MUC1介导的半枝莲总黄酮配伍5-氟尿嘧啶协同抗胰腺癌作用机制研究

The intrinsic resistance is the major cause of chemotherapy failure and poor prognosis of pancreatic cancer. Recent studies showed that aberrant MUC1 expression was closely correlated with intrinsic resistance of pancreatic cancer, and interfering MUC1 expression can significantly increase the sensitivity to 5-fluorouracil (5-FU) of pancreatic cancer cells. Our previous large scale screening study for 5-FU synergistic agents showed that Scutellaria Barbata total flavonoids (SBF) could synergistically increase the antitumor effects of 5-FU both in vitro and in vivo, which might be related to MUC1 expression, and the exact mechanism remains to be illustrated. Thus, this project is focused on the mechanism by which the two main functional areas of MUC1, MUC1 tandem repeats (MUC1-TR) and MUC1 cytoplasmic tail (MUC1-CT), mediate the synergistic anti-pancreatic cancer effects of SBF and 5-FU? First, pancreatic cancer cell lines stablely experssing different functional areas of MUC1 will be established. Then, the effects of SBF and 5-FU on the MUC1-TR mediated physical barrier mechanism, the MUC1-CT mediated apoptosis protection mechanism, and the MUC1-CT mediated proliferation mechanism will be studied in vitro and in vivo. This project is expected to find a new way to improve the chemotherapy of pancreatic cancer. The outcome of this study will not only provide scientific theoretical basis for the development of MUC1 targeting synergistic agents, but also provide new insights into the application of combined traditional Chinese medicine and chemotherapeutic drugs in clinical therapy of pancreatic cancer.

胰腺癌的天然耐药性是导致胰腺癌患者化疗失败和预后差的主要因素。近年研究表明，MUC1的异常表达与胰腺癌的天然耐药和化疗敏感性密切相关。项目前期的大规模抗肿瘤增效剂筛选研究发现，半枝莲总黄酮（SBF）与5-氟尿嘧啶（5-FU）以不同比例配伍具有协同抗胰腺癌作用，且与MUC1的表达有关，但具体的调节机制尚不清楚。故，本项目拟在前期研究基础上，通过构建表达MUC1不同功能区域{即胞外串联重复序列（MUC1-TR）和胞质尾区域（MUC1-CT）}的胰腺癌稳转细胞株，研究SBF与5-FU单药及配伍对MUC1不同功能区域的影响，并重点从MUC1-TR介导的物理屏障机制、MUC1-CT介导的凋亡保护机制和MUC1-CT介导的增殖机制等方面进行体内外分子机制研究。本项目不仅可以为以MUC1为靶点的抗肿瘤增效剂的开发提供理论依据，还可以为胰腺癌的中西药配伍化疗提供一种新的安全有效的思路。

胰腺癌恶性程度高、发展快，是预后最差的恶性肿瘤之一。常用的化疗药物如吉西他滨、5-氟尿嘧啶（5-FU）等治疗效果很不理想。我们前期发现半枝莲总黄酮（SBF）与5-FU以不同比例配伍体内外均可产生协同抗肿瘤作用，其机制可能与干扰MUC1的表达有关。本课题选用不同MUC1表达水平的胰腺癌细胞株Panc-1 (MUC1low)和Capan-1(MUC1high)，Isobol-curves曲线法评价了SBF配伍5-FU的抗肿瘤效果，结果表明SBF与5-FU以80:1混合时表现出更为明显的协同作用。Capanc-1和panc-1细胞中CI值分别为0.42和0.30。胰腺癌移植瘤裸鼠模型结果表明SBF配伍5-FU后抗肿瘤效果大大增强，抑瘤率可以达到53%。进一步的机制研究表明，SBF配伍5-FU抗肿瘤的效果与降低细胞内MUC1水平、调节caspase-3活性、bax/bcl-2比率、p53及bid表达和诱导凋亡有关。我们还发现SBF配伍5-FU可影响肿瘤细胞中5-FU代谢相关的DPD酶，提示SBF可能是通过抑制DPD，增加细胞内5-FU含量，而增强其抗肿瘤活性的。.我们用Isobol-curves 曲线法评价了SBF中几种重要的抗肿瘤活性成分配伍5-FU对胰腺癌细胞株的抗肿瘤效果，结果表明相比其他黄酮类有效成分，木犀草素的增效效果较好。进一步胰腺癌移植瘤裸鼠模型研究表明木犀草素配伍5-FU后抗肿瘤效果大大增强，抑瘤率可以达到45%。机制研究表明，木犀草素配伍5-FU抗肿瘤的效果与降低细胞内MUC1水平、影响DPD酶、调节bax/bcl-2比率、p53表达及PARP的裂解和诱导凋亡有关。这表明SBF对5-FU增效的主要成分可能是木犀草素，这将为半枝莲作为抗肿瘤辅助用药的研究奠定基础。.此外，我们对研究中的新问题进行了延伸，主要是对MUC1糖基化对胰腺癌细胞生长转移的影响进行了更进一步的研究。发现干扰MUC1表明糖链后对胰腺癌的恶性生物学行为如增殖、转移等均有明显的影响，且干扰糖基化后对5-FU的敏感性大大增强。此外，我们研究中还发现SBF可以影响胰腺癌细胞的糖酵解，我们将会进行进一步研究。