自噬参与丹栀逍遥散诱导乳腺癌细胞死亡的机制研究

Autophagic death plays a crucial regulation role in the occurrence and development of tumor. Danzhi Xiaoyao Powder not only exhibited significant inhibition of the progression as well as induction the apoptosis of breast cancer cells (MCF-7), but also revealed that Danzhi Xiaoyao Powder could inhibit the proliferation of MCF-7 cell (human breast adenocarcinoma cell line) in a dose-and time-dependent manner in our previous studies. The MDC staining showed autophagic cell death during the course. Therefore we propose the hypothesis that autophagic death is invoved in Danzhi Xiaoyao Powder inhibiting the proliferation of MCF-7 cells. In vivo studies, tumor-inhibition index was observed, the autophagic cell death and apoptosis were counted by electron microscope, TUNEL method was applied to detect the apoptosis and the correlated proteins of autophagy were analyzed by immunohistochemistry method. In vitro experiments, MCF-7 cells were treated by drug-contained serum. MTT method, AO/EB double-labeled staining, MDC fluorescent staining, transfection technique, flow cytometry and Western blotting were applied to prove the hypothesis. On one hand all of the above researches aim to discuss the effectiveness and mechanism of autophagic death of Danzhi Xiaoyao Powder in treating tumor. On the other hand, our studies will investigate the effect of autophagic death of Traditional Chinese Medicinal complex formula in antineoplastic aspects. It is of great significance in enriching the theory of Traditional Chinese Medicine in anti-tumor.

自噬性死亡在肿瘤发病过程中具有重要的作用。我们在前期体外实验研究中发现丹栀逍遥散明显抑制乳腺癌细胞MCF-7的生长，诱导细胞发生凋亡，具有时间-剂量依赖性的特点，罗丹明染色证明与线粒体途径有关。同时，MDC染色初步证明这个过程中伴有细胞自噬性死亡的发生。因此我们提出"自噬参与了丹栀逍遥散诱导乳腺癌细胞死亡"的假说。体内实验通过抑瘤指数观察，电镜计数自噬和凋亡体数，TUNEL检测细胞凋亡，免疫组化法检测自噬相关蛋白LC3；体外实验通过含药血清作用于MCF-7乳腺癌细胞，MTT法测定细胞生长抑制率，AO/EB双染色进一步观察证实细胞凋亡情况，MDC荧光染色观察自噬变化，转染和免疫荧光技术检测对自噬关键蛋白Beclin1表达影响，流式细胞术分析凋亡、细胞周期和自噬的变化；体内外免疫印迹法检测自噬相关蛋白表达。旨在探讨自噬性死亡对肿瘤的影响，阐明自噬在中药复方抗肿瘤中的作用，丰富中医药抗肿瘤理论。

自噬与肿瘤的发生有着密切的关系，在肿瘤发病过程中具有重要的作用。在前期体外实验研究中，课题组发现丹栀逍遥散明显抑制乳腺癌细胞MCF-7的增殖，诱导细胞发生凋亡，同时，单丹磺酰戊二胺（MDC）染色初步证明这个过程中伴有细胞自噬性死亡的发生。因此提出假说：“丹栀逍遥散诱导乳腺癌肿瘤细胞死亡过程中，自噬参与了该过程，是其重要机制。”通过体内实验结合体外实验的方法，运用HE染色、荧光染色、流式细胞术、免疫组化、免疫印迹等方法技术，明确了丹栀逍遥散可以通过凋亡和自噬诱导乳腺癌细胞死亡，能够上调LC3、Beclin1自噬关键蛋白水平，阐明了该方通过自噬诱导乳腺癌细胞死亡的作用机制，通过上调p-PTEN蛋白活性，并下调Akt/mTOR通路关键分子p-Akt、Rheb、p-mTOR蛋白的活性，从而抑制Akt/mTOR信号通路的传导，负向调控乳腺癌肿瘤细胞发生自噬性死亡。揭示了中医疏肝健脾，清热养血治法防治乳腺癌的合理性，丰富了中医药防治肿瘤理论，从而更好地发挥中医药治疗肿瘤的特色为临床服务。