RESUMO
Although 3D cell culture models are considered to reflect the physiological microenvironment and exhibit high concordance with in vivo conditions, one disadvantage has been that cell proliferation is slower in 3D culture as compared to 2D culture. However, the signaling differences that lead to this slower proliferation are unclear. Here, we conducted a cell-based high-throughput screening study and identified novel small molecules that promote cell proliferation, particularly under 3D conditions. We found that one of these molecules, designated GA-017, increases the number and size of spheroids of various cell-types in both scaffold-based and scaffold-independent cultures. In addition, GA-017 also enhances the ex vivo formation of mouse intestinal organoids. Importantly, we demonstrate that GA-017 inhibits the serine/threonine protein kinases large tumor suppressor kinase 1/2, which phosphorylate Yes-associated protein and transcriptional coactivator with PDZ-binding motif , key effectors of the growth- and proliferation-regulating Hippo signaling pathway. We showed that GA-017 facilitates the growth of spheroids and organoids by stabilizing and translocating Yes-associated protein and transcriptional coactivator with PDZ-binding motif into the cell nucleus. Another chemical analog of GA-017 obtained in this screening also exhibited similar activities and functions. We conclude that experiments with these small molecule large tumor suppressor kinase inhibitors will contribute to further development of efficient 3D culture systems for the ex vivo expansion of spheroids and organoids.
Assuntos
Via de Sinalização Hippo , Animais , Proliferação de Células/efeitos dos fármacos , Via de Sinalização Hippo/efeitos dos fármacos , Camundongos , Organoides/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismoRESUMO
Anticancer drug discovery efforts have used 2-D cell-based assay models, which fail to forecast in vivo efficacy and result in a lower success rate of clinical approval. Recent 3-D cell culture models are expected to bridge the gap between 2-D and in vivo models. However, 3-D cell culture methods that are available for practical anticancer drug screening have not yet been fully attained. In this study, we screened several polymers for their ability to suspend cells or cell spheroids homogeneously in a liquid medium without changing the viscosity behavior, and identified gellan gum (FP001), as the most potent polymer. FP001 promoted cell dispersion in the medium and improved the proliferation of a wide range of cancer cell lines under low attachment conditions by inhibiting the formation of large-sized spheroids. In addition, cancer cells cultured with FP001-containing medium were more susceptible to inhibitors of epidermal growth factor (EGF) signaling than those cultured under attachment conditions. We also showed that ligands of the EGF receptor family clearly enhance proliferation of SKOV3 ovarian carcinoma cells under anchorage-independent conditions with FP001. Consistent with this result, the cells grown with FP001 showed higher EGF receptor content compared with cells cultured under attachment conditions. In conclusion, we developed a novel 3-D cell culture system that is available for high throughput screening of anticancer agents, and is suitable for evaluation of molecular-targeted anticancer drugs. Three-dimensional cell culture using FP001 will be of value in the development of useful technologies for anticancer drug discovery.
Assuntos
Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Humanos , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50-150 °C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.
Assuntos
Materiais Revestidos Biocompatíveis , Animais , Adesão Celular , Materiais Revestidos Biocompatíveis/química , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Nus , Microscopia Eletrônica de VarreduraRESUMO
Three-dimensional (3D) cultures of cancer cells in liquid without extracellular matrix (ECM) offer in vitro models for metastasising conditions such as those in vessels and effusion. However, liquid culturing is often hindered by cell adhesiveness, which causes large cell clumps. We previously described a liquid culture material, LA717, which prevents nonclonal cell adhesion and subsequent clumping, thus allowing clonal growth of spheroids in an anchorage-independent manner. Here, we examined such liquid culture cancer spheroids for the acquisition of apical-basal polarity, sensitivity to an Akt inhibitor (anticancer drug MK-2206) and interaction with ECM. The spheroids present apical plasma membrane on the surface, which originated from the failure of polarisation at the single-cell stage and subsequent defects in phosphorylated ezrin accumulation at the cell boundary during the first cleavage, failing internal lumen formation. At the multicellular stage, liquid culture spheroids presented bleb-like protrusion on the surface, which was enhanced by the activation of the PI3K/Akt pathway and reduced by PI3K/Akt inhibitors. Liquid culture spheroids exhibited slow proliferation speed and low endogenous pAkt levels compared with gel-cultured spheroids and 2D-cultured cells, explaining the susceptibility to the Akt-inhibiting anticancer drug. Subcutaneous xenografting and in vitro analysis demonstrated low viability and adhesive property of liquid culture spheroids to ECM, while migratory and invasive capacities were comparable with gel-cultured spheroids. These features agree with the low efficacy of circulating tumour spheroids in the settling step of metastasis. This study demonstrates the feature of anchorage-independent spheroids and validates liquid cultures as a useful method in cancer spheroid research.
