低氧诱导活性氧和硫氧还蛋白1表达异常对口腔鳞状细胞癌细胞侵袭能力的影响

The influence of aberrant expression of reactive oxygen species and Trx-1 on the invasion of oral squamous cell carcinoma cells induced by hypoxia

目的探讨低氧条件下,活性氧(ROS)与硫氧还蛋白1(Trx-1)在口腔鳞状细胞癌(OSCC)细胞系中的表达情况,及其对OSCC细胞侵袭能力的影响。方法在常氧及低氧条件下培养人OSCC细胞系CAL33和HSC6细胞,2,7-二氯二氢荧光素二醋酸酯(DCFH-DA)法检测ROS水平,Western blot检测Trx-1表达水平,采用独立样本t检验方法进行统计分析。特异性抑制Trx-1后检测ROS的表达。Transwell细胞侵袭实验检测不同状态下CAL33细胞的侵袭能力变化,结果用单因素方差分析统计。结果低氧条件下,CAL33和HSC6细胞的ROS在6 h出现峰值,分别是各自对照组的(1.52±0.08)、(1.81±0.11)倍,后逐渐下降;而Trx-1随低氧诱导时间表达持续上调(P_(CAL33)=0.002,P_(HSC6)=0.0001)。特异性抑制Trx-1可显著上调CAL33和HSC6细胞的ROS表达至(2.78±0.26)、(7.29±0.83)倍,差异有统计学意义(t=13.4,P=0.0001)。与常氧比较,低氧可促进OSCC细胞的侵袭能力(P=0.001);特异性抑制Trx-1可抑制低氧环境中OSCC细胞侵袭能力,且这一趋势可被乙酰半胱氨酸(NAC)逆转(F=137.66,P=0.0001)。结论低氧状态下,ROS与Trx-1的表达异常可促进OSCC的侵袭能力。特异性抑制Trx-1可通过激活ROS介导的信号通路抑制OSCC细胞侵袭。

Objective To understand expression of ROS/Trx-1 feedback loop that induced by hypoxia and their effects on the invasion of oral squamous cell carcinoma（OSCC）cells. Methods CAL33 and HSC6 cells（two cell lines of OSCC）were cultured under the normoxic and hypoxic condition. Then the expression of ROS and Trx-1 were detected by 2,7-dichlorodihydrofluoresceindiacetate（DCFH-DA）assay and Western blot,respectively. Subsequently,the ROS level was tested after specially inhibiting the Trx-1 expression. The results were analyzed with student-t test. Transwell assays were used to investigate the invasion ability of OSCC cells under different conditions. The results were analyzed with One-Way ANOVA. Results The ROS level of CAL33 and HSC6 cells induced by hypoxia quickly increased and reached a peak at 6 h,with 1.52 ± 0.08 and 1.81 ± 0.11 folds of their respective control,and then slowly went down after 12 h. On the other hand,the protein level of Trx-1 was gradually increased until 24 h（P_（CAL33）= 0.002,P_（HSC6）= 0.0001）. The ROS level recovered and maintained at a relatively high level when incubating CAL33 and HSC6 cells with PX-12 for 24 h,up to 2.78 ± 0.26 and 7.29 ± 0.83 folds of their control（t = 13.4,P = 0.0001）. The hypoxia significantly promoted invasion ability of CAL33 and HSC6 cells compared with the normoxia（P = 0.001）. The invasion numbers of CAL33 and HSC6 cells were reduced in the presence of PX-12 under hypoxia. However,pretreatment with the antioxidant NAC could markedly reduce the inhibitive effect of PX-12 on invasion（F = 137.66,P = 0.0001）. Conclusions The expressiondifferences between ROS and Trx-1 enhance the invasion of OSCC cells. Specially inhibition of Trx-1 suppresses the OSCC cells invasion in ROS-dependent manner under hypoxia.