辛伐他汀对肺癌细胞株Lewis增殖侵袭能力及放疗增敏的影响

Effects of simvastatin on the proliferation, invasion and radiosensitivity in Lewis lung cancer cell line

目的 探讨辛伐他汀在抑制肺癌细胞株增殖和侵袭中的作用及机制，以及其对放疗增敏的影响。方法 采用四甲基偶氮唑蓝法检测辛伐他汀对Lewis细胞增殖的抑制效应，采用Matrigel侵袭和迁移实验检测辛伐他汀对Lewis细胞的侵袭和迁移能力的影响，采用p38活性试剂盒检测p38活性，采用Western blot检测磷酸化p38（p-p38）、丝裂原活化蛋白激酶磷酸酶1（MKP-1）、Ras相似物A （RhoA）和基质金属蛋白酶2（MMP-2）的表达。建立C57BL/6小鼠肺癌Lewis细胞株移植瘤模型，采用随机数字表法随机分为对照组、辛伐他汀组、单纯放疗组和联合治疗组（辛伐他汀＋放疗）。接种后27 d，处死小鼠，比较小鼠移植瘤质量、体积和肺转移结节数。结果 10 μmol/L辛伐他汀组、20 μmol/L辛伐他汀组、30 μmol/L辛伐他汀组和对照组的增殖率分别为（87.0±9.0）%、（76.5±8.1）%、（67.0±7.3）%和100%，侵袭细胞数分别为（251±26）个/视野、（207±20）个/视野、（132±19）个/视野和（298±30 ）个/视野，p38活性分别为（83.1±8.8）%、（70.2±8.2）%、（59.0±6.4）%和100%，p-p38的相对表达水平分别为（76.2±6.7）%、（56.4±5.4）%、（36.5±3.2）%和100%，RhoA的相对表达水平分别为（80.1±5.3）%、（55.3±6.2）%、（38.6±4.8）%和100%，MMP-2的相对表达水平分别为（89.6±8.6）%、（51.9±4.7）%、（42.7±3.1）%和100%，MKP-1的表达水平分别为（136.5±12.2）%、（168.8±15.3）%、（187.7±13.4）%和100%，差异均有统计学意义（均P〈0.05）。对照组、辛伐他汀组、单纯放疗组和联合治疗组肺转移结节数分别为（6.24±1.09）个、（5.09±1.16）个、（3.12±0.68）个和（2.65±0.38）个，移植瘤质量分别为（3.07±0.71）g、（2.43±0.53）g、（1.96±0.62）g和（1.12±0.43）g，差异均有统计学意义（均P〈0.05）。单纯放疗组、联合治疗组和辛伐他汀组的肿瘤抑制率分别为39.0%、48.1%和26.5%，差异有统计学意义（P〈0.05）。结论 辛伐他汀可抑制p38活性及p-p38的表达，上调MKP-1的表达，抑制肺癌Lewis细胞株的增殖能力。通过下调RhoA和MMP-2的表达，抑制其侵袭和迁移能力。辛伐他汀联合放疗能抑制小鼠肿瘤的生长和转移，起到协同增敏作用。

Objective To investigate the effects of simvastatin on proliferation, invasion and radiosensitivity of mouse Lewis lung cancer cell line in vitro.Methods The inhibitory effects of simvastatin on proliferation of Lewis lung cancer cells were detected by MTT assay. Matrigel invasion and migration assay was used to determine the invasion and motility ability of the Lewis cells. P38 activity was measured by p38 activity detection kit, and the expressions of p-p38, MKP-1, RhoA and MMP-2 were analyzed by Western blot. Lung cancer xenograft model was established in C57BL/6 mice. The mice were randomly divided into control group, simvastatin group, radiotherapy alone group and combined treatment group. The mice were killed 27 days after inoculation. The tumor mass, volume and lung metastatic nodules in the mice were compared.Results The cell proliferation rates of 0 μmol/L, 10 μmol/L, 20 μmol/L and 30 μmol/L simvastatin groups were 100%, （87.0±9.0）%, （76.5±8.1）% and （67.0±7.3）%, respectively （P〈0.05）. Invasive cell numbers of the above groups were 298±30, 251±26, 207±20 and 132±19 per field, respectively （P〈0.05）. The intracellular p38 activities were 100%, （83.1±8.8）%, （70.2±8.2）% and （59.0±6.4）%, respectively. The relative expressions of p-p38 were 100%, （76.2±6.7）%, （56.4±5.4）% and （36.5±3.2）%, respectively. The expressions of RhoA were 100%, （80.1±5.3）%, （55.3±6.2）% and （38.6±4.8）%, respectively. The expressions of MMP-2 were 100%, （89.6±8.6）%, （51.9±4.7）% and （42.7±3.1）%, respectively, while the expressions of MKP-1 were 100%, （136.5±12.2）%, （168.8±15.3）% and （187.7±13.4）%, respectively （all P〈0.05）. Lung metastatic nodules and mass in the control, simvastatin, radiotherapy group and combined treatment groups were 6.24±1.09, 3.07±0.71 g, 5.09±1.16, 2.43±0.53 g, 3.12±0.68, 1.96±0.62 g and 2.65±0.38, 1.12±0.43 g, respectively （all P〈0.05）. The tumor inhibition rates were 39.0%, 48.1% and 26.5%, respectively, in the radiotherapy alone, combined treatment and simvastatin groups （all P〈0.05）.Conclusions Simvastatin inhibits the proliferation of Lewis cell line by inhibiting the activity of p38 and expression of p-p38. Meanwhile, simvastatin reduces the invasion and motility of Lewis cell line through down-regulating the expression of RhoA and MMP-2. When combined with radiotherapy, simvastatin can inhibit tumor growth and metastasis, and improve the treatment efficacy of radiotherapy synergistically.