褪黑素联合顺铂、甲氨蝶呤对骨肉瘤SaOS-2细胞增殖的影响

Effects of Melatonin Combined with Cis-platinum or Methotrexate on the Proliferation of Osteosarcoma Cell Line SaOS-2

目的探讨褪黑素(Mel)联合顺铂(DDP)、甲氨蝶呤(MTX)对骨肉瘤细胞系Sa OS-2增殖的影响,以及Mel与DDP、MTX是否具有协同抗肿瘤作用。方法 Mel、DDP、MTX、Mel+DDP、Mel+MTX作用于Sa OS-2细胞后,CCK-8法检测药物对细胞活性的影响,应用Compusyn软件分析药物的合并效应,流式细胞术分析细胞周期分布及细胞凋亡率。结果与对照组相比,Mel、DDP、MTX单独均能显著降低Sa OS-2细胞活性(P均<0.05),且呈剂量-效应关系。与单用DDP或MTX相比,Mel与DDP或MTX联合应用能显著降低Sa OS-2细胞的活性(P均<0.05)。1 mmol/L的Mel与6.67、16.67、33.33、66.66μmol/L的DDP合用时,联合用药指数(CI)分别为1.18、1.21、1.09、0.84,与0.1、0.5、1、2、4 mmol/L MTX合用后,CI分别为0.88、0.88、0.83、0.78、0.81。与对照组相比,Mel组与Mel+MTX组的G1期细胞比例显著增多(P均<0.05),S期细胞比例显著减少(P均<0.05);MTX组的S期细胞比例显著增多(P均<0.05)。药物作用组的细胞凋亡率均显著高于对照组(P均<0.05),联合用药组的细胞凋亡率显著高于单药组(P均<0.05)。结论 Mel在体外能抑制Sa OS-2细胞的活性,阻滞细胞周期于G1期,诱导凋亡,发挥抗肿瘤效应,与较低浓度DDP呈拮抗效应,而与MTX、较高浓度DDP联合应用时呈协同抗肿瘤效应。

Objective To evaluate the effects of melatonin（ Mel） combined with cis-platinum（ DDP）or methotrexate（ MTX） on the proliferation of osteosarcoma cell line Sa OS-2,and to explore whether Mel combined with DDP or MTX could play a synergistic antitumor effect. Methods Sa OS-2 was treated with Mel alone or Mel combined with DDP or MTX. Cell counting kit-8 assay was used to measure the cell activities. Combination index（ CI） value was used to evaluate the combined effects： CI 1 indicating synergetic effect,CI = 1 additive,and CI 1 antagonistic. Flow cytometry was used to analyze cell cycle distribution and cell apoptosis. Results After treated with Mel（ 0. 5,1,2,4,5 mmol / L）, DDP（ 6. 67,16. 67,33. 33,66. 66μmol / L） or MTX（ 0. 1,0. 5,1,2,4 mmol / L） alone,Sa OS-2 cell activities decreased in a dose-dependent manner（ all P 〈0. 05）. The activities of Sa OS-2 cell treated with both Mel（ 1 mmol / L） and DDP or MTX were significantly lower than that of DDP or MTX alone（ all P 〈0. 05）. CI values of cells exposed to 1 mmol / L Mel plus 6. 67,16. 67,33. 33,and 66. 66 μmol / L DDP were 1. 18,1. 21,1. 09,and 0. 84,respectively,and CI values of cells exposed to 1 mmol / L Mel plus 0. 1,0. 5,1,2,and 4 mmol / L MTX were 0. 88,0. 88,0. 83,0. 78,and 0. 81,respectively. The G1-stage cells were increased and the S-stage cells were reduced when the cells were treated with Mel（ 1 mmol / L） alone or combined with MTX（ 0. 5 mmol / L）（ P 〈0. 05）. The Sstage cells were increased when the cells treated with MTX（ 0. 5 mmol / L）（ P 〈0. 05）. The apoptotic cells were increased when they treated with Mel（ 1 mmol / L） alone or combined with DDP（ 16. 67 μmol / L） or MTX（ 0. 5 mmol /L）（ P 〈0. 05）. When the cells were treated with Mel combined with DDP or MTX,the apoptotic cells were more than that of DDP or Mel alone（ P 〈0. 05）. Conclusions Mel can inhibit Sa OS-2 cells activity,block the cell cycle at G1-stage,and induce apoptosis. Mel has an antagonistic effect with lower concentration of DDP but a synergistic effect with MTX or higher concentration of DDP.