鬼臼乙叉苷对人鼻咽癌细胞CNE_2的细胞毒作用和对DNA的断裂作用

CYTOTOXIC AND DNA BREAKAGE EFFECT OF ETOPOSIDE ON HUMAN NASOPHARYNGEAL CANCER CELL LINE CNE_2

作者用噻唑蓝还原(MTT)法测定了鬼臼乙叉苷(鬼臼乙叉甙,VP-16)对人体鼻咽癌细胞CNE2的毒性作用,在药物浓度为2.5,5.0,10.0,20.0,40.0,80.0和160.0 ug/ml的条件下,连续4次实验,VP-16对CNE2细胞的生长抑制率分别为3.3±3.7％,15.2±3％,36.l±l0％,54±17％,68.4％士13％,76.7士9％和84.2±8％,其半数抑制浓度(IC_(50))为17.0 ug/ml。CNE2细胞分别在含有20.0,100.0和200.0ug/ml VP-16的培养液中孵育24h,细胞的DNA发生了断裂。DNA断裂量随药物浓度的增加而增加。将CNE2细胞放入含200.0ug/ml VP-16的培养液中孵育6h,12h和24 h,发现各时间点的DNA均发生了断裂。断裂程度随时间的增长而增加,未经药物处理的对照细胞的DNA未见断裂现象。经用50.0,100.0,200.0和400.Oug/VP-16处理的CNE_2细胞的拓扑异构酶Ⅱ能诱导超螺旋pBR322质粒DNA断裂成线状DNA,断裂程度与药物的浓度相平行。

The cytotoxic effect of DNA topoisomerase Ⅱ inhibitor, etoposide (VP-16), on human nasopharyngeal cancer cell line(CNE2) was determined using MTT assay method. The average inhibition rates of CNE2 cell growth in four experiments were 3.3±3.7, 15.2±3.0, 36.1±10, 54 ±17, 68.4113, 76.7±9 and 84.2±8% under the concentrations of 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 160.0μg/ml of VP-16, respectively. The fifty percent inhibition concentration(IC50) was calculated to be 17 μg/ml. After CNE2 cells were incubated in culture media containing 20, 100 and 200 μg/ml of VP-16 for 24 hours at 37@@, the cell DNA was extracted and electrophoresed on 0.7% agarose gel. It was found that VP-16 could induce DNA breakage. The degree of DNA breakage increased with increasing concentrations of VP-16. After CNE2 cells were incubated in medium containing 200 μg/ ml of VP-16 for 6,12 and 24 hours, respectively, the cell DNA was extracted and electrophoresed onD0.7% agarose gel. It was found that the degree of DNA breakage increased with increasing time of VP-16 treatment. DNA breakage of control untreated cells in different time points did not occur. DNA topoisomerase Ⅱ from CNE2 cells could induce supercoiled pBR322 plasmid DNA cleavage under concentrations of 50, 100, 200 and 400 μg/ml of VP-16. In the presence of VP-16, topoisomerase Ⅱ can induce the change of Form Ⅰ (double chain supercoiled DNA) to Form Ⅱ (double closed chain open circular DNA) or Form Ⅲ (linear DNA). The amount of linear (Form Ⅲ) DNA increased with increasing concentrations of VP-16.