安罗替尼对宫颈癌细胞增殖和迁移与侵袭的影响及作用机制

Effect and mechanism of anlotinib on proliferation,migration and invasion of cervical cancer cells

目的 研究安罗替尼对宫颈癌细胞增殖、迁移和侵袭的影响及作用机制。方法 取对数生长期宫颈癌SiHa细胞,采用细胞计数试剂盒-8(CCK-8)实验根据半数抑制浓度(IC50)确定安罗替尼实验浓度,将SiHa细胞分为对照组(0μmol/L)、低浓度组(5μmol/L)、中浓度组(10μmol/L)、高浓度组(20μmol/L)。以平板克隆实验、细胞划痕实验和Transwell侵袭实验检测安罗替尼对SiHa细胞增殖、迁移和侵袭的影响。以Western blot法检测血管内皮生长因子(VEGF)、基质金属蛋白酶(MMP)-9、上皮钙黏附素(E-cadherin)、无翅型MMTV整合位点家族成员1(Wnt1)和β-连环蛋白(β-catenin)的表达水平。结果 CCK-8实验显示安罗替尼能够剂量依赖性抑制SiHa细胞的增殖,作用24、48和72 h的IC50分别为(26.96±2.25)、(13.59±1.13)和(9.27±0.75)μmol/L。平板克隆实验显示,处理SiHa细胞48 h后,对照组、低浓度组、中浓度组和高浓度组细胞克隆形成率分别为(62.05±7.88)%、(39.96±5.10)%、(24.02±4.98)%和(15.14±3.03)%。与对照组相比,低、中、高浓度组SiHa细胞克隆率显著降低,差异均有统计学意义(P均<0.05)。随着药物剂量的增加,SiHa细胞克隆率也逐渐减小,具有剂量依赖性,差异有统计学意义(P<0.05)。细胞划痕实验显示,对照组、低浓度组、中浓度组细胞迁移率分别为(72.03±5.40)%、(56.98±6.55)%和(41.33±7.02)%。与对照组相比,低浓度组、中浓度组SiHa细胞迁移率显著下降,差异均有统计学意义(P均<0.05),随着药物剂量的增加,SiHa细胞迁移率也逐渐减小,差异有统计学意义(P<0.05)。Transwell细胞侵袭实验结果显示,对照组、低浓度组、中浓度组和高浓度组侵袭细胞数分别为(180±10)、(125±10)、(78±8)和(30±5)个,差异均有统计学意义(P均<0.05)。Western blot结果显示,与对照组相比,低浓度组和高浓度组的E-cadherin蛋白表达升高,VEGF、MMP-9、Wnt1和β-catenin的蛋白表达降低,差异均有统计学意义(P均<0.05)。结论 安罗替尼可能通过Wnt/β-catenin通路发挥抑制SiHa细胞增殖、迁移和侵袭的作用。

Objective To study the effect and mechanism of anlotinib on proliferation,migration and invasion of cervicalcancer cells.Methods Si Ha cells of cervical cancer were cultured to logarithmic growth period for the determination of theinhibitory concentration 50%(IC50)of anlotinib by cell couting kit-8(CCK-8)assay. Si Ha cells were divided into controlgroup(0 μmol/L),low-dose experimental group(5 μmol/L),middle-dose experimental group(10 μmol/L)and high-doseexperimental group(20 μmol/L). The effects of anlotinib on proliferation,migration and invasion of Si Ha cells weredetected by cell plate cloning test,cell scratch test and transwell invasion test,respectively. Western blot was used to detectthe protein expressions of vascular endothelial growth factor(VEGF)、matrix metalloproteinase(MMP)-9、epithelial cadherin(E-cadherin),wingless type mmtv integration site family member 1(Wnt1)and β-catenin.Results CCK-8 assay showedthat anlotinib could inhibit the proliferation of Si Ha cells in a dose-dependent manner,and the IC50of 24,48 and 72 h were(26.96±2.25)μmol/L,(13.59±1.13)μmol/L and(9.27±0.75)μmol/L,respectively. Cell plate cloning experiment showed that the rates of cell clone formation were(62.05±7.88)%,(39.96±5.10)%,(24.02±4.98)% and(15.14±3.03)% in thecontrol,low,medium and high concentration groups,respectively,after 48 h of drug treatment of Si Ha cells. Comparedwith the control group,the cloning rate of Si Ha cells was significantly lower in the low,medium and high concentrationgroups,and the differences were statistically significant(all P<0.05). With the increase of drug dose,the clonogenic rateof Si Ha cells also decreased gradually,and the difference was statistically significant(P<0.05). The cell scratch assayshowed that the migration rates of cells in the control,low concentration and medium concentration groups were(72.03±5.40)%,(56.98±6.55)% and(41.33±7.02)%,respectively. Compared with the control group, the migration rate of Si Hacells in the low and medium concentration groups decreased significantly, and the difference was statistically significant(allP<0.05). The migration rate of Si Ha cells decreased gradually with the increase of drug dose, and the difference wasstatistically significant(P<0.05). Transwell cell invasion assay showed that the number of invaded cells in the control, low,medium and high concentration groups were(180±10),(125±10),(78±8)and(30±5),respectively, and the differenceswere statistically significant(all P<0.05). Western blot results showed that compared with the control group,anlotinibexperimental groups could increase the protein expression of E-cadherin,and reduce the protein expression of VEGF,MMP-9,Wnt1 and β-catenin(all P<0.05).ConclusionAnlotinib might be through the Wnt/β-catenin signaling pathway to inhibitthe proliferation,migration and invasion of Si Ha cells.