地黄管食通口服液对食管癌细胞凋亡及Wnt通路β-catenin和c-myc的影响
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摘要
目的:观察地黄管食通口服液诱导人食管癌Eca109细胞及放疗后模型大鼠食管癌细胞的凋亡作用并探讨其分子机制。
方法:体外实验采用不同浓度的地黄管食通口服液直接作用于人食管癌细胞(Eca-109),并使用四甲基偶氮唑蓝(MTT)、TUNEL及流式细胞术,观察不同浓度的地黄管食通口服液对体外培养的Eca109细胞的生长活力和细胞凋亡的影响。体内实验通过对化学诱癌的大鼠模型,常规放疗后采用不同浓度的地黄管食通口服液灌服模型动物五周后,探讨该药对放疗后食管癌模型大鼠的影响,用TUNEL方法检测该药对食管癌细胞凋亡的影响,免疫组化检测对β-catenin、c-myc的影响,Western-blot检测Wnt通路的关键蛋白β-catenin的表达,RT-PCR检测Wnt通路下游靶基因c-myc的变化。
结果:①地黄管食通口服液能抑制体外培养的人食管癌Eca109细胞生长,这种作用呈时间、浓度依赖性增强。②地黄管食通口服液可以诱导Eca109细胞发生凋亡改变,随着药物浓度的增加,凋亡指数增加,这种作用呈浓度依赖性,方差分析F值为158.35(p<0.01)。③地黄管食通口服液体内实验可以诱导食管癌放疗后模型大鼠食管癌细胞凋亡,这种作用呈剂量依赖性,方差分析F值为133.95(p<0.01)。④地黄管食通口服液治疗后,各组食管组织胞浆、胞核β-catenin、c-myc蛋白的表达减弱。高剂量组与低剂量有显著性差异(p<0.05),与空白组比较无显著性差异(p>0.05)。⑤地黄管食通口服液能显著抑制食管癌放疗后大鼠食管癌细胞β-catenin的表达,方差分析F值为78.59(p<0.01);管食通高剂量组与低剂量组、六味组比较有显著性差异(p<0.05),它还能下调c-myc基因的转录,方差分析F值为89.67(p<0.01),地黄管食通口服液高剂量能抑制c-mycmRNA的转录,与六味组有显著性差异(p<0.05)。
Objective : To observe the inhibiting effect of Dihuang Guanshitong oral liquid (DGOL)on the human esophageal carcinoma 109 cell(Eca109) and the esophageal carcinoma cell in model rats after radiotherapy, and to study its mechanism.Methods: The human esophageal carcinoma 109 cell(Eca-109) cultured in vitro were treated by different concentration of Dihuang Guanshitong oral liquid.The MTT, TUNEL and flow cytometry (FCM) techniques were used to observe the effect of different concentration of Dihuang Guanshitong oral liquid on cell growth and cell apoptosis of Eca109 in vitro. After radiotherapy, the chemical carcinogenesis model rats were treated orally with different concentration of "Dihuang Guanshitong oral liquid" and "control drugs" for 5 weeks to explore the effect of Dihuang Guanshitong oral liquid on the esophageal carcinoma tissues of model rats . The effect of this drug on apoptosis were detected by TUNEL;on expression of P-catenin ,the key protein of Wnt, by immunohistochemistry (IHC) and Western-blot;on expression of c-myc, the target gene on the downstream of Wnt by IHC and RT-PCR.Results: ① Dihuang Guanshitong oral liquid can inhibit the growth of cultured esophageal carcinoma cell Eca109 in a time and concentration- dependent manner. ② Dihuang Guanshitong oral liquid can induce change of cell apoptosis with bioequivalence F of 158.35(p<0.01). The apoptotic index increase with the enhancement of drug concentration,which indicates concentration-dependence. ? Dihuang Guanshitong oral liquid can induce apoptosis of esophageal carcinoma cell in model rats after radiotherapy with bioequivalence F of 133.95(p<0.01) ,show
concentration- dependence on cell apoptosis inducement. ?After treating with Dihuang Guanshitong oral liquid, expression of P-catenin and c-myc protein in cytoplasm and cell nucleolus of every group is decreased.There is significant difference between the low dosage group and the high dosage group(p<0.05), but no significant difference with the normal group(p> 0.05).?Dihuang Guanshitong oral liquid can inhibit the P-catenin expression of esophageal carcinoma cell in model rats with bioequivalence F of 78.59(p<0.01). There is significant difference between the high dosage group and the low dosage group (p<0.05), and significant difference with liuwei group (p<0.05).Then it downregulated the transcription of c-myc with bioequivalence F of 89.67(p<0. 01). There is significant difference between the high dosage group and liuwei group (p<0.05).Conclusion: Dihuang Guanshitong oral liquid can inhibit proliferation of EcalO9 and induce apoptosis of EcalO9 in vitro. Former in vivo test approved that the apoptotic inducement of Dihuang Guanshitong oral liquid on esophageal carcinoma cell was related with increase of p53 through inhibiting gene mutation of p53 and decrease of bcl-2, c-jun and cyclinDl.In this test, the result showed that this apoptotic inducement was related to the down expression of P-catenin and c-myc protein in cytoplasm and cell nucleolus. Dihuang Guanshitong oral liquid can inhibit the expression of P-catenin and decrease the transcription of c-myc of esophageal carcinoma cells in model rats after radiotherapy , which may be one of mechanisms of the apoptotic inducement and esophageal carcinoma suppression of this drug.
方法:体外实验采用不同浓度的地黄管食通口服液直接作用于人食管癌细胞(Eca-109),并使用四甲基偶氮唑蓝(MTT)、TUNEL及流式细胞术,观察不同浓度的地黄管食通口服液对体外培养的Eca109细胞的生长活力和细胞凋亡的影响。体内实验通过对化学诱癌的大鼠模型,常规放疗后采用不同浓度的地黄管食通口服液灌服模型动物五周后,探讨该药对放疗后食管癌模型大鼠的影响,用TUNEL方法检测该药对食管癌细胞凋亡的影响,免疫组化检测对β-catenin、c-myc的影响,Western-blot检测Wnt通路的关键蛋白β-catenin的表达,RT-PCR检测Wnt通路下游靶基因c-myc的变化。
结果:①地黄管食通口服液能抑制体外培养的人食管癌Eca109细胞生长,这种作用呈时间、浓度依赖性增强。②地黄管食通口服液可以诱导Eca109细胞发生凋亡改变,随着药物浓度的增加,凋亡指数增加,这种作用呈浓度依赖性,方差分析F值为158.35(p<0.01)。③地黄管食通口服液体内实验可以诱导食管癌放疗后模型大鼠食管癌细胞凋亡,这种作用呈剂量依赖性,方差分析F值为133.95(p<0.01)。④地黄管食通口服液治疗后,各组食管组织胞浆、胞核β-catenin、c-myc蛋白的表达减弱。高剂量组与低剂量有显著性差异(p<0.05),与空白组比较无显著性差异(p>0.05)。⑤地黄管食通口服液能显著抑制食管癌放疗后大鼠食管癌细胞β-catenin的表达,方差分析F值为78.59(p<0.01);管食通高剂量组与低剂量组、六味组比较有显著性差异(p<0.05),它还能下调c-myc基因的转录,方差分析F值为89.67(p<0.01),地黄管食通口服液高剂量能抑制c-mycmRNA的转录,与六味组有显著性差异(p<0.05)。
Objective : To observe the inhibiting effect of Dihuang Guanshitong oral liquid (DGOL)on the human esophageal carcinoma 109 cell(Eca109) and the esophageal carcinoma cell in model rats after radiotherapy, and to study its mechanism.Methods: The human esophageal carcinoma 109 cell(Eca-109) cultured in vitro were treated by different concentration of Dihuang Guanshitong oral liquid.The MTT, TUNEL and flow cytometry (FCM) techniques were used to observe the effect of different concentration of Dihuang Guanshitong oral liquid on cell growth and cell apoptosis of Eca109 in vitro. After radiotherapy, the chemical carcinogenesis model rats were treated orally with different concentration of "Dihuang Guanshitong oral liquid" and "control drugs" for 5 weeks to explore the effect of Dihuang Guanshitong oral liquid on the esophageal carcinoma tissues of model rats . The effect of this drug on apoptosis were detected by TUNEL;on expression of P-catenin ,the key protein of Wnt, by immunohistochemistry (IHC) and Western-blot;on expression of c-myc, the target gene on the downstream of Wnt by IHC and RT-PCR.Results: ① Dihuang Guanshitong oral liquid can inhibit the growth of cultured esophageal carcinoma cell Eca109 in a time and concentration- dependent manner. ② Dihuang Guanshitong oral liquid can induce change of cell apoptosis with bioequivalence F of 158.35(p<0.01). The apoptotic index increase with the enhancement of drug concentration,which indicates concentration-dependence. ? Dihuang Guanshitong oral liquid can induce apoptosis of esophageal carcinoma cell in model rats after radiotherapy with bioequivalence F of 133.95(p<0.01) ,show
concentration- dependence on cell apoptosis inducement. ?After treating with Dihuang Guanshitong oral liquid, expression of P-catenin and c-myc protein in cytoplasm and cell nucleolus of every group is decreased.There is significant difference between the low dosage group and the high dosage group(p<0.05), but no significant difference with the normal group(p> 0.05).?Dihuang Guanshitong oral liquid can inhibit the P-catenin expression of esophageal carcinoma cell in model rats with bioequivalence F of 78.59(p<0.01). There is significant difference between the high dosage group and the low dosage group (p<0.05), and significant difference with liuwei group (p<0.05).Then it downregulated the transcription of c-myc with bioequivalence F of 89.67(p<0. 01). There is significant difference between the high dosage group and liuwei group (p<0.05).Conclusion: Dihuang Guanshitong oral liquid can inhibit proliferation of EcalO9 and induce apoptosis of EcalO9 in vitro. Former in vivo test approved that the apoptotic inducement of Dihuang Guanshitong oral liquid on esophageal carcinoma cell was related with increase of p53 through inhibiting gene mutation of p53 and decrease of bcl-2, c-jun and cyclinDl.In this test, the result showed that this apoptotic inducement was related to the down expression of P-catenin and c-myc protein in cytoplasm and cell nucleolus. Dihuang Guanshitong oral liquid can inhibit the expression of P-catenin and decrease the transcription of c-myc of esophageal carcinoma cells in model rats after radiotherapy , which may be one of mechanisms of the apoptotic inducement and esophageal carcinoma suppression of this drug.
引文
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