应用纳米乳剂载入紫杉醇与PI3K/mTOR双通路抑制剂BEZ235抗结肠癌的增效作用机制研究

Colon cancer is one of most common cancer in the world, with drug resistance is the major challenges to effective treatments. To overcome it, combination therapy with different chemotherapeutics is common practice. Our early study demonstrated that paclitaxel together with BEZ235 exhibited a synergetic inhibition effect on colon cancer cell growth. Further, Nanoemulsion (NE) loaded paclitaxel and BEZ235 were more effective than the free drug and a combination treatment of both NE-drugs increased the efficiency of the treatments. Given that paclitaxel and BEZ235 react to different aspects of cell division of cancer cells, we assumed that they might have a synergistic effect on inhibiting cancer cell growth of colon cancer through induced apoptosis and anti-multidrug resistance. However, this combination therapy has not been documented in colon cancer. In addition, combination treatment with both NE-delivered BEZ235 and NE-delivered paclitaxel has not been reported in any cancer. Therefore, bases on the early study, we tended to investigate whether the combination treatment with both BEZ235 and paclitaxel are work on drug resistant colon cancer cell line and animal model, and decrease the dosage and cytotoxicity, followed by a study of NE-loaded drugs. Beside it, we prepare to observe the change of tumor morphology, using RT-PCR, Weston blot and immunohistochemical tools to detect the expression of colon cancer cell proliferation, apoptosis, drug resistance, PI3K/mTOR pathway related key genes and tumor stem cells marker after combination treatment. Therefore, we would like to explain the molecular mechanism of synergistic inhibition on colon cancer cell growth, provide an opportunity for the clinical application of NE loaded paclitaxel and BEZ235, and found a more low toxic and effective treatment method for colon cancer.

结肠癌是最常见的恶性肿瘤之一，肿瘤耐药是治疗的巨大挑战。多药联合是常见克服耐药的疗法。前期研究证明紫杉醇和PI3K/ mTOR双信号通路抑制剂BEZ235对结肠癌细胞生长具有良好的协同抑制作用，应用纳米乳剂装载双药后敏感性更高，我们推测联用BEZ235与紫杉醇可能协同作用于结肠癌细胞的多个靶点，抗耐药并促进细胞凋亡，但国内外均未见相同报道。故本项目拟在前期研究基础上，通过细胞与裸鼠成瘤实验，深入研究联用BEZ235与紫杉醇是否在结肠癌耐药细胞株、动物体内实验中发挥协同作用，降低毒副反应，并应用纳米乳剂载入双药后观察其是否进一步增敏增效，及其对肿瘤形态学的改变，并应用RT-PCR、WB、免疫组化等检测其对肿瘤组织增殖、凋亡、耐药、PI3K/mTOR通路关键基因以及肿瘤干细胞主要标记表达的影响，探讨其作用机制，为纳米乳剂紫杉醇-BEZ235在结肠癌的临床应用奠定基础，提供更低毒有效的治疗方法。

结肠癌是全世界中最常见的癌症之一，耐药性是与肿瘤相关的死亡的重要原因。纳米粒子的联合治疗和药物递送已显示可有效克服许多癌症中的耐药性。我们先前曾报道载有纳米乳（NE）的紫杉醇（PTX）和BEZ235可以协同抑制结肠癌细胞的生长。本研究在前期工作基础上，探讨应用纳米乳剂运载的紫杉醇（NE-PTX）和BEZ235在体外和体内能否克服肿瘤耐药并协同抑制结肠癌耐药细胞HCT116-LOHP和HT29-DDP的生长。主要的方法为CCK8试剂盒测定体外药物对细胞活力的影响，用β-微管蛋白免疫荧光染色检测细胞形态变化，用western blotting检测耐药相关与抗凋亡蛋白，并检测、比较各组药物在裸鼠体内进行体内移植2种耐药性结肠癌细胞的抗肿瘤生长疗效。实验结果显示两种结肠癌耐药细胞系均对PTX敏感，但对BEZ235不敏感。 PTX与BEZ235联合可协同抑制两种细胞系的增殖。载有纳米乳液的PTX（NE-PTX）将PTX的IC50降低至游离PTX的约2/5，表明NE-PTX具有很高的抑制功效。当NE-PTX与低浓度的BEZ235（50 nM）结合使用时，IC50降低至游离PTX的约2/3。此外，与单药治疗和对照组相比，NE-PTX+ BEZ235治疗可增加细胞凋亡，降低Pgp、ABCC1与Bcl-2的表达，并显著减轻肿瘤重量与体积。这些结果均表明负载纳米乳液的PTX+BEZ235可以有效克服耐药性并改善对癌细胞增殖和肿瘤生长的抑制作用，为治疗结肠癌耐药性患者提供了一种可能的新方法。