Effect of methotrexate packaged by tumor derived microparticles combined with radiotherapy on proliferation of cancer stem cells in lung adenocarcinoma in vitro / 肿瘤研究与临床

ABSTRACT

Objective To explore the effect of methotrexate packaged by tumor derived microparticles (T-MP MTX) combined with radiotherapy on lung cancer stem cell (CSC) in vitro. Methods T-MP MTX was prepared from non-small cell lung cancer A549 cells. Proliferative changes of A549 cells, bronchial epithelial cells H460 and 16HBE cells treated by T-MP MTX were assayed by MTT method. Cell cycles of A549 cells in blank group and T-MP MYX group were examined by fluorescence activated cell sorting (FACS). The effect of T-MP MTX combined with radiotherapy on CSCs was assessed by tumor sphere formation experiment and animal experiment. The expressions of stemness relative genes (such as β-catenin, Nanog, SOX-2 and KLF4) were measured by Western blot. Results T-MP MTX dose-dependently inhibited the cell growth in A549 cells, but didn't in H460 cells and 16HBE cells. The S cycle ratio of A549 cells in blank group and T-MP MYX group measured by FACS were (15.83±3.14)%and (47.47±6.69)%, respectively. S cycle ratio of T-MP MYX group was notably higher compared with that of blank group (t=7.411, P=0.002). Further study revealed that the number of tumor sphere in blank group, control group, 2 Gy group, 4 Gy group and 6 Gy group was (268.9±22.4), (172.4±18.7), (48.3±5.1), (16.3±3.5) and (5.1±3.1), respectively. The number of tumor sphere in other groups was decreased compared with that in blank group (F=228.291, P=0.000). The numbers of tumor sphere in 2 Gy group, 4 Gy group and 6 Gy groups was also reduced compared with that in control group. Importantly, the number of tumor sphere in these groups were decreased dramatically as the dose of radiotherapy increased (F=95.142, P=0.000). The results of tumor sphere volume were similar with the number of tumor sphere. Western blot experiment showed that T-MP MTX treatment in A549 cells decreased the expression of stemness relative genes (β-catenin, Nanog, SOX-2 and KLF4), and its role was reinforced when radiotherapy was combined. Animal experiment implied that activity of luciferase in T-MP MTX group was decreased compared with that in blank group (P=0.000), and the activity of luciferase in T-MP MTX plus 2 Gy group was reduced significantly (t=6.887, P=0.002). Conclusions T-MP MTX has a potential to sensibilize radiotherapy, and it will synergistically inhibit the proliferation of CSCs when combined with radiotherapy. Moreover, its mechanism may be related with T-MP MTX activating CSCs from hypometabolism state and blocking process of cell cycle.