ABSTRACT
OBJECTIVE: Weightlessness simulation due to the simulated microgravity has been shown to considerably affect behavior of tumor cells. It is aim of this study to evaluate characteristics of human breast cancer cells in this scaffoldfree 3D culture model. MATERIALS AND METHODS: In this experimental study, the cells were exposed to simulated microgravity in a randompositioning machine (RPM) for five days. Morphology was observed under phase-contrast and confocal microscopy. Cytofilament staining was performed and changes in expression level of cytofilament genes, proliferation/differentiation genes, oncogenes and tumor suppressor genes were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), followed by western blot confirmation. RESULTS: After five days, distinct spheroid formation was observed. Rearrangement of the cytoskeleton into spherical shape was visible. VIM gene expression was significantly up-regulated for adherent cells and spheroids (3.3x and 3.6x respectively, P<0.05 each). RHOA also showed significant gene up-regulation for adherent cells and spheroids (3.2x and 3.9x respectively, P<0.05 each). BRCA showed significant gene up-regulation in adherent cells and spheroids (2.1x and 4.1x respectively, P<0.05 each). ERBB2 showed significant gene up-regulation (2.4x, P<0.05) in the spheroids, but not in the adherent cells. RAB27A showed no significant alteration in gene expression. MAPK) showed significant gene up-regulation in adherent cells and spheroids (3.2x, 3.0x, P<0.05 each). VEGF gene expression was down-regulated under simulated microgravity, without significance. Alterations of gene expressions could be confirmed on protein level for vimentin and MAPK1. Protein production was not increased for BRCA1, human epidermal growth factor receptor 2 (HER2) and VEGF. Contradictory changes were determined for RHOA and its related protein. CONCLUSION: Microgravity provides an easy-to handle, scaffold-free 3D-culture model for human breast cancer cells. There were considerable changes in morphology, cytoskeleton shape and gene expressions. Identification of the underlying mechanisms could provide new therapeutic options.
ABSTRACT
We studied the behavior of Ewing's Sarcoma cells of the line A673 under simulated microgravity (s-µg). These cells express two prominent markers-the oncogene EWS/FLI1 and the chemokine receptor CXCR4, which is used as a target of treatment in several types of cancer. The cells were exposed to s-µg in a random-positioning machine (RPM) for 24 h in the absence and presence of the CXCR4 inhibitor AMD3100. Then, their morphology and cytoskeleton were examined. The expression of selected mutually interacting genes was measured by qRT-PCR and protein accumulation was determined by western blotting. After 24 h incubation on the RPM, a splitting of the A673 cell population in adherent and spheroid cells was observed. Compared to 1 g control cells, EWS/FLI1 was significantly upregulated in the adherent cells and in the spheroids, while CXCR4 and CD44 expression were significantly enhanced in spheroids only. Transcription of CAV-1 was upregulated and DKK2 and VEGF-A were down-regulated in both, adherent in spheroid cells, respectively. Regarding, protein accumulation EWS/FLI1 was enhanced in adherent cells only, but CD44 decreased in spheroids and adherent cells. Inhibition of CXCR4 did not change spheroid count, or structure. Under s-µg, the tumor marker EWS/FLI1 is intensified, while targeting CXCR4, which influences adhesion proteins, did not affect spheroid formation.
Subject(s)
Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS/genetics , Receptors, CXCR4/genetics , Sarcoma, Ewing/genetics , Cell Adhesion/radiation effects , Cell Line, Tumor , Cell Proliferation/radiation effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Sarcoma, Ewing/pathology , Signal Transduction/radiation effects , Spheroids, Cellular/radiation effects , Weightlessness SimulationABSTRACT
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.
