Medical Research Center,the Affiliated Changzhou No.2 People’s Hospital of Nanjing Medical University
目的：研究莱菔硫烷（Sulforaphane, SFN）对小鼠肺癌细胞系LLC细胞的生长抑制作用，并探讨其作用机制。方法：CCK8 法鉴定SFN对LLC细胞增殖率的影响；流式细胞术检测SFN对LLC细胞周期和细胞凋亡的影响；DCFH-DA探针检测SFN对LLC细胞株ROS表达的影响；Western Blot检测SFN对LLC细胞Nrf2蛋白和ERK信号通路的影响。最后通过皮下注射建立C56BL/6小鼠肺癌肿瘤模型，建模后实验组及对照组分别给予SFN及PBS灌胃处理，检测SFN治疗对小鼠肿瘤的影响。结果：细胞实验证实SFN能显著抑制小鼠LLC细胞的增殖(P<0.05)，促进LLC细胞的凋亡，随着SFN浓度的增加，LLC细胞晚期凋亡比例逐渐升高(P<0.05)。细胞周期分析提示12.5 μM和25.0 μM的SFN处理可减少LLC细胞G1期比例 (P<0.05)，增加G2/M期的细胞比例 (P<0.01)，周期相关基因CyclinD1、CDK4、CDK6、CyclinB1和CDK1的表达显著下降(P<0.01)。Western blot提示SFN促进LLC细胞ERK磷酸化以及增加Nrf2表达(P<0.05) 。流式检测DCFH-DA探针显示SFN能显著提高LLC细胞内ROS水平。动物实验结果发现SFN处理组小鼠肿瘤大小显著小于对照组(P<0.05）。结论：体内及体外实验证实SFN触发LLC细胞内ERK磷酸化，使得细胞内Nrf2表达增加，细胞内ROS水平升高，阻滞细胞周期，从而加速LLC细胞的凋亡。SFN可能会是一种安全且有效的抗癌药物。
Objective: To explore the inhibitory effect and mechanism of sulforaphane on proliferation and migration of mouse lung cancer cells. Methods: CCK8 method was used to identify the effect of SFN on LLC proliferation. Flow cytometry technology was used to detect the effect of SFN on cell cycle and apoptosis of LLC cells. DCFH-DA probe was detected the ROS expression in LLC cells. The effect of SFN on ERK signaling pathway in LLC cells was detected by Western Blot. Finally, C56BL/6 mouse lung cancer tumor model was established by subcutaneous injection. After modeling, the experimental group and the control group were given SFN and PBS gavage treatment respectively, to detect the effect of SFN treatment on mouse tumors. Results: Cell experiments confirmed that SFN can significantly inhibit the proliferation and promote the apoptosis of LLC cells(P<0.05). With the increase of SFN concentration, the proportion of late apoptosis of LLC cells gradually increased (P<0.05). Cell cycle analysis showed that 12.5 μM/L and 25.0 μM/L SFN treatment reduced the proportion in G1 phase (P<0.05) and increased the proportion of LLC cells in G2/M phase (P<0.01). The expression of cycle-related genes CyclinD1, CDK4, CDK6, CyclinB1 and CDK1 decreased significantly(P<0.01). Western blot suggested that SFN promoted ERK phosphorylation and increased Nrf2 expression in LLC cells (P<0.05). The flow test results showed that SFN could significantly increase ROS level in LLC cells. The results of animal experiments showed that the tumor volume of SFN treated mice was significantly smaller than that of control group (P<0.05). Conclusion: In vivo and in vitro experiments confirmed that SFN triggered ERK phosphorylation, which increased the expression of Nrf2, improved the expression level of ROS and blocked cell cycle, thus accelerating apoptosis and inhibiting the growth of tumor cells. Sulforaphane may be a safe and effective anti-cancer drug.