CSC properties of HCC sphere cells by promoting nuclear accumulation of ß-Catenin. Inhibition of SCD1 interfered with sphere formation, down-regulated expression of CSC-related markers, and reduced ß-Catenin nuclear accumulation. CONCLUSIONS: Sphere-forming culture can effectively enrich subpopulations with stem-cell properties, which are maintained through activation of the PPARα-SCD1 axis. Therefore, we suggest that targeting the SCD1-related CSC machinery might provide a novel insight into HCC treatment.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Estearoil-CoA Dessaturase/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Autorrenovação Celular , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , PPAR alfa/metabolismo , Transdução de Sinais , Estearoil-CoA Dessaturase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Tumour Biol ; 41(8): 1010428319869101, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31423948

RESUMO

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24- cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Animais , Antígeno CD24/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Embrião de Galinha , Membrana Corioalantoide/citologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/genética , Células Tumorais Cultivadas
14.
J Vet Med Sci ; 81(9): 1238-1248, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31308293

RESUMO

Cancer consists of heterogeneous cells that contain a small population of cells that possess stem cell properties; these cells, referred to as cancer stem cells (CSCs) or tumor-initiating cells, are involved in tumor progression and metastasis. Using a sphere-forming assay, canine mammary CSCs were found to be similar to human breast CSCs. Metabolic reprogramming has been recognized as a hallmark of various cancers. However, the significance of cellular metabolism in CSCs remains unclear. The aim of this study was to define the metabolic characteristics of CSCs derived from canine mammary tumors and gain an understanding of the maintenance of stemness. We identified metabolite profiles of canine mammary adenocarcinoma cell lines using gas chromatography-mass spectrometry. Metabolites were extracted from both adherent and sphere-forming cells derived from three cell lines. Sphere-forming cells were separated from adherent cells using an orthogonal, partial least-squares discriminant analysis. Sphere-forming cells were found to contain high levels of the amino acids alanine, glycine and proline compared with adherent cells. They also had high levels of palmitoleate, palmitate and dihomo-gamma-linolenic acid compared with adherent cells. In a sphere-forming assay, palmitate increased the number of spheres for all cell lines. These results indicate that the sphere-forming cells derived from canine mammary adenocarcinoma cell lines have specific metabolic profiles that may be useful for the development of CSC-specific therapies targeting metabolic pathways and potential stemness biomarkers; these results also clarify the maintenance of stemness in canine mammary CSCs.


Assuntos
Adenocarcinoma/veterinária , Neoplasias Mamárias Animais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Aminoácidos/metabolismo , Animais , Linhagem Celular Tumoral , Cães , Ácidos Graxos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Neoplasias Mamárias Animais/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Palmitatos/farmacologia
15.
Adv Mater ; 31(36): e1901166, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322299

RESUMO

Many 3D in vitro models induce breast cancer spheroid formation; however, this alone does not recapitulate the complex in vivo phenotype. To effectively screen therapeutics, it is urgently needed to validate in vitro cancer spheroid models against the gold standard of xenografts. A new oxime-crosslinked hyaluronan (HA) hydrogel is designed, manipulating gelation rate and mechanical properties to grow breast cancer spheroids in 3D. This HA-oxime breast cancer model maintains the gene expression profile most similar to that of tumor xenografts based on a pan-cancer gene expression profile (comprising 730 genes) of three different human breast cancer subtypes compared to Matrigel or conventional 2D culture. Differences in gene expression between breast cancer cultures in HA-oxime versus Matrigel or 2D are confirmed for 12 canonical pathways by gene set variation analysis. Importantly, drug response is dependent on the culture method. Breast cancer cells respond better to the Rac inhibitor (EHT-1864) and the PI3K inhibitor (AZD6482) when cultured in HA-oxime versus Matrigel. This study demonstrates the superiority of an HA-based hydrogel as a platform for in vitro breast cancer culture of both primary, patient-derived cells and cell lines, and provides a hydrogel culture model that closely matches that in vivo.


