- 學位論文
RGD小分子藥物合併低劑量紫杉醇誘導人類神經膠母細胞瘤凋亡
Combination of RGD Compound and Low-Dose Paclitaxel Induces Apoptosis in Human Glioblastoma Cells
摘要
根據世界衛生組織最新公布的癌症報告，腦癌發生率占所有癌症的1.8%(直腸癌9.2%，乳癌25.2%)，看似不高的發生率，但是將近100%的最終致死率佔癌症前10名 。行政院衛生署癌病登記統計，台灣每年約有600位原發性惡性腦瘤新病例，佔所有惡性腫瘤發生個案的1.03%。人類多型性神經膠母細胞瘤(glioblastoma multiforme, GBM)，約佔所有惡性腦瘤60%，是最常見的成人原發惡性腦瘤。目前，臨床對於人類多型性神經膠母細胞瘤的治療方式為放射線治療、外科手術切除，以及合併化學藥物治療。然而，人類多型性神經膠母細胞瘤生長速率快，具高度組織浸潤性(infiltrative)，導致治療效率不佳。 組合蛋白(integrin)表現在細胞表面，負責細胞訊號傳遞的穿膜蛋白質受器，由α及β鏈組成的蛋白二聚體。Integrin-αvβ3活化主要參與腫瘤組織血管新生(neovasculature / angiogenesis)及腫瘤細胞轉移。目前已知生化合成的RGD (Arg-Gly-Asp)胜肽小分子藥物可以與integrin-αvβ3專一性結合，進而達到抑制血管新生的效果。但是，腫瘤細胞專一性受體-integrin-αvβ3的表現量不足會影響RGD小分子藥物的標靶治療效率。本研究以低劑量癌症治療藥物紫杉醇(Paclitaxel, PTX)進行人類多型性神經膠母細胞瘤U87MG細胞株前處理，以誘導integrin-αvβ3在腫瘤細胞上的表現增加，接著應用單環結構c(RGDyK)與雙環結構E[c(RGDyK)]2兩種生化合成之RGD小分子藥物進行標靶治療，以期達到增加RGD小分子藥物的治療效率。 人類多型性神經膠母細胞瘤U87MG細胞，給予12小時10 nM低劑量紫杉醇前處理，顯著增加integrin-αvβ3受器表現，提供更多與RGD胜肽結合位置。接著給予RGD胜肽處理後，可觀察到U87MG細胞存活率下降。另外，在低劑量紫杉醇前處理合併RGD胜肽處理之組別，參與細胞凋亡的半胱天蛋白酶(caspase) -3，-8，-9基因表現量明顯高於單一給予紫杉醇或RGD胜肽處理的組別。給予caspase抑制劑，觀察到U87MG細胞經由合併PTX及E[c(RGDyK)]2處理後的外在細胞凋亡路徑caspase-3，-8，-9被專一性抑制。 本研究發現低劑量紫杉醇的前處理可以促進人類多型性神經膠母細胞瘤U87MG細胞integrin-αvβ3表現，進而增加與RGD胜肽專一性鍵結，提高RGD胜肽小分子標靶藥物促進U87MG細胞凋亡之成效。
並列摘要
According to the latest World Health Organization report, the incidence of brain tumor constitutes 1.8% of all cancer cases lower than other cancers (e.g., colorectal cancer, 9.2%; breast cancer, 25.2%); however, the final mortality close to 100% is firmly in the top 10 cancers. Currently, the standard therapeutic strategies for GBM include palliative care, radiation therapy, and surgery in combination with anti-cancer drugs. However, this malignant brain tumor is well known for its highly invasive behavior and typically responds poorly to conventional cytotoxic therapy. Integrins are a family of transmembrane adhesion proteins that mediate cell adhesion and intracellular signaling. Integrin-αvβ3 is expressed on the surface of human glioblastoma multiforme (GBM) cells, and can be further induced by chemical or bio-mechanical stress. As a primary receptor of extracellular matrix adhesion molecules, integrin-αvβ3 acts as a crucial transducer to regulate cell signaling to die. The Arg-Gly-Asp (RGD) motif-containing peptides are specifically bound to integrin-αvβ3, and potential to inhibit neovasculature underlying competition to normal extracellular matrix proteins. This study employed two types of RGD peptides, cyclic RGD (c(RGDyK)) and bi-cyclic RGD (E[c(RGDyK)]2), to human GBM U87MG cells with the combination of low-dose Paclitaxel (PTX) pre-treatment to examine augmentation of therapeutic activity for RGD peptide-induced apoptosis. The docking simulation represented that both c(RGDyK) peptide and E[c(RGDyK)]2 peptide had better avidity and specificity to integrin-αvβ3 attachment (Ligscore 296.04 vs. 245.89). Human GBM U87MG cells were treated with RGD peptides in the absence or presence of initial exposure to low-dose 10 nM PTX. Results showed that integrin-αvβ3 expressing on the surface of U87MG cells was induced by 10 nM PTX pre-treatment for 12 hrs. Additionally, the U87MG cells pretreated with PTX and followed by RGD peptides exhibited greater expression of caspases-3, -8 and -9 genes than those merely treated with single agent of PTX or RGD peptide. Furthermore, the caspase-3, -8 and -9 inhibitor presented significant protection against E[c(RGDyK)]2 peptide induced U87MG programmed cell death. The increased expression of PTX-induced integrin-αvβ3 was correlated with the enhanced apoptosis in U87MG cells. This study proposes a novel method of targeting integrin-αvβ3 with RGD peptides in combination with low-dose PTX pre-treatment to improve the efficiency of human GBM treatment.