硒代胱氨酸对宫颈癌HeLa细胞放射增敏作用体外研究

目的放射治疗是中晚期宫颈癌的主要治疗方法,但患者个体放疗敏感性的差异导致宫颈癌预后差别明显。硒代胱氨酸(selenocystine,SeC)在肿瘤防治中扮演了重要的角色。本研究探讨SeC对人宫颈癌HeLa细胞放射敏感性的影响,为宫颈癌的放射治疗提供新的增敏靶点。方法宫颈腺癌细胞系HeLa(HPV16 E6阳性,p53突变)购于中国科学院上海细胞库。分别将0、5、10和20μmol/L SeC与宫颈癌HeLa细胞共孵育,然后再采用不同剂量X射线照射,实验分组为正常对照组、单纯SeC处理组、单纯照射组和照射组+SeC处理组。MTT法检测细胞生长抑制情况,克隆形成实验检测细胞放射敏感性并采用"单击多靶模型"拟合细胞存活曲线,计算放射生物学参数,流式细胞仪检测细胞凋亡,蛋白质印迹检测细胞凋亡蛋白的表达。对各组数据进行正态性和方差齐性的条件检验,相关性采用方差分析。结果细胞生长抑制实验结果显示,加入0、5、10和20μmol/L的SeC与HeLa细胞孵育24h后,各组细胞抑制率分别为0、22.68%、41.71%和74.19%,与0μmol/L组相比,F=306.45,P<0.05。计算得到的IC50为14.83μmol/L,HeLa细胞的生长抑制率与SeC的作用浓度呈剂量依赖关系。采用SeC 5μmol/L联合X射线共同作用于HeLa细胞后,克隆形成实验结果显示,在相同SeC浓度下(0或5μmol/L),随着照射剂量的增加(0、2、4、6和8Gy),HeLa细胞的克隆形成率分别是(84.34±2.21)%、(77.56±1.90)%、(46.89±2.09)%、(28.67±2.06)%和(10.89±1.76)%,当加入5μmol/L的SeC后,其克隆形成率分别是(70.98±2.08)%、(59.45±2.10)%、(30.67±2.53)%、(14.29±1.76)%和(3.72±2.18)%,差异有统计学意义,F=504.48,P<0.01,资料整体满足正态性检验,W=0.922,P=0.201;采用两因素析因设计的方差分析,显示2种处理方法的主效应均差异均有统计学意义,FSeC=21.22,P<0.001;FGy=139.21,P<0.001;而且二者间存在协同的交互效应,FSeC×Gy=38.38 P<0.001。在相同照射剂量下,加入SeC组的细胞克隆存活率均低于单纯照射组,F=20.81,P<0.01。单纯X射线照射组Do值为2.18±0.03,Dq值为1.17±0.04,N值为1.36±0.05;5μmol/L SeC联合X线照射组Do值为1.45±0.04,Dq值为0.31±0.01,N值为1.19±0.03,SER值为1.52±0.01。SeC联合X射线组与单纯X射线组比较,P<0.05。流式细胞仪检测细胞凋亡,单独X射线照射剂量为0、2、4和8Gy时,细胞凋亡分数依次为(2.51±0.06)%、(6.76±0.03)%、(14.51±0.15)%和(21.41±0.08)%;当加入5μmol/L的SeC再联合X射线照射,各组细胞凋亡分数依次为(10.89±0.36)%、(15.18±0.21)%、(31.19±0.08)%、(45.53±0.28)%。SeC联合X射线对HeLa细胞存在交互作用,细胞凋亡率增加,F=21.52,P<0.01。在相同照射剂量下,SeC联合放射组细胞凋亡分数与单纯照射组相比,差异有统计学意义,F=45.34,P<0.01。蛋白质印迹assay检测结果显示,当SeC联合X射线处理HeLa细胞后,细胞凋亡蛋白PARP蛋白、磷酸化的p53蛋白表达明显增加,P=0.034。结论 SeC对HeLa细胞具有放射增敏作用,其机制可能与促进细胞凋亡有关。SeC有望成为宫颈癌放射治疗新的增敏靶点。