Assuntos
Neoplasias da Mama/patologia , Transformação Celular Neoplásica , Ácido Hialurônico/química , Hidrogéis/química , Hidrogéis/farmacologia , Oximas/química , Esferoides Celulares/efeitos dos fármacos , Animais , Benchmarking , Linhagem Celular Tumoral , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Esferoides Celulares/patologia
16.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340547

RESUMO

BACKGROUND: Lung cancer cells are known to change proliferation and migration under simulated microgravity. In this study, we sought to evaluate cell adherence, apoptosis, cytoskeleton arrangement, and gene expression under simulated microgravity. METHODS: Human lung cancer cells were exposed to simulated microgravity in a random-positioning machine (RPM). Cell morphology and adherence were observed under phase-contrast microscopy, cytoskeleton staining was performed, apoptosis rate was determined, and changes in gene and protein expression were detected by real-time PCR with western blot confirmation. RESULTS: Three-dimensional (3D)-spheroid formation was observed under simulated microgravity. Cell viability was not impaired. Actin filaments showed a shift in alignment from longitudinal to spherical. Apoptosis rate was significantly increased in the spheroids compared to the control. TP53, CDKN2A, PTEN, and RB1 gene expression was significantly upregulated in the adherent cells under simulated microgravity with an increase in corresponding protein production for p14 and RB1. SOX2 expression was significantly upregulated in the adherent cells, but protein was not. Gene expressions of AKT3, PIK3CA, and NFE2L2 remained unaltered. CONCLUSION: Simulated microgravity induces alteration in cell adherence, increases apoptosis rate, and leads to upregulation of tumor suppressor genes in human lung cancer cells.


Assuntos
Apoptose/genética , Adesão Celular/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Ausência de Peso , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Simulação de Ausência de Peso/instrumentação , Simulação de Ausência de Peso/métodos
17.
Biomed Pharmacother ; 117: 109051, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31177062

RESUMO

The inhibitory roles of limonin have been revealed in various tumors. However, the roles and related mechanism of limonin in hepatocellular carcinoma (HCC) progression are still confused. Here, we collected non-adherent spheroids formed by HCC cells and found that the proportion of spheroids in G0 phase was remarkably higher than that in HCC cells. Additionally, limonin increased EdU incorporation without affecting apoptosis in spheroids. Notably, upon limonin treatment, the proportion of spheroids in G0 was decreased and S/G2/M increased. Furthermore, we found that limonin attenuated the stemness of HCC cells, characterized as the decreased ALDH1 activity, stemness marker expression and spheroid formation ability. Moreover, an integrated transcriptional analysis based on RNA-sequencing data was employed to gain mechanistic insight into limonin functioning, and we found that the most significant pathways identified centered on PI3K/Akt signaling. qRT-PCR and western blot obtained the consistent results. Overall, these data suggest that limonin attenuates the stemness of HCC cells by reducing cellular quiescence through activating PI3K/Akt signaling.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Ciclo Celular , Limoninas/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Hep G2 , Humanos , Limoninas/farmacologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
18.
Mater Sci Eng C Mater Biol Appl ; 102: 863-875, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31147058

RESUMO

The frequent occurrence of multidrug resistance (MDR) in solid tumors is the major obstacle for nano-drug delivery systems (nDDS) to realize the successful cancer chemotherapy. Herein, we had prepared pH-responsive nanogels via cross-linking TPGS-grafted soy protein with an acid-labile ortho ester cross-linker (OEAM) to realize the efficient intracellular drugs release and accumulation, and subsequently enhance therapeutic effect in MDR tumor cells. These nanogels displayed a uniform size (~200 nm) and morphology, and the introduction of ortho ester bonds endowed nanogels stability in neutral environment and acid-degradability in acidic conditions. Cisplatin (CDDP) was successfully loaded into nanogels, which exhibited an accelerated drug release at low pH. The modification of TPGS efficiently improved cellular internalization and drug accumulation in A549/DDP cells by inhibiting the function of drug efflux pumps (MRP2 and ATP7A/7B), leading to higher cytotoxicity and apoptosis. Moreover, TPGS-grafted nanogels also showed better drug accumulation and penetration in tumor-like spheroids, and then remarkably inhibited tumor growth owing to the rapid drug release in acidic organelles. As a result, the TPGS-grafted and pH-sensitive soy protein nanogels have a great potential as a drugs carrier for the efficient cancer treatment.


