Sphk1/S1P/NF-кB反馈环路介导的ABL-N抗大肠癌机制研究

There is substantial evidence that the Sphk1/S1P/NF-кB feedback loop is involved in colorectal cancer and some researches support the concept that the activation of Sphk1/S1P/NF-кB feedback loop limits the effectiveness of anti-cancer drug treatment. Acetylbritannilactone (ABL) is a sesquiterpene lactone abundant in Inula Britannica L, a traditional Chinese medicinal herb. In the course of our search for cytotoxic ABL analogues, we synthesized the compound ABL-N, which was synthesized to improve efficacy and pharmacologic characteristics by substitution at C-6 of ABL. In the preliminary studies, it is demonstrated that ABL-N and oxaliplatin interact synergistically to suppress cancer cell growth and induce cell apoptosis via NF-кB-dependent mechanism. Thus, it is suggested that the decrease in colorectal cancer viability by ABL-N may be resulted from inhibition of Sphk1/S1P/NF-кB pathway. In summary, the purpose of this study is designed to evaluate the efficacy of ABL-N on the Sphk1/S1P/NF-кB feedback loop related to colorectal cancer cell apoptosis, and investigate the mechanism of the combination treatment of ABL-N and oxaliplatin on cancer cells. This research will offer a potential therapeutic strategy of ABL-N in the prevention and treatment of colorectal cancer.

Sphk1/S1P/NF-кB反馈环路介导着大肠癌的发生，影响着肿瘤对药物的敏感性。我们从中草药欧亚旋覆花中分离出大花旋覆花内酯（ABL）并对其6位羟基进行修饰，筛选得到衍生物ABL-N。前期研究中我们发现ABL-N与化疗药物奥沙利铂具有协同抗肿瘤作用，且可以通过抑制NF-кB的活化诱导肿瘤细胞发生凋亡。据此我们推测ABL-N可能是通过阻断Sphk1的激活，下调S1P的合成及TRAF2的泛素化调节活性，从而降低NF-кB的活化。本项目拟以人大肠癌细胞为研究对象，应用腺病毒转染、激酶活性分析、泛素化水平检测、免疫印迹等技术，揭示ABL-N对大肠癌细胞中Sphk1/S1P/NF-кB反馈环路的直接作用，探讨ABL-N诱导大肠癌细胞凋亡的机制以及与奥沙利铂的协同作用。此研究将为ABL-N的研发和大肠癌的综合治疗提供新的研究思路和作用靶点。

Sphk1的表达及活性高低调节着S1P的合成，与其下游NF-кB信号通路共同介导着肠道炎症反应和大肠癌的发生，影响着肿瘤对药物的敏感性。我们从中草药欧亚旋覆花中分离出大花旋覆花内酯（ABL）并对其6位羟基进行修饰，筛选得到衍生物ABL-N，本项目中我们研究了ABL-N对大肠癌细胞中Sphk1/S1P/NF-кB通路的影响并探讨ABL-N诱导大肠癌细胞凋亡的机制以及与奥沙利铂的协同作用。首先，我们观察了不同大肠癌细胞Sphk1的表达及活化的差异，以此制备高表达和低表达Sphk1的大肠癌细胞，并且采用不同浓度的奥沙利铂处理大肠癌细胞并检测细胞活力的改变，发现p-Sphk1表达及活化与大肠癌细胞对奥沙利铂的药物敏感性密切相关；随后，我们联用Sphk1抑制剂和奥沙利铂后，检测大肠癌细胞对奥沙利铂药物敏感性的改变。结果显示两者联用后高表达和低表达Sphk1的大肠癌细胞活力下降皆有协同作用，并且两者联用后能够协同抑制Sphk1上游转录因子NF-κB的激活，进而减少Sphk1的活化，同时下调细胞中S1P的表达。采用Sphk1的特异siRNA转染高表达Sphk1的细胞后，原本对奥沙利铂耐药的大肠癌细胞对奥沙利铂恢复了药物敏感性，提示靶向抑制Sphk1可以显著提高奥沙利铂的抗大肠癌作用并逆转大肠癌细胞的耐药性；最后，我们观察了ABL-N、奥沙利铂单用以及两者联用对奥沙利铂耐药大肠癌细胞凋亡的影响，发现两者联用协同促进耐药大肠癌细胞凋亡，减少Sphk1的活化。同时我们还发现ABL-N和奥沙利铂联用能够协同抑制耐药大肠癌细胞种植瘤的生长，伴随着动物模型血清中S1P浓度的下降。