Radiosensitization effect of SeC on HeLa cells in vitro

OBJECTIVE Radiotherapy is the main treatment for advanced cervical cancer.However,the difference in individual patients’ radiotherapy sensitivity leads to obvious difference in the prognosis of cervical cancer.How to ensure and even enhance the efficiency and effectiveness of radiant sensitizers involved in the clinical curement so far as radiotherapy we mentioned above has been a crucial target of such cutting-edge researches.Selenocystine(SeC)plays an important role in tumor prevention and treatment.This study aimed to explore the influence of SeC on the radiosensitivity of human cervical cancer HeLa cells to provide a new sensitization target for cervical cancer radiotherapy.METHODS Different doses of SeC were co-incubated with HeLa cells,followed by X-ray irradiation.The experimental groups were divided into normal control group,only SeC treatment group,only irradiation group and irradiation group combined with SeC treatment group.Cytotoxic effect of SeC on cell viability was determined by MTT assay.Radiosensitization effect of SeC on HeLa cells was detected by clonal formation assay,Clonal formation rate(PE)=(colony number/cell inoculation number)×100%,survival score(SF)=irradiated clonal formation rate/control group clonal formation rate.The single-hit multi target model was used to stimulate the dose response curve of survival and to calculate radiosensitization parameter.The apoptosis of HeLa cells was analyzed with flow cytometry.The expressions of p53 and PARP were detected by Western blotting assay.RESLUTS The results of cell growth inhibition experiment showed that the cell inhibition rates of each group were0,22.68%,41.71% and 74.19% after 24 hof incubation with different doses of 0,5,10 and 20μmol/L SeC on HeLa cells.Compared with the 0μmol/L group(F=306.45,P<0.05),the calculated IC50 was 14.83μmol/L.The growth inhibition rate of HeLa cells presented a dose-dependent relationship with the action concentration of SeC.The results of clonal formation experiment showed that the clonal formation rate of HeLa cells was(84.34±2.21)%,(77.56±1.90)%,(46.89±2.09)%,(28.67±2.06)%,(10.89±1.76)% while the radiation dose was 0,2,4,6 and 8 Gy.With the increase of radiation dose,the clonal formation rate of HeLa cells was significantly reduced(F=504.48,P<0.01).After the addition of 5μmol/L SeC,the clone formation rate at the same exposure dose was reduced to(70.98±2.08)%,(59.45±2.10)%,(30.67±2.53)%,(14.29±1.76)% and(3.72±2.18)%,respectively(F=504.48,P<0.01).The results indicated that the clonal survival rate of the SeC group was lower than that of the single irradiation group(F=20.81,P<0.01).The cell survival curve was fitted according to the "single-click multi-target model",and the radiobiological parameters were calculated:Do=2.18±0.03,Dq=1.17±0.04,N=1.36±0.05;Do=1.45±0.04,Dq=0.31±0.01,N=1.19±0.03,SER=1.52±0.01(SeC combined X ray group vs only X ray group,P<0.05).Cell apoptosis was detected by flow cytometry.When the X-ray radiation dose was 0,2,4 and 8 Gy,the apoptosis fraction was 2.51±0.06,6.76±0.03,14.51±0.15,and 21.41±0.08,respectively.When 5μmol/L of SeC was added to joint X-ray irradiation and the radiation dose was 0,2,4,8 Gy,the apoptosis fraction of each group was 10.89±0.36,15.18±0.21,31.19±0.08,45.53±0.28,when SeC combined with X-ray had interaction with Hela cells,and the apoptosis rate increased(F=21.52,P<0.01).At the same radiation dose,the difference in apoptosis fraction between the SeC combined radiation group and the single radiation group was statistically significant(F=45.34,P<0.01).The expression of apoptotic protein PARP and phosphorylated p53 was significantly increased compared with the control group after the SeC combined with X-ray treatment of HeLa cells.CONCLUSIONS Different concentrations of SeC can inhibit the growth of HeLa cells.SeC combined with X-ray irradiation can significantly enhance the radiosensitivity of HeLa cells,increase the apoptosis rate of HeLa cells,and up-regulate the expression of apoptosis-related proteins PARP and phosphorylated p53,compared with X-ray irradiation alone.This indicated that SeC had a radiosensitization effect on HeLa cells,and the mechanism may be related to the promotion of apoptosis.SeC is expected to be a new sensitization target for cervical cancer radiotherapy.