Methods: EA cells were treated with conditioned media (CM) of LS 174T-γ-Syn or LS 174T-PrP, and their proliferation, invasion, migration, adhesion and ability to form angiogenic tubes were assessed using a range of biological assays. To investigate plausible background mechanisms in conferring the properties of EA cells above, nitrite oxide (NO) levels were measured and the expression of angiogenesis-related factors was assessed using a human angiogenesis antibody array.
Results: EA proliferation was significantly inhibited by LS 174T-PrP CM whereas its telomerase activity was reduced by CM of LS 174T-γ-Syn or LS 174T-PrP, as compared to EA incubated with LS 174T CM. Besides, LS 174T-γ-Syn CM or LS 174T-PrP CM inhibited EA invasion and migration in Boyden chamber assay. Furthermore, LS 174T-γ-Syn CM significantly inhibited EA migration in scratch wound assay. Gelatin zymography revealed reduced secretion of MMP-2 and MMP-9 by EA treated with LS 174T-γ-Syn CM or LS 174T-PrP CM. In addition, cell adhesion assay showed lesser LS 174T-γ-Syn or LS 174T-PrP cells adhered onto EA, as compared to LS 174T. In tube formation assay, LS 174T-γ-Syn CM or LS 174T-PrP CM induced EA tube formation. Increased NO secretion by EA treated with LS 174T-γ-Syn CM or LS 174T-PrP CM was also detected. Lastly, decreased expression of pro-angiogenic factors like CXCL16, IGFBP-2 and amphiregulin in LS 174T-γ-Syn CM or LS 174T-PrP CM was detected using the angiogenesis antibody array.
Discussion: These results suggest that overexpression of γ-Syn or PrPCcould possibly be involved in colorectal cancer-induced angiogenesis by inducing an endothelial proliferation-differentiation switch. NO could be the main factor in governing this switch, and modulation on the secretion patterns of angiogenesis-related proteins could be the strategy of colorectal cancer cells overexpressing γ-Syn or PrPCin ensuring this transition.
MATERIALS AND METHODS: Adipose stromal cells were cultured in three different types of medium: (i) F12/DMEM (FD) supplemented with 10% FBS from passage 0 (P0) to P6; (ii) FD supplemented with 2% FBS at P6; and (iii) FD supplemented with 2% FBS plus 50 ng/ml of VEGF at P6. Morphological changes and growth rate of ASCs were recorded. Changes in stemness, angiogenic and endogenic genes' expressions were analysed using Real-Time PCR.
RESULTS: Adipose stromal cells changed from fibroblast-like shape when cultured in 10% FBS medium to polygonal when cultured in 2% FBS plus VEGF-supplemented medium. Their growth rate was lower in 2% FBS medium, but increased with addition of VEGF. Real-Time PCR showed that ASCs maintained most of their stemness and angiogenic genes' expression in 10% FBS at P1, P5 and P6, but this increased significantly in 2% FBS at P6. Endogenic genes expression such as PECAM-1, VE chaderin and VEGFR-2 decreased after serial passage in 10% FBS, but increased significantly at P6 in 2% FBS. Addition of VEGF did not cause any significant change in gene expression level.
CONCLUSION: Adipose stromal cells had greater angiogenic potential when cultured in reduced serum conditions. VEGF did not enhance their angiogenic potential in 2% FBS-supplemented medium.
RESULTS: Different production media were tested for lipase production by a newly isolated thermophilic Geobacillus sp. strain ARM (DSM 21496 = NCIMB 41583). The maximum production was obtained in the presence of peptone and yeast extract as organic nitrogen sources, olive oil as carbon source and lipase production inducer, sodium and calcium as metal ions, and gum arabic as emulsifier and lipase production inducer. The best models for optimization of culture parameters were achieved by multilayer full feedforward incremental back propagation network and modified response surface model using backward elimination, where the optimum condition was: growth temperature (52.3 degrees C), medium volume (50 ml), inoculum size (1%), agitation rate (static condition), incubation period (24 h) and initial pH (5.8). The experimental lipase activity was 0.47 Uml(-1) at optimum condition (4.7-fold increase), which compared well to the maximum predicted values by ANN (0.47 Uml(-1)) and RSM (0.476 Uml(-1)), whereas R2 and AAD were determined as 0.989 and 0.059% for ANN, and 0.95 and 0.078% for RSM respectively.
CONCLUSION: Lipase production is the result of a synergistic combination of effective parameters interactions. These parameters are in equilibrium and the change of one parameter can be compensated by changes of other parameters to give the same results. Though both RSM and ANN models provided good quality predictions in this study, yet the ANN showed a clear superiority over RSM for both data fitting and estimation capabilities. On the other hand, ANN has the disadvantage of requiring large amounts of training data in comparison with RSM. This problem was solved by using statistical experimental design, to reduce the number of experiments.