Department of General Surgery, Zhongda Hospital, Southeast University Medical School, Nanjing, 210009, Jiangsu, China.
Department of Pharmacology, School of Medicine and Life Science, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China.
Apoptosis. 2018 Jun;23(5-6):356-374. doi: 10.1007/s10495-018-1460-0.
Recent studies have confirmed that IL-6/GP130 targets are closely associated with tumor growth, metastasis and drug resistance. 5-Fluorouracil (5-FU) is the most common chemotherapeutic agent for colon cancer but is limited due to chemoresistance and high cytotoxicity. Bazedoxifene (BZA), a third-generation selective estrogen receptor modulator, was discovered by multiple ligand simultaneous docking and drug repositioning approaches to have a novel function as an IL-6/GP130 target inhibitor. Thus, we speculated that in colon cancer, the anti-tumor efficacy of 5-FU might be increased in combination with IL-6/GP130 inhibitors. CCK8 assay and colony formation assay were used to detect the cell proliferation and colony formation. We measured the IC value of 5-FU alone and in combination with BZA by cell viability inhibition. Cell migration and invasion ability were tested by scratch migration assays and transwell invasion assays. Flow cytometric analysis for cell apoptosis and cell cycle. Quantitative real-time PCR was used to detect Bad, Bcl-2 and Ki-67 mRNA expression and western blotting (WB) assay analyzed protein expression of Bad/Bcl-2 signaling pathway. Further mechanism study, WB analysis detected the key proteins level in IL-6/GP130 targets and JAK/STAT3, Ras/Raf/MEK/ERK, and PI3K/AKT/mTOR signaling pathway. A colon cancer xenograft model was used to further confirm the efficacy of 5-FU and BZA in vivo. The GP130, P-STAT3, P-AKT, and P-ERK expression levels were detected by immunohistochemistry in the xenograft tumor. BZA markedly potentiates the anti-tumor function of 5-FU in vitro and in vivo. Conversely, 5-FU activation is reduced following exogenous IL-6 treatment in cells. Further mechanistic studies determined that BZA treatment enhanced 5-FU anti-tumor activation by inhibiting the IL-6/GP130 signaling pathway and the phosphorylation status of the downstream effectors AKT, ERK and STAT3. In contrast, IL-6 can attenuate 5-FU function via activating IL-6R/GP130 signaling and the P-AKT, P-ERK and P-STAT3 levels. This study firstly verifies that targeting IL-6/GP130 signaling can increase the anti-tumor function of 5-FU; in addition, this strategy can sensitize cancer cell drug sensitivity, implying that blocking IL-6/GP130 targets can reverse chemoresistance. Therefore, combining 5-FU and IL-6/GP130 target inhibitors may be a promising approach for cancer treatment.
最近的研究证实，IL-6/GP130 靶点与肿瘤生长、转移和耐药性密切相关。氟尿嘧啶（5-FU）是结肠癌最常用的化疗药物，但由于耐药性和高细胞毒性而受到限制。Bazedoxifene（BZA），第三代选择性雌激素受体调节剂，通过多配体同时对接和药物再定位方法发现具有作为 IL-6/GP130 靶标抑制剂的新功能。因此，我们推测在结肠癌中，5-FU 的抗肿瘤功效可能会因与 IL-6/GP130 抑制剂联合使用而增加。CCK8 检测和集落形成实验检测细胞增殖和集落形成。我们通过细胞活力抑制测定单独使用和联合使用 BZA 时的 5-FUIC 值。划痕迁移实验和 Transwell 侵袭实验检测细胞迁移和侵袭能力。流式细胞术分析细胞凋亡和细胞周期。实时定量 PCR 检测 Bad、Bcl-2 和 Ki-67 mRNA 表达，Western blot（WB）分析 Bad/Bcl-2 信号通路蛋白表达。进一步的机制研究，WB 分析检测 IL-6/GP130 靶点和 JAK/STAT3、Ras/Raf/MEK/ERK、PI3K/AKT/mTOR 信号通路的关键蛋白水平。建立结肠癌异种移植模型进一步证实 5-FU 和 BZA 在体内的疗效。免疫组织化学检测异种移植瘤中 GP130、P-STAT3、P-AKT 和 P-ERK 的表达水平。BZA 显著增强 5-FU 在体外和体内的抗肿瘤作用。相反，细胞外 IL-6 处理后，5-FU 的激活减少。进一步的机制研究表明，BZA 通过抑制 IL-6/GP130 信号通路和下游效应子 AKT、ERK 和 STAT3 的磷酸化状态增强 5-FU 的抗肿瘤激活作用。相反，IL-6 可以通过激活 IL-6R/GP130 信号和 P-AKT、P-ERK 和 P-STAT3 水平来减弱 5-FU 的功能。本研究首次验证靶向 IL-6/GP130 信号可以增加 5-FU 的抗肿瘤功能；此外，该策略可以增强癌细胞对药物的敏感性，表明阻断 IL-6/GP130 靶点可以逆转耐药性。因此，联合使用 5-FU 和 IL-6/GP130 靶标抑制剂可能是癌症治疗的一种有前途的方法。