Assuntos
Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos , Espaço Intracelular/química , Metacrilatos/química , Polietilenoglicóis/química , Polietilenoimina/química , Proteínas de Soja/química , Vitamina E/química , Células A549 , Trifosfato de Adenosina/metabolismo , Apoptose , Endocitose , Humanos , Hidrodinâmica , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial , Tamanho da Partícula , Espectroscopia de Prótons por Ressonância Magnética , Esferoides Celulares/patologia
19.
Curr Med Sci ; 39(3): 403-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209810

RESUMO

Accumulation of lactate in tumor has been linked to poor prognosis of oral squamous cell carcinoma (OSCC), but the underlying mechanism remained largely uncertain. Previous studies have suggested that presence of cancer stem cells (CSCs) closely correlated with cellular malignancy of OSCC. Here, using 3D organoid culture model, we investigated whether lactate promoted CSCs phenotype in primary OSCC cells. We generated organoids using fresh OSCC specimens and verified that organoids recapitulated histopathology and cellular heterogeneity of parental tumor. Organoids were then transfected with a Wnt reporter to visualize Wnt activity. The sphere forming assay demonstrated that high Wnt activity functionally designated CSCs population in OSCC cells. Further investigations indicated that lactate treatment promoted Wnt activity and increased the expression of CSCs (i.e. CD133+ cells) in organoids. Moreover, silencing monocarboxylate transporter 1 (MCT1), the prominent path for lactate uptake in human tumor with siRNA significantly impaired organoid forming capacity of OSCC cells. Together, our study demonstrated that lactate can promote CSCs phenotype of OSCC, and MCT1 may be a therapeutic target against OSCC growth.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Ácido Láctico/farmacologia , Transportadores de Ácidos Monocarboxílicos/genética , Neoplasias Bucais/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Simportadores/genética , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Idoso , Proteína Axina/genética , Proteína Axina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Via de Sinalização Wnt/genética
20.
Mol Carcinog ; 58(10): 1754-1769, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31215708

RESUMO

We have previously shown that nearly half of mesothelioma patients have tumors with low autophagy and that these patients have a significantly worse outcome than those with high autophagy. We hypothesized that autophagy may be beneficial by facilitating immunogenic cell death (ICD) of tumor cells following chemotherapy. An important hallmark of ICD is that death of tumor cells is preceded or accompanied by the release of damage-associated molecular pattern molecules (DAMPs), which then can stimulate an antitumor immune response. Therefore, we measured how autophagy affected the release of three major DAMPs: high mobility group box 1 (HMGB1), ATP, and calreticulin following chemotherapy. We found that autophagy in three-dimensional (3D) models with low autophagy at baseline could be upregulated with the cell-permeant Tat-BECN1 peptide and confirmed that autophagy in 3D models with high autophagy at baseline could be inhibited with MRT 68921 or ATG7 RNAi, as we have previously shown. In in vitro 3D spheroids, we found that, when autophagy was high or upregulated, DAMPs were released following chemotherapy; however, when autophagy was low or inhibited, DAMPs release was significantly impaired. Similarly, in ex vivo tumors, when autophagy was high or upregulated, HMGB1 was released following chemotherapy but, when autophagy was low, HMGB1 release was not seen. We conclude that autophagy can be upregulated in at least some tumors with low autophagy and that upregulation of autophagy can restore the release of DAMPs following chemotherapy. Autophagy may be necessary for ICD in this tumor.


Assuntos
Autofagia/genética , Proteína HMGB1/genética , Mesotelioma/tratamento farmacológico , Trifosfato de Adenosina/genética , Alarminas/genética , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Proteínas Relacionadas à Autofagia/genética , Proteína Beclina-1/genética , Calreticulina/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade Celular/genética , Mesotelioma/genética , Mesotelioma/patologia , Interferência de RNA , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia
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