OBJECTIVES: This study has aimed to establish optimum conditions to generate and characterize MSC from human umbilical cord (UC-MSC).
MATERIALS AND METHODS: To optimize MSC population growth, basic fibroblast growth factor (bFGF) was utilized in culture media. Effects of bFGF on expansion kinetics, cell cycle, survival of UC-MSC, cytokine secretion, expression of early stem-cell markers and immunomodulation were investigated.
RESULTS: bFGF supplementation profoundly enhanced UC-MSC proliferation by reducing population doubling time without altering immunophenotype and immunomodulatory function of UC-MSC. However, cell cycle studies revealed that bFGF drove the cells into the cell cycle, as a higher proportion of cells resided in S phase and progressed into M phase. Consistent with this, bFGF was shown to promote expression of cyclin D proteins and their relevant kinases to drive UC-MSC to transverse cell cycle check points, thus, committing the cells to DNA synthesis. Furthermore, supplementation with bFGF changed the cytokine profiles of the cells and reduced their apoptotic level.
CONCLUSION: Our study showed that bFGF supplementation of UC-MSC culture enhanced the cells' growth kinetics without compromising their nature.
METHODS: A WEHI-3 cell line was used to evaluate the cytotoxicity of BM by MTT. AO/PI and Hoechst 33342 dyes, Annexin V, multiparametric cytotoxicity 3 by high content screening (HCS); cell cycle tests were used to estimate the features of apoptosis and BM effects. Caspase 3 and 9 activities, ROS, western blot for Bcl2, and Bax were detected to study the mechanism of apoptosis. BALB/c mice injected with WEHI-3 cells were used to assess the apoptotic effect of BM in vivo.
RESULTS: BM suppressed the growth of WEHI-3 cells at an IC50value of 14 ± 3 μg/mL in 24 h. The ROS production was increased inside the cells in the treated doses. Both caspases (9 and 3) were activated in treating WEHI-3 cells at 24, 48 and 72 h. Different signs of apoptosis were detected, such as cell membrane blebbing, DNA segmentation and changes in the asymmetry of the cell membrane. Another action by which BM could inhibit WEHI-3 cells is to restrain the cell cycle at the G1/G0 phase. In the in vivo study, BM reduced the destructive effects of leukaemia on the spleen and liver by inducing apoptosis in leukaemic cells.
CONCLUSION: BM exerts anti-leukaemic properties in vitro and in vivo.
MATERIALS AND METHODS: Considering the ability of SCS to also promote the activity of the antiestrogen, tamoxifen, we further examined the effect of SCS in modulating cell cycle progression and related proteins in MCF-7 and MDA-MB-231 cells alone and in combination with tamoxifen. Expression of cell cycle- related transcripts was analysed based on a previous microarray dataset.
RESULTS: SCS significantly caused G1 arrest of both types of cells, similar to tamoxifen and this was associated with modulation of cyclin D1, p21 and p53. In combination with tamoxifen, the anticancer effects involved downregulation of ERα protein in MCF-7 cells but appeared independent of an ER-mediated mechanism in MDA-MB-231 cells. Microarray data analysis confirmed the clinical relevance of the proteins studied.
CONCLUSIONS: The current data suggest that SCS growth inhibitory effects are similar to that of the antiestrogen, tamoxifen, further supporting the previously demonstrated cytotoxic and apoptotic actions of both agents.
OBJECTIVE: The effects of Brequinar Sodium (BQR) and 4SC-101 on lymphoblastoid cell lines were investigated.
METHODS: DHODH expression and cell proliferation inhibition of lymphoblastoid and lymphoma cell lines were analyzed using Western blot analysis and XTT assay, respectively. JC-1 probe and ATP biochemiluminescence kit were used to evaluate the mitochondrial membrane potential and ATP generation in these cell lines. Furthermore, we explored the cell cycle progression using Muse™ Cell Cycle Kit.
RESULTS: Ramos, SUDHL-1 and RPMI-1788 cells are fast-growing cells with equal expression of DHODH enzyme and sensitivity to DHODH inhibitors that showed that the inhibition of DHODH was not cancer-specific. In ATP depletion assay, the non-cancerous RPMI-1788 cells showed only a minor ATP reduction compared to Ramos and SUDHL-1 (cancer) cells. In the mechanistic impact of DHODH inhibitors on non-cancerous vs cancerous cells, the mitochondrial membrane potential assay revealed that significant depolarization and cytochrome c release occurred with DHODH inhibitors treatment in Ramos but not in the RPMI-1788 cells, indicating a different mechanism of proliferation inhibition in normal cells.
CONCLUSION: The findings of this study provide evidence that DHODH inhibitors perturb the proliferation of non-cancerous cells via a distinct mechanism compared to cancerous cells. These results may lead to strategies for overcoming the impact on non-cancerous cells during treatment with DHODH inhibitors, leading to a better therapeutic window in patients.