RESUMEN
Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-ß promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-ßreceptor I (TGFßR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-ß-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFßR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 µM and 1.21 µM, respectively, while IC50 of SB431542 was 2.38 µM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales/métodos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Imidazoles/farmacología , Indoles/farmacología , Benzamidas/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dioxoles/farmacología , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Humanos , Proyectos Piloto , Esferoides Celulares , Factor de Crecimiento Transformador beta2/farmacologíaRESUMEN
Three-dimensional (3D) cell culture is beneficial for physiological studies of tumor cells, due to its potential to deliver a high quantity of cell culture information that is representative of the cancer microenvironment and predictive of drug responses in vivo. Currently, gel-associated or matrix-associated 3D cell culture is comprised of intricate procedures that often result in experimental complexity. Therefore, we developed an innovative anti-cancer drug sensitivity screening technique for 3D cell culture on NanoCulture Plates (NCP) by employing the imaging device BioStation CT. Here, we showed that the human breast cancer cell lines BT474 and T47D form multicellular spheroids on NCP plates and compared their sensitivity to the anti-cancer drugs trastuzumab and paclitaxel using the BioStation CT. The anticancer drugs reduced spheroid migration velocity and suppressed spheroid fusion. In addition, primary cells derived from the human breast cancer tissues B58 and B61 grown on NCP plates also exhibited similar drug sensitivity. These results were in good agreement with the conventional assay method using ATP quantification. We confirmed the antitumor effects of the drugs on cells seeded in 96-well plates using the BioStation CT imaging technique. We expect this method to be useful in research for new antitumor agents and for drug sensitivity tests in individually-tailored cancer treatments.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales/métodos , Imagen de Lapso de Tiempo/métodos , Tomografía Computarizada por Rayos X/métodos , Neoplasias de la Mama/patología , Movimiento Celular , Femenino , Humanos , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Chemoresistance is one of the most critical prognostic factors in osteosarcoma, and elucidation of the molecular backgrounds of chemoresistance may lead to better clinical outcomes. Spheroid cells resemble in vivo cells and are considered an in vitro model for the drug discovery. We found that spheroid cells displayed more chemoresistance than conventional monolayer cells across 11 osteosarcoma cell lines. To investigate the molecular mechanisms underlying the resistance to chemotherapy, we examined the proteomic differences between the monolayer and spheroid cells by 2D-DIGE. Of the 4762 protein species observed, we further investigated 435 species with annotated mass spectra in the public proteome database, Genome Medicine Database of Japan Proteomics. Among the 435 protein species, we found that 17 species exhibited expression level differences when the cells formed spheroids in more than five cell lines and four species out of these 17 were associated with spheroid-formation associated resistance to doxorubicin. We confirmed the upregulation of cathepsin D in spheroid cells by western blotting. Cathepsin D has been implicated in chemoresistance of various malignancies but has not previously been implemented in osteosarcoma. Our study suggested that the spheroid system may be a useful tool to reveal the molecular backgrounds of chemoresistance in osteosarcoma.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/metabolismo , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Proteoma/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Electroforesis en Gel Bidimensional , Humanos , Osteosarcoma/patología , Proteoma/análisis , Proteómica , Esferoides CelularesRESUMEN
Here, we report the highly efficient in vitro differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (MPCs) using a novel nanotechnology-based culture plate, nanoculture plate(®) (NCP). The NCP contains uneven microfabrications with diameters of â¼2-3 µm arranged in a honeycomb pattern on its culture surface, which is devoid of animal-derived protein sources. When human MPCs were subjected to three-dimensional (3D) culture using an NCP, they rapidly formed adhesive spheroids. We showed that adipogenic differentiation in NCP-mediated 3D cultures led to more rapid accumulation of triglycerides than that in two-dimensional cultures. During adipogenesis in 3D cultures, the rapid and intense induction of adipocyte-specific gene expressions, such as peroxisome proliferator-activated receptor γ (PPAR-γ), CCAAT-enhancer-binding protein α (C/EBP-α), adipocyte protein 2 (aP2), and adiponectin was observed. Together, these results indicate that this 3D culture system is suitable for the differentiation of human MPCs into adipogenic lineage, and could be applicable to adipose tissue engineering under xeno-free condition.
Asunto(s)
Adipocitos/citología , Adipogénesis/fisiología , Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/citología , Nanotecnología/instrumentación , Esferoides Celulares/citología , Andamios del Tejido , Adipocitos/fisiología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Línea Celular , Diseño de Equipo , Humanos , Células Madre Mesenquimatosas/fisiología , Miniaturización , Esferoides Celulares/fisiologíaRESUMEN
The major Smad pathways serve in regulating the expression of genes downstream of TGFbeta signals. In this study, we examined the effects of sustained Smad7 expression in cultured cells. Interestingly, Smad7 caused various mesenchymal cells, including NIH3T3 fibroblast and ST2 bone-marrow stromal cells, to undergo a marked morphological alteration into a flattened cell shape, but kept them alive for as long as 60 days. Furthermore, Smad7 arrested the proliferation of the cells even before they reached confluence. These cells became quiescent in G0/G1 phase and accumulated a hypophosphorylated form of retinoblastoma. The cytostatic effect of Smad7 was closely associated with a preceding decrease in the levels of G1 cyclins, such as cyclin D1 and cyclin E. Accordingly, ectopic cyclin E was able to overcome the Smad7-induced arrest of proliferation. These results indicate that Smad7 functions upstream of G1 cyclins and suggest a novel role for Smad7 as an antiproliferative factor. In contrast to the growth of mesenchymal cells, that of epithelial cells was little susceptible to Smad7. The present findings raise the possibility that a link between Smad7 and the G1 to S phase transition may also contribute to the cell cycle control by certain Smad7-inducing stimuli in a cell-type-dependent fashion.
Asunto(s)
Ciclinas/antagonistas & inhibidores , Ciclinas/biosíntesis , Fase G1/fisiología , Mesodermo/fisiología , Fase de Descanso del Ciclo Celular/fisiología , Proteína smad7/fisiología , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Proliferación Celular , Ciclina G , Ciclina G1 , Ciclinas/genética , Fase G1/genética , Ratones , Células 3T3 NIH , Fosforilación , Fase de Descanso del Ciclo Celular/genética , Proteína de Retinoblastoma/metabolismo , Células 3T3 SwissRESUMEN
The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. We identified a Pax6 protein-responsive element termed LE9 (52 bp in length) within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein formed in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activated LE9 synergistically with Pax6. A scanning mutagenesis experiment indicated that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins formed a complex when they were expressed together. Based on these findings, we propose a model in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein.