肺岩宁颗粒干预细胞周期G_1/S检测点调控信号抗Lewis肺癌细胞增殖的研究

Effect of Feiyanning Granule in Antagonizing Lewis Lung Cancer Cell Proliferation through Cell Cycle G_1/S Checkpoint Dominating Signaling Intervention

目的观察肺岩宁颗粒对Lewis肺癌小鼠肿瘤生长、肿瘤细胞周期分布,以及G1/S检测点调控信号RB-E2F1生物轴的影响。方法采用C57BL/6小鼠,造模并随机分为6组:模型组、顺铂组、肺岩宁煎剂组(煎剂组)、肺岩宁颗粒低剂量组(低剂量组,0·3g)、肺岩宁颗粒中剂量组(中剂量组,0·6g)、肺岩宁颗粒高剂量组(高剂量组,1·2g)。造模后顺铂组1、3、5天腹腔注射顺铂0·1mg,其他各组分别灌胃给药14天,观察各组治疗后抑瘤率、瘤重、体重,流式细胞术检测细胞周期时相分布,RT-PCR测定肿瘤组织视网膜母细胞瘤蛋白(RB)-腺病毒E2启动子结合转录因子(E2F1)的mRNA表达,Western blot检测RB、E2F1蛋白表达。结果各用药组瘤重低于模型组(P<0·05,P<0·01),中剂量组体重明显高于模型组和顺铂组(P<0·05,P<0·01)。流式细胞术发现中剂量组的G0/G1比例较模型组、顺铂组和煎剂组显著增多(P<0·01),癌细胞增殖指数明显降低(P<0·01,P<0·05)。RT-PCR显示中剂量组E2F1mRNA水平明显低于模型组、顺铂组和煎剂组(P<0·01);中剂量组RBmRNA表达明显高于模型组、顺铂组和煎剂组(P<0·01)。Western blot分析显示中剂量组较模型组和顺铂组明显抑制E2F1蛋白表达(P<0·05),并较模型组、顺铂组和煎剂组明显增加RB蛋白表达(P<0·05)。结论肺岩宁颗粒抑制Lewis肺癌肿瘤生长与干预细胞周期时相分布有关,能使癌细胞较多的阻滞在细胞周期G0/G1期,通过抑制E2F1,增强RB表达,从而干预细胞周期G1/S检测点信号通路中RB-E2F1生物轴实现抗肺癌增殖。

Objective To observe the effect of Feiyanning Granule （FYN） on tumor growth and cell cycle distribution in mice with Lewis lung cancer, as well as its influence on G1/S cell cycle checkpoint dominating signaling RB-E2F1 bio-axis. Methods Modeled C57BL/6 mice were randomly divided into 6 groups： the model group （A）, the DDP treated group （B） peritoneally injected with cisplatin 0.1 mg on dl, d3 and d5 after modeling, and the 4 FYN treated groups （C-F）, administered via gastrogavage with FYN Decoction, and FYN Granule in small-, median- and high- dose respectively for 14 days. The tumour inhibiting rate, tumour weight, and body weight of mice were observed after treatment; cell cycle distribution was detected by flow cytometry （ FCM）, RB- E2FlmRNA expressions in tumour tissue were analyzed by RT-PCR, and their protein expressions by Western blot. Results Tumour weight in the 5 treated groups was lower than that in the model group （P 〈 0.05, P 0.01）. Body weight in group E was significantly higher than that in group A and B （P 〈0.05, P〈0.01 ）. FCM analysis showed the proportion of G0/G1 phase was higher in group E than in group A, B and C （P〈0.01）, and cancer cell proliferation index （P1） in group E was lower than in group B （ P 〈0. 05, P 〈0. 01 ）. RT-PCR showed mRNA level of E2F1 was lower, but that of RB was significantly higher in group E than those in group A, B and C respectively （P〈0.01）. Western blot analysis showed the protein expression of E2F1 was lower in clrout） E and B than that in group A （P 〈0.05）, while the protein expression of Rb in group E was higher than that in group A, B and C （ P 〈 0.05）. Conclusion The effect of FYN in inhibiting Lewis lung cancer growth was related to its intervention on cancer cell cycle distribution which blocks most tumor cells in G0/G1 phase. Moreover, FYN can reduce MDM2 expression, enhance P53 expression to influence cell cycle G1/S checkpoint dominating signaling, so as to achieve the effect of antagonizing lung cancer cell proliferation.