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目的:探讨西妥昔单抗(C225)对乳腺癌MDA-MB-231细胞株的放射增敏作用及其机制。方法:体外培养人乳腺癌MDA-MB-231细胞。使用活细胞计数试剂盒(CCK-8)检测浓度分别为1、5、25、125和625nmol/L C225对乳腺癌MDA-MB-231细胞株的增殖抑制率,计算C225的半数抑制浓度(IC50),并将20%IC50作为后续实验的浓度;观察C225联合X射线照射对MDA-MB-231细胞株增殖抑制的影响,并评价两者的联合效应。采用克隆形成实验检测MDA-MB-231细胞存活率,观察C225对细胞放射增敏性的影响,按多靶单击模型拟合细胞存活曲线,计算反映放射敏感性指标的细胞存活分数(SF2)、平均致死剂量(D0)、准阈剂量(Dq)和放射增敏比(SER)。流式细胞仪检测细胞凋亡及细胞周期情况。结果:1)1、5、25、125、625nmol/L C225对细胞的增殖抑制率分别为(3.66±0.26)%、(16.48±1.78)%、(35.41±2.46)%、(47.84±1.67)%和(62.49±2.94)%,C225作用于细胞48h的IC50为(151±2.98)nmol/L。2、4、6和8Gy的照射对细胞的增殖抑制率分别为31.12%、47.95%、59.12%和68.54%;当30nmol/L的C225与上述剂量的照射联合使用,细胞增殖抑制率分别为66.27%、82.03%、89.86%和93.03%,两者表现出协同作用,P<0.05。2)克隆形成实验显示,C225+照射组与单纯照射组相比,克隆形成能力下降,SF2、D0及Dq均下降(P<0.05),放射增敏比(SERD0)为1.41。3)细胞凋亡实验结果显示,C225+0、2、4、6、8Gy照射组凋亡率分别为(11.76±0.81)%、(21.46±1.40)%、(38.23±1.76)%、(44.01±1.23)%和(50.41±1.87)%,均高于单独照射组相应剂量点的凋亡率,分别为(2.94±0.58)%、(10.72±0.34)%、(21.14±1.08)%、(24.12±0.98)%和(30.05±2.64)%,P<0.05。C225联合照射明显增强了照射诱导的细胞凋亡。4)细胞周期分布显示,单独使用C225使细胞阻滞于G0/G1期,并减少S期细胞比例,C225作用后G0/G1期及S期细胞比例分别为(58.35±1.66)%和(31.12±0.23)%,P<0.05;单独照射使细胞阻滞于G2/M期,G2/M期细胞比例为(28.25±1.55)%,P<0.05;C225与照射联合作用后,G0/G1期细胞比例进一步升高,为(59.90±2.21)%,S期细胞比例进一步降低,为(13.67±1.34)%,P<0.05。结论:C225对MDA-MB-231细胞具有放射增敏作用,其机制可能与C225增强放射线对细胞的生长抑制作用、抑制肿瘤细胞亚致死性损伤的修复、增加细胞凋亡和诱导细胞G0/G1期阻滞有关。
Objective: To investigate the radiosensitization effect of cetuximab (C225) on breast cancer cell line MDA-MB-231 and its mechanism. Methods: Human breast cancer MDA-MB-231 cells were cultured in vitro. Proliferation inhibition rates of breast cancer cell line MDA-MB-231 were detected by live cell counting kit (CCK-8) at concentrations of 1, 5, 25, 125 and 625nmol / ), And 20% IC50 was taken as the concentration of the follow-up experiment. The effect of C225 combined with X-ray irradiation on the proliferation inhibition of MDA-MB-231 cell line was observed, and the combined effect was evaluated. The survival rate of MDA-MB-231 cells was determined by clone formation assay. The effect of C225 on cell radiosensitivity was observed. Cell survival curve was fitted according to multi-target click model and cell survival score (SF2) reflecting radiation sensitivity index was calculated. , Average lethal dose (D0), quasi-threshold dose (Dq) and radiosensitization ratio (SER). Flow cytometry was used to detect apoptosis and cell cycle. The inhibitory rates of C225 on the proliferation of cells were (3.66 ± 0.26)%, (16.48 ± 1.78)%, (35.41 ± 2.46)%, (47.84 ± 1.67) % And (62.49 ± 2.94)% respectively. The IC50 of C225 treated for 48h was (151 ± 2.98) nmol / L. The inhibitory rates of 2,4,6 and 8Gy on cell proliferation were 31.12%, 47.95%, 59.12% and 68.54%, respectively. When 30nmol / L C225 was used in combination with the above doses, the cell proliferation inhibition rates were 66.27 %, 82.03%, 89.86% and 93.03%, respectively. Both of them showed synergistic effect, P <0.05. 2) The clonogenic assay showed that the ability of clonogenic ability of C225 + (P <0.05), and the ratio of SER (SER) was 1.41. 3) The results of apoptosis showed that the apoptotic rates of C225 + 0, 2, 4, 6, 8 Gy group were (11.76 ± 0.81)% , (21.46 ± 1.40)%, (38.23 ± 1.76)%, (44.01 ± 1.23)% and (50.41 ± 1.87)%, respectively, which were all higher than those of the corresponding irradiation group (2.94 ± 0.58) %, (10.72 ± 0.34)%, (21.14 ± 1.08)%, (24.12 ± 0.98)%, and (30.05 ± 2.64)%, P <0.05. Combination of C225 significantly enhanced irradiation-induced apoptosis. 4) The cell cycle distribution showed that C225 alone blocked cells in G0 / G1 phase and decreased the proportion of S phase cells. The percentage of cells in G0 / G1 phase and S phase after C225 treatment were (58.35 ± 1.66)% and (31.12 ± 0.23)%, P <0.05. The cells were arrested in G2 / M phase by irradiation alone, the proportion of cells in G2 / M phase was (28.25 ± 1.55)%, P <0.05; (59.90 ± 2.21)%, the proportion of cells in S phase was further decreased (13.67 ± 1.34)%, P <0.05. Conclusion: C225 has a radiosensitizing effect on MDA-MB-231 cells. The mechanism may be that C225 enhances the growth inhibitory effect of radiation on cells, inhibits the repair of sublethal damage of tumor cells, increases apoptosis and induces G0 / G1 Period of block related.