ABSTRACT
Objective:To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) and their conditioned medium on proliferation, migration and apoptosis of human non-small cell lung cancer (NSCLC) polyploid A549 cells.Methods:A549 cells in logarithmic phase were selected. After induction treatment with 1 μmol/L docetaxel for 24 h, DMEM/F12 medium with 10% fetal bovine serum was used to culture the cells for 3 d, finally the polyploid A549 cells model was successfully established. After finishing the separation and culture of hUC-MSC, hUC-MSC conditioned medium was prepared. Normally cultured polyploid A549 cells were treated as the control group, conditioned medium cultured polyploid A549 cells were treated as the conditioned medium group. hUC-MSC was co-cultured with polyploid A549 cells, and the ratio of the total number of cells was 2:1 and 5:1, respectively, which were recorded as MSC 1 group and MSC 2 group. Cells in each group were continually cultured for 48 h or 72 h. Proliferation and apoptosis of polyploid A549 cells in each group were detected by using flow cytometry, cell migration ability was detected by using Transwell assay, and the expressions of migration and apoptosis-related proteins were detected by using Western blotting.Results:Polyploid A549 cells model was successfully established and hUC-MSC was cultured separately. The result of cell proliferation detected by flow cytometry showed that at 48 h, the mean fluorescence intensity of the control group, conditioned medium group, MSC 1 group and MSC 2 group was 1 695±305, 2 020±85, 1 259±35 and 1 356±33, respectively, and the difference was statistically significant ( F = 14.00, P < 0.05); at 72 h, the mean fluorescence intensity of the control group, conditioned medium group, MSC 1 group and MSC 2 group was 1 052±77, 1 309±24, 864±201 and 1 103±237, respectively, and the difference was statistically significant ( F = 3.90, P > 0.05). The result of Transwell assay showed that at 48 h, the number of cell migration in the control group, conditioned medium group, MSC 1 group and MSC 2 group was 52±9, 57±12, 68±8 and 75±11, respectively, and the difference was statistically significant( F = 32.16, P < 0.05); the number of cell migration in each experimental group was all higher than that in the control group (all P < 0.05). The percentage of apoptotic cells in the control group, conditioned medium group, MSC 1 group and MSC 2 group was (15.53±4.27)%, (13.77±1.75)%, (3.60±0.50)% and (2.33±0.06)%, respectively, and the difference was statistically significant ( F = 182.36, P < 0.05); there was no statistically significant difference between the control group and conditioned medium group ( P > 0.05); there were statistically significant differences between MSC 1 group and the control group, MSC 2 group and the control group (both P < 0.05). Western blotting results showed that compared with the control group, the expression of migration-related protein matrix metallopeptidase 9 (MMP-9) was increased, the expression of pro-apoptotic protein bax was reduced, the expression of anti-apoptotic protein bcl-xL was increased in conditioned medium group, MSC 1 group and MSC 2 group. Conclusions:hUC-MSC can improve the migration and anti-apoptotic ability of polyploid A549 cells, suggesting that hUC-MSC may affect the survival of tumor cells during the process of chemotherapy damage and repair.
