姜黄素逆转上皮间质转化介导的结肠癌5-FU耐药机制

Epithelial－mesenchymal transition (EMT) plays important roles in the chemotherapy resistance in colorectal cancer. Interventions targeting EMT has been proposed as efficient approaches for the prevention and treatment of chemotherapy resistance. Our preliminary results found that curcumin could efficiently inhibit tumor cell growth both in vitro and in vivo. In addition, curcumin inhibits EMT and the activity of Wnt signaling via upregulating the expression of NKD2, an antagonist of Wnt signaling. Therefore, we proposed that curcumin promotes NKD2 expression to suppress carcinogenesis via inhibiting EMT and Wnt signaling. We would prove this hypothesis in this study by exploring the effect of curcumin on the regulation and function of NKD2 as well as other genes related with EMT and carcinogenesis and inhibits the resistance of 5-FU. By doing so, our study would provide insights to understand the mechanism of human carcinogenesis and develop more efficient anti-cancer drugs from traditional Chinese drugs.

结肠癌发病率死亡率近年在中国急剧增加。化疗耐药是结肠癌预后不佳的主要原因之一。因此，寻找抗耐药的策略是结肠癌治疗的重点。本课题组前期研究中证实中药单体姜黄素诱导去甲基化酶TET1表达，抑制EMT标记物细胞内水平，抑制耐药性结肠癌细胞生长。由此，我们推测姜黄素通过TET1上调NKD2而抑制Wnt信号通路，从而抑制上皮间质转化（EMT）介导的肿瘤细胞耐药性。为验证该假说，本项目将通过肠癌细胞、肿瘤动物模型等实验来：1) 明确5-FU耐药细胞中，姜黄素能够抑制EMT介导的5-FU耐药；2)证实TET1-NKD2-Wnt信号通路抑制EMT介导的结肠癌细胞5-FU耐药；3)阐明姜黄素通过TET1调控NKD2来抑制Wnt活性，从而抑制EMT介导的5-FU化疗耐药。本项目将有助于阐明结肠癌5-FU耐药的分子机制，为姜黄素的临床运用提供坚实的理论及实验基础。

5-氟尿嘧啶是结肠化疗的基石药物，5-氟尿嘧啶耐药是结肠癌(CRC)预后不佳的主要原因之一，因此寻找有效预防化疗耐药的策略一直是CRC防治研究的重点。本研究通过结肠癌细胞实验及移植瘤模型实验，证实姜黄素能够抑制5-FU耐药的结肠癌细胞增值，促进其凋亡并抑制细胞的上皮间质转化进程，且呈浓度同时姜黄素能够逆转。并通过蛋白质免疫印迹法（WB）、实时荧光定量PCR（RT-PCR）及重亚硫酸测序分别从蛋白、基因以及甲基化修饰层面证实姜黄素可以通过去甲基化酶TET1调控NKD2基因去甲基化，诱导其表达，进而抑制Wnt信号介导的上皮间质转化，最终逆转5-FU介导的耐药。通过慢病毒转染敲低TET1后，发现NKD2的表达受到明显抑制，其去甲基化进程受到抑制，由此可以证实TET1与NKD2存在相互作用。同时TET1的转录因子Pax-6也受到姜黄素的直接作用从而活化TET1，通过染色质免疫共沉淀技术证实了Pax-6与TET1的直接作用。上述体外研究证实了姜黄素调控Pax-6的转录促使TET1上调，由此介导NKD2的去甲基化，从而抑制Wnt信号介导的上皮间质转化进程，最终逆转结肠癌5-FU耐药。在体外的研究中也证实了姜黄素能够上调转移瘤中NKD2、TET1的表达，同时抑制上皮间质转化。综上所诉，本研究证实了姜黄素逆转结肠癌5-FU耐药的分子生物学机制，着眼于上皮间质转化，从表观遗传学调控入手，丰富了中药单体逆转化疗耐药的深层次机制，为解决临床肿瘤5-FU耐药提供新的靶点和新的思路，同时为推广中药单体临床辅助治疗的应用奠定基础，将有效地提高临床结肠癌5-FU耐药患者的生存率。