Mechanisms of rapamycin on regulating rat mesangial cell proliferation and cell cycle through mTORC1/p70S6K pathways / 药学学报

To explore the affect and mechanisms of rapamycin on mesangial cell proliferation and cell cycle, rat mesangial cells (HBZY-1) were cultured and divided into the six groups: normal; normal with platelet derived growth factor (PDGF) 20 ng·mL-1; PDGF + rapamycin 1, 10, 100, 1 000 nmol·L-1. The cell proliferation was measured by MTT in 24 and 48 h; flow cytometry was used to detect the cell cycle phase. Western blot was performed to determine cyclin D1,cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), p27, p70S6K/p-p70S6K protein expression. The p27 mRNA was detect by Real-time PCR. The results showed that rapamycin significantly suppressed PDGF induced glomerular mesangial cells (MCs) proliferation in a dose and time-dependent manner, but with the dose increased (1 to 1 000 nmol·L-1), the time dependence gradually weakened. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G0/G1 phase. PDGF at 20 ng·mL-1 significantly increased the expression of cyclin D1, cyclin E and CDK2, CDK4 (P < 0.05), but rapamycin did not affect the expression of cyclin D1, cyclin E and CDK2, CDK4. Rapamycin can significantly inhibited p70S6K phosphorylation, up-regulated the expression of p27 protein and mRNA. Collectively, rapamycin has the effect of inhibiting the glomerular mesangial cells proliferation of mesangial cells by regulating the transcription of p27 mRNA, increasing its protein expression through the mTORC1/p70S6K pathway, resulting in decreased activity of cyclin-CDK, and blocking cell cycle in G0/G1 phase.

Inhibition of proliferation of breast cancer cells by down-regulation of cyclin d1 and survivin protein expression by erk inhibitor u0126 / 中国药理学通报

AimTo investigate the inhibitory effect of ERK signaling pathway inhibitor UO126 on breast cancer cell proliferation and explore its specific regulatory mechanism. Methods Cultured human breast cancer cell MCF-7, MDA-MB231, the cell were divided into different groups and intervened. MTT was used to measure the cell proliferation; Flow cytometry was employed to test cell cycle and cell apoptosis; Western blot was applied to test p-ERK/ERK, cyclin D1, survivin and cleaved caspase-3 protein expression. Results The results of MTT showed that the inhibitory rate of MCF-7 and MDA-MB231 cells increased significantly after U0126 intervention for 24 h and 48 h(P < 0.01). Cell cycle and apoptosis were detected by MCF-7 and MDA-MB231 cells treated with U0126 for 24 h. The proportion of cells in G0/G1 phase was significantly higher than that in control group, and the proportion of cells in S and G2 phase decreased(P < 0.05). The apoptotic rate in intervention group was significantly higher than that in non-intervention group, and the difference was significant(P < 0.01); U0126 treatment of human breast cancer cells could block ERK phosphorylation, increase cyclin D1 protein expression, inhibit survivin and up-regulate cleaved caspase3 expression, which were significantly different from control group(P < 0.05). Conclusions U0126 blocks ERK signaling pathway in MCF-7, MDA-MB231, down-regulates the cyclin D1, and blocks cell cycle in G0/G1 phase, then it inhibits the expression of survivin and increases cleaved caspase-3, promotes cell apoptosis, and inhibits the proliferation of breast cancer cell MCF-7, MDA-MB231.