ABSTRACT
Objective:To study the migration of polyploid tumor cells induced by docetaxel, the characteristics of epithelial-mesenchymal transition, and the killing effect of immune cells on them, in order to explore the potential role of polyploid tumor cells in tumor recurrence.Methods:The human non-small cell lung cancer A549 cells were treated with 1 μmol/L docetaxel for 24 h, and the cells were collected as Doc 1 d group. After drug removal, the cells were cultured in fresh and complete medium for 3 or 5 days, then the cells were collected as Doc 3 d group or Doc 5 d group respectively. The A549 cells were treated with DMSO for 24 h as control group. Immunofluorescence staining was used to detect cell morphology, flow cytometry was used to analyze cell ploidy, scratch test was used to detect cell migration, Western blotting was used to detect the expression of epithelial-mesenchymal transition related proteins, and lactate dehydrogenase release method was used to evaluate the killing activity of cytokine-induced killer (CIK) cells.Results:Compared with the control group, most of the cells in the Doc 1 d group, Doc 3 d group and Doc 5 d group were apoptotic, a few of the surviving cells were significantly larger, and the nucleus was polynuclear. The proportions of polyploid cell subset (DNA content > 4N) in the control group, Doc 1 d group, Doc 3 d group and Doc 5 d group were (1.93±0.55)%, (22.97±2.37)%, (51.30±12.51)% and (67.87±8.31)% respectively, and the difference among the four groups was statistically significant ( F=26.521, P<0.001). The proportion of polyploid cell subset in Doc 1 d group, Doc 3 d group and Doc 5 d group was significantly higher than that in the control group (all P<0.001). With the prolongation of withdrawal time, the proportion of polyploid cell subset in Doc 3 d group and Doc 5 d group was significantly higher than that in Doc 1 d group ( P=0.009; P=0.004). After 24 h and 48 h culture, the wound healing rates of the control group were both 100%, and the wound healing rates of the Doc 3 d group were (39.10±2.12)% and (46.13±5.32)% respectively, with no significant difference ( t=2.126, P=0.051). Compared with the control group at 24 h and 48 h, the cell migration abilities of Doc 3 d group were significantly lower ( t=49.756, P<0.001; t=30.825, P<0.001). Compared with the control group, the expression of E-cadherin protein decreased gradually in the Doc 1 d group, Doc 3 d group and Doc 5 d group, the expression of Vimentin protein increased gradually, and the expressions of Snail protein and N-cadherin protein did not change significantly. The killing efficiencies of CIK cells against the cells of the control group, Doc 3 d group and Doc 5 d group were (27.27±1.91)%, (17.87±2.35)%, (9.47±0.51)% respectively, and the difference was statistically significant ( F=11.294, P<0.001). The killing efficiency of Doc 3 d group and Doc 5 d group was significantly lower than that of the control group ( P=0.004; P<0.001). The killing efficiency of Doc 5 d group was significantly lower than that of Doc 3 d group ( P=0.003). Conclusion:The migration ability of polyploid tumor cells induced by docetaxel is weakened, but epithelial-mesenchymal transition is likely to occur, and the killing effect of immune cells on them is reduced.
ABSTRACT
Objective To study the effect of metformin on the growth of megakaryocytic leukemia cell line Dami and to explore the molecular mechanisms of the inhibitory effect of metformin on the proliferation of Dami. Methods The Dami cells were cultured and divided into control and 1,2,4,8,16 and 32 mmol·L-1 metformin groups.Then MTT test was performed to detect the inhitory rate of proliferation of Dami cells after treated with different concentrations of metformin. Flow cytometry was used to examine the distribution of cell cycle, and Western blotting was carried out to analyze the expressions of Cdc2 and CylinB1 and the phosphorylation of Cdc2. Results The MTT results showed that compared with control group,the inhibitory rates of proliferation of the Dami cells in 32 mmol·L-1 metformin groups at 0,24,48,72 and 96 h (35.1%±2.3%,49.7%±5.1%, 78.85±0.9%,79.1%± 3.0%%,and 85.2%± 3.2%)were significantly increased(P<0.01),Furthermore, after metformin treatment for 72 h,the inhibitory rates of proliferation of the Dami cells in 1,2,4,8,16 and 32 mmol·L-1 metformin groups were (33.8 ± 0.3)%,(51.9 ± 0.2)%,(59.4 ± 1.6)%,(65.5 ± 2.0)%, (75.5±0.9)%,and (79.1±3.0)%,respectively. Metformin inhibited the growth of Dami cells in a time-and dose-dependent manner. The flow cytometry results results revealed that compared with control group, the percentages of Dami cells in G2/M phase in 1,2 and 4 mmol·L-1 metformin groups were increased from (26.0± 0.5)% to (38.5 ± 1.5 )%, (48.4 ± 1.1 )%, and (58.2 ± 2.7 )%;there was significant difference in the percentages of Dami cells in G2/M phase between control group and 4 mmol·L-1 metformin group (P<0.01). Western blotting analysis showed that compared with control group, the expressions of Cdc2 and CyclinB were evidently reduced, the phosophorylation of Cdc2 at Tyr1 5 was up-regulated, and the phosphorylation at Thr1 6 1 was down-regulated.Conclusion Metformin can inhibit the growth of Dami cells and induce G2/M arrest,and its mechanism may be related to inhibiting the activation of Cdc2/CyclinB1 complex.