骨肉瘤细胞脱逸化疗诱导的细胞衰老可重新增殖成瘤细胞

Osteosarcoma cells can reproduce into tumorigenic cells by escaping chemotherapy-induced cell senescence

目的探讨骨肉瘤细胞(OS)能否脱逸化疗至细胞衰老和再增殖;评价逃逸衰老的细胞成瘤能力,从新的角度解释骨肉瘤复发的机制。方法多柔比星(DOX)诱导骨肉瘤细胞U2OS和MG63制备衰老的细胞模型,SA-Gal染色法检测细胞衰老;Western blot检测衰老相关分子。更换成不含DOX的培养基培养75 d,细胞计数检测细胞增殖和计算脱逸率。而后分为3组:对照组、衰老细胞组和脱逸衰老细胞组,琼脂糖克隆实验检测集落形成和裸鼠成瘤实验检测成瘤能力。结果超过90%的U2OS和MG63呈SA-Gal染色阳性;衰老相关的细胞分子p-p53、p-Rb和p-γH2AX明显升高(P<0.05)。75 d后,观察到有细胞重新增殖(脱逸率百万分之一),具有集落形成和裸鼠成瘤能力。结论衰老的骨肉瘤细胞能够脱逸细胞衰老状态重新增殖并具有成瘤能力。

Objective To explore whether osteosarcoma cells can escape from cell senescence and reproduce,and to evaluate the oncogenic ability of senescent-escaped cells.To explain the mechanism of osteosarcoma recurrence from a new perspective.Methods Doxorubicin(DOX)was used to develop aging model of U2OS and MG63.Cell senescence was detected by SA-Gal staining and senescence-related molecules were detected by Western blot.The cells were replaced with DOX-free medium and cultured for 75 days.Cell proliferation and escape rate were measured by cell counting.Then they were divided into three groups:parent cell,senescent cell,escaped-senescence cell.Agarose cloning assay was used to detect cloning formation and nude mice tumorigenicity test was used to detect tumorigenicity.Results In DOX-induced U2OS or MG63 cells,senescence specific marker SA-β-Gal positive cells emerged 2-day after stimulation and reached the plateau on day 4 with more than 90%SA-β-Gal-positive cells.Effects of DOX on the activities of other SIPS-related regulators in OS cells were also measured at protein level and the expression of senescent regulators p-p53,p-Rb,and p-γ-H2AX were all increased significantly.Senescent OS cells were cultured in medium without DOX.The numbers of cells kept unchanged until day 75 and then began to increase(one-millionth of the escape rate).The reproducing cells gained even higher tumorigenic capability.Conclusions The senescent osteosarcoma cells escape from senescence and gain even higher tumorigenic capability to form new colonies eventually in recurrent tumor.