含甲氨蝶呤的肿瘤源性微粒联合放疗体外对肺腺癌干细胞增殖的影响

Effect of methotrexate packaged by tumor derived microparticles combined with radiotherapy on proliferation of cancer stem cells in lung adenocarcinoma in vitro

目的：探讨肿瘤微粒包装的甲氨蝶呤（T-MP MTX）联合放疗体外对肺腺癌干细胞增殖的影响。方法制备非小细胞肺癌A549细胞来源的T-MP MTX。MTT法检测不同浓度T-MP MTX作用的A549、H460细胞及支气管上皮细胞株16HBE细胞增殖变化；流式细胞术检测空白组、T-MP MTX组A549细胞周期；肿瘤球形成实验和动物实验检测空白组、T-MP MTX组（对照组）及T-MP MTX联合放疗组（2 Gy组、4 Gy组、6 Gy组）肿瘤干细胞增殖情况；Western blot检测肿瘤干性相关基因β-catenin、Nanog、SOX-2、KLF4等表达变化。结果四甲基偶氮唑盐（MTT）检测结果显示，T-MP MTX呈浓度依赖性地抑制A549细胞增殖，对H460细胞及16HBE细胞增殖无明显抑制作用。流式细胞仪检测结果显示，空白组和T-MP MTX组中S期细胞比例分别为（15.83±3.14）%和（47.47±6.69）%，T-MP MTX组较空白组明显增高（t＝7.411，P＝0.002）。肿瘤球实验结果发现，空白组、对照组、2 Gy组、4 Gy组、6 Gy组肿瘤球的数量分别为（268.9±22.4）、（172.4±18.7）、（48.3±5.1）、（16.3±3.5）、（5.1±3.1）个，组间差异有统计学意义（F＝228.291，P＝0.000）；与空白组比较，对照组、2 Gy组、4 Gy组、6 Gy组肿瘤球的数量均减少，差异均有统计学意义（均P＜0.05）；与对照组比较，2 Gy组、4 Gy组、6 Gy组肿瘤球数减少（均P＜0.05），且随放疗剂量增高，减少幅度增大（F＝95.142，P＝0.000）。肿瘤球体积分析结果与肿瘤球数量对比结果相似。Western blot检测结果显示，T-MP MTX单独处理A549细胞后β-catenin、KLF4、Nanog、SOX-2等肿瘤干细胞相关基因表达水平有所下降，而T-MP MTX联合放疗处理后明显加强这种抑制作用。动物实验结果显示T-MP MTX组移植瘤荧光素酶活性较空白组下降（P＝0.000），而T-MP MTX＋2 Gy组移植瘤荧光值下降更为明显（t＝6.887，P＝0.002）。结论T-MP MTX可能具有较高的放疗增敏作用，与放疗联合能协同抑制肺腺癌干细胞增殖，其机制可能与激活低代谢状态的肿瘤干细胞和阻断细胞周期进程有关。

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）＆amp;nbsp;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.