Assuntos
Adesão Celular/genética , Técnicas de Cultura de Células , Neoplasias/genética , Esferoides Celulares/patologia , Animais , Linhagem Celular Tumoral , Polaridade Celular/genética , Matriz Extracelular/genética , Humanos , Camundongos , Metástase Neoplásica , Neoplasias/patologia , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/genética , Esferoides Celulares/metabolismo , Transplante HeterólogoRESUMO
Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell-cell communication by transporting proteins, lipids, and nucleic acids. As the involvement of EVs in diseases has become apparent, druggable regulators of EV secretion are required. However, the lack of a highly sensitive EV detection system has made the development of EV regulators difficult. We developed an ELISA system using a high-affinity phosphatidylserine-binder TIM4 to capture EVs and screened a 1567-compound library. Consequently, we identified one inhibitor and three activators of EV secretion in a variety of cells. The inhibitor, apoptosis activator 2, suppressed EV secretion via a different mechanism and had a broader cellular specificity than GW4869. Moreover, the three activators, namely cucurbitacin B, gossypol, and obatoclax, had broad cellular specificity, including HEK293T cells and human mesenchymal stem cells (hMSCs). In vitro bioactivity assays revealed that some regulators control EV secretion from glioblastoma and hMSCs, which induces angiogenesis and protects cardiomyocytes against apoptosis, respectively. In conclusion, we developed a high-throughput method to detect EVs with high sensitivity and versatility, and identified four compounds that can regulate the bioactivity of EVs.
Assuntos
Apoptose/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Células K562 , Camundongos , Células NIH 3T3 , Células THP-1RESUMO
Cells grown in three-dimensional (3D) cultures are more likely to have native cell-cell and cell-matrix interactions than in 2D cultures that impose mechanical constraints to cells. However, most 3D cultures utilise gel matrix which, while serving as a scaffold, limits application due to its solid and opaque nature and inconsistency in cell exposure to exogenous signals. In 3D culture without gel matrix, cells tend to adhere to each other and form clumps with necrotic zone at the centre, making them unsuitable for analyses. Here we report that addition of low-molecular-weight agar named LA717 to culture media allows cells to grow as dispersed clonal spheroids in 3D. LA717 maintains cells dispersed and settled to the bottom of the medium while keeping the medium clear with little additional viscosity, making it suitable for microscopic observation. Importantly, cancer spheroids formed in LA717-containing medium show higher sensitivity to anti-cancer drugs such as Trametinib and MK-2206 that are not as effective in 2D. Because of the small and consistent size of spheroids, cell viability and drug toxicity are readily detectable in automated imaging analysis. These results demonstrate that LA717 offers a novel 3D culture system with great in vivo reflection and practicality.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células A549 , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HCT116 , Células HeLa , Células Hep G2 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Piridonas/farmacologia , Pirimidinonas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacosRESUMO
The Lys(gaY) of Lactobacillus gasseri JCM 1131(T) phage phigaY endolysin was purified to homogeneity using the Escherichia coli/His.Tag system. Zymographic and spectrophotometric assays showed that Lys(gaY) lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37 degrees C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys(gaY) were involved in cell-lytic activity: in the beta/alpha(gaY) domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b(gaY) domain, Y272 and W284. In addition, deletion analysis demonstrated that the beta/alpha(gaY) domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys(gaY). These mutational experiments suggested that beta/alpha(gaY) (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b(gaY) promotes beta/alpha(gaY) possibly through cell-wall binding function. The purified His-tagged SH3b(gaY) domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys(gaY), (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131(T), L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys(gaY), and (iv) impeded autolysis of L. gasseri JCM 1131(T) in buffer systems. A truncated protein HDeltaSH3b(gaY) lacking in N-terminal 21 residues (from S217 to E237) of SH3b(gaY) and an amino-acid substituted protein HSH3b(gaY)G (W284G) lost the activities of HSH3b(gaY), showing that the N-terminal region and W284 probably play important roles in the SH3b(gaY) function(s).
