RESUMEN
BACKGROUND/AIM: Tumour cells of the profile CD44+/CD24low/- have a high tumorigenic potential. Salinomycin can specifically inhibit the growth of these cells. Herein, we investigated the effects of salinomycin on the viability and migration of triple negative breast cancer cells. MATERIALS AND METHODS: We analysed two cell lines: i) triple-negative MDA-MB 231 breast cancer cells and ii) a cytokeratin 18-transfected, re-differentiated subclone of the MDA-MB 231 cell line. The viability was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, and the migration was determined using 24-h videography. The expression of oestrogen receptor was determined using immunohistochemistry. RESULTS: Salinomycin reduces all migration parameters in MDA-MB 231 cells. A significant correlation was found between increasing salinomycin concentrations and loss of cell viability, which was significantly less noticeable in the transfected control cells. CONCLUSION: With salinomycin there is a specific inhibition of MDA-MB 231 cells. Since MDA-MB 231 has over 90% cells with the profile CD44+/CD24low/-, these might represent a possible point of attack for salinomycin.
Asunto(s)
Queratina-18/genética , Piranos/farmacología , Receptores de Estrógenos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Queratina-18/metabolismo , Transfección , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
BACKGROUND: Radiotherapy is administered in more than 60% of all solid tumors. Most patients are cured but a significant number develops local recurrences or distant metastases. The question arises if irradiation might influence the metastatic process. In the present study we examined whether the adhesion of glioblastoma or breast cancer cells to endothelial cells, an important step in metastasis, is affected by photon irradiation. METHODS: U-87 MG, U-373 MG and MDA-MB-231 cancer cells as well as primary human endothelial cells were irradiated with 0, 2, 4, or 8 Gy photons at a dose rate of 5 Gy/min. The adhesion of cancer cells to endothelial cells was tested either with the Vybrant based assay via fluorescent labelling or with an ibidi pump system able to mimic the physiological blood flow in vitro. In addition, the impact of FAK (focal adhesion kinase) inhibitor PF-573, 228 on the adhesion of non-irradiated and irradiated tumor cells was analyzed. Adhesion related and regulated proteins were analyzed by Western blotting. RESULTS: The cellular adhesion was increased after irradiation regardless of which cell type was irradiated. The FAK-inhibitor was able to reduce the adhesion of non-irradiated cells but also the irradiation-induced increase in adhesion of tumor cells to endothelium. Adhesion related proteins were enhanced after irradiation with 4 Gy or 8 Gy in both cells types. The increased adhesion after irradiation is accompanied by the phosphorylation of src (Y416), FAK (Y397) and increased expression of paxillin. CONCLUSION: Irradiation with photons in therapeutic doses is able to enhance the interaction between tumor cells and endothelial cells and by that might influence important steps of the metastatic process.
Asunto(s)
Neoplasias de la Mama/patología , Adhesión Celular , Endotelio Vascular/patología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Rayos gamma , Glioblastoma/patología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/radioterapia , Proliferación Celular , Endotelio Vascular/enzimología , Endotelio Vascular/efectos de la radiación , Inhibidores Enzimáticos/farmacología , Femenino , Glioblastoma/enzimología , Glioblastoma/radioterapia , Humanos , Células Tumorales CultivadasRESUMEN
Glioblastoma is a primary brain tumor with a poor prognosis despite of many treatment regimens. Radiotherapy significantly prolongs patient survival and remains the most common treatment. Slit2 and Robo1 are evolutionarily conserved proteins involved in axon guidance, migration, and branching of neuronal cells. New studies have shown that Slit2 and Robo1 could play important roles in leukocyte chemotaxis and glioblastoma cell migration. Therefore, we investigated whether the Slit2/Robo1 complex has an impact on the motility of glioblastoma cells and whether irradiation with therapeutic doses modulates this effect. Our results indicate that photon irradiation increases the migration of glioblastoma cells in vitro. qPCR and immunoblotting experiments in two different glioblastoma cell lines (U-373 MG and U-87 MG) with different malignancy revealed that both Slit2 and Robo1 are significantly lower expressed in the cell populations with the highest motility and that the expression was reduced after irradiation. Overexpression of Robo1 significantly decreased the motility of glioblastoma cells and inhibited the accelerated migration of wild-type cells after irradiation. Immunoblotting analysis of migration-associated proteins (fascin and focal adhesion kinase) and of the epithelial-mesenchymal-transition-related protein vimentin showed that irradiation affected the migration of glioblastoma cells by increasing vimentin expression, which can be reversed by the overexpression of Slit2 and Robo1. Our findings suggest that Robo1 expression might counteract migration and also radiation-induced migration of glioblastoma cells, a process that might be connected to mesenchymal-epithelial transition.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Vimentina/metabolismo , Adhesión Celular/efectos de la radiación , Línea Celular Tumoral , Movimiento Celular/efectos de la radiación , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Vimentina/genética , Rayos X , Proteínas RoundaboutRESUMEN
AIM: To observe and document the migration of living cells by time-lapse videography, we constructed a low-budget system based on a common inverted microscope. MATERIALS AND METHODS: Long-term observation of six-well plates is enabled through maintenance of cell culture conditions (5% CO2 in air at 37°C). Points of interest can be revisited in definable intervals with <1 µm repositioning error. Digital photographs from each programmed time point are paired with environmental data and combined into a record. RESULTS: We used this new chamber to observe the migration of various cell lines. The design represents a good compromise between low cost and good precision. Detailed analyses verified that the environmental conditions were appropriately maintained, enabling long-term observation of viable cells. The stimulating influence of irradiation with photons (radiotherapy) on cellular motility of glioblastoma cells is presented. CONCLUSION: This study demonstrates that useful videographic systems can be constructed at low cost.