Assuntos
Fagos Bacilares/enzimologia , Endopeptidases/química , Endopeptidases/genética , Lactobacillus/virologia , Sequência de Aminoácidos , Fagos Bacilares/genética , Análise Mutacional de DNA , Endopeptidases/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Signaling by thrombopoietin (TPO) in complex with its receptor, c-MPL, is critical for hematopoietic stem cell (HSC) homeostasis and platelet generation. Here we show that TA-316, a novel chemically synthesized c-MPL agonist (CMA), is useful for ex vivo platelet generation from human-induced pluripotent stem (iPS) cell-derived immortalized megakaryocyte progenitor cell lines (imMKCLs). Moreover, the generation is clinically applicable, because self-renewal expansion and platelet release is tightly controllable. TA-316 but not eltrombopag, another CMA, promoted both the self-renewal and maturation of imMKCLs, leading to more than a twofold higher platelet production than that achieved with recombinant human TPO (rhTPO). Interestingly, TA-316 seemed to favor MK-biased differentiation from bone marrow CD34+ HSC/progenitors and imMKCLs through the upregulation of vascular endothelial growth factor A and fibroblast growth factor 2. This result suggests TA-316 could facilitate the development of an efficient and useful system to expand platelets from imMKCLs.
RESUMO
Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.
Assuntos
Células-Tronco Pluripotentes/citologia , Polímeros/química , Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Cariotipagem , Microscopia Eletrônica de Transmissão , Células-Tronco Pluripotentes/metabolismo , Teratoma/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
INTRODUCTION: Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and to differentiate into all blood cell lineages, and are currently the foundation of HSC transplantation therapy. A variety of methods have recently been explored to find a way to expand hematopoietic stem and progenitor cells (HSCs/PCs) ex vivo in order to improve the efficiency and outcome of HSC transplantation. AREAS COVERED: Recent studies of HSCs/PCs have led to the development of new ways to detect and purify HSCs/PCs and have also revealed several intrinsic and extrinsic factors that control the molecular signals fundamental to self-renewal and differentiation of HSCs. These findings have provided new approaches for expanding HSCs/PCs ex vivo utilizing protein factors and small-molecule compounds (SMCs) and have also demonstrated promising outcomes in clinical trials. EXPERT OPINION: Although further technical innovation is still needed, elucidation of the whole picture of signaling pathways critical to HSCs/PCs and manipulation of such pathways by SMCs could establish efficient, cost-effective, riskless and robust methods for ex vivo expansion of HSCs/PCs. With these efforts, more sophisticated HSC transplantation would be possible in the near future.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Separação Celular/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34(+)CD38(-) HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34(+)CD38(-) HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34(+) HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not. CONCLUSIONS/SIGNIFICANCE: Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs.
Assuntos
Inibidores Enzimáticos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Histona Acetiltransferases/antagonistas & inibidores , Terpenos/farmacologia , ADP-Ribosil Ciclase 1/metabolismo , Acetilação/efeitos dos fármacos , Animais , Antígenos CD34/metabolismo , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Inibidores Enzimáticos/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Histona Acetiltransferases/metabolismo , Humanos , Camundongos , Camundongos SCID , Terpenos/químicaRESUMO
OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Receptores de Trombopoetina/agonistas , Trombopoese/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/transplante , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Subunidade beta Comum dos Receptores de Citocinas/genética , Subunidade beta Comum dos Receptores de Citocinas/fisiologia , DNA Complementar/genética , Avaliação Pré-Clínica de Medicamentos , Sangue Fetal/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/fisiologia , Leucemia Mieloide/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Quimera por Radiação , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/fisiologia , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/efeitos dos fármacos , Trombopoetina/farmacologiaRESUMO
Four xanthocillins (1-4), including a new compound 4, were isolated from cultured marine fungus Basipetospora sp. as thrombopoietin (TPO) mimics. Compounds 1-4 promoted the proliferation of a TPO-sensitive human leukemia cell line, UT-7/TPO, and UT-7/EPO-mpl, genetically engineered to express c-Mpl, a receptor for TPO in dose-dependent manners. However, the proliferation of UT-7/EPO, a parental cell line of UT-7/EPO-mpl that was devoid of TPO receptor, was not affected by them. Thrombopoietic action of compound 1 was nearly as potent as that of TPO, inducing cell proliferation at a concentration ranging from 1 to 100nM. Compound 1 also induced the phosphorylation of several proteins, including Janus kinase 2 (Jak2), signal transducers, and activators of transcription-3 (STAT3) and STAT5 in the UT-7/EPO-mpl cell line, but not in the UT-7/EPO cell line. These data indicated that xanthocillins are putative agonists for c-Mpl, as their cellular actions were analogous to those of TPO.