探討小分子化合物DRH-9增強化療藥物Vinorelbine在人類非小細胞肺癌之抗癌機轉研究

Abstract

肺癌不但是全球最常被診斷出的惡性腫瘤類型，更是造成癌症致死率高居不下的主要原因之一，而大多數肺癌患者都屬於非小細胞肺癌(non-small cell lung cancer, NSCLC)。因此，開發能被應用於治療非小細胞肺癌的治療策略極為重要。化學治療是臨床上最常用的療法之一，即便目前有多種化療藥物，但是晚期非小細胞肺癌患者的五年生存率仍低於10%。此外，化學治療劑的毒性和副作用限制了化學藥品的使用。因此，合併化學治療是一種可以降低化學治療藥物的劑量及毒性並同時增加其抗癌活性的潛力療法。從我們合作團隊合成的一系列新化合物進行篩選後，我們發現化合物DRH-9對於PDE5抑制活性降低許多，並且在與臨床上用於轉移性非小細胞肺癌患者的一線化學藥物溫諾平(vinorelbine)合併使用時，擁有很高的增敏活性。有趣的是，DRH-9本身不具有強烈細胞毒性，因此，並不會影響細胞存活，卻在sulforhodamine B試驗中顯著地增強溫諾平對非小細胞肺癌細胞株NCI-H460的抗癌作用，大幅將抑制百分之五十癌細胞生長所需的藥物濃度(GI50)從5.16±0.31 nM降低至2.41±0.11 nM。而以流式細胞儀分析的CFSE染色試驗和集落形成試驗結果均反映DRH-9和溫諾平合併療法對癌細胞增殖的強烈抑制作用。此外，細胞週期分佈試驗和細胞死亡酵素免疫分析試驗的結果皆顯示DRH-9顯著地加強溫諾平誘導的細胞凋亡。雖然DRH-9與微管蛋白並無直接相互作用關係，但DRH-9增強溫諾平使微管不穩定的效果，並促進異常紡錘體之有絲分裂細胞生成。除此之外，DRH-9和溫諾平的合併療法顯著地增加停滯於有絲分裂期的細胞族群以及相關蛋白質的表現量，包括細胞週期蛋白B1 (cyclin B1)、有絲分裂相關蛋白磷酸化以及Bcl-2磷酸化。也因而進一步活化下游的內源性細胞凋亡途徑，並最終導致更多的癌細胞走向凋亡。總結來說，此研究數據顯示DRH-9極具有潛力成為是一種有低PDE5抑制活性，並且對治療非小細胞肺癌之溫諾平誘導的抗癌作用具有高增敏能力的化學增敏劑。

Carcinoma of lung is the most frequently diagnosed malignancy and the leading cause of cancer mortality worldwide, and most patients are categorized as non-small cell lung cancer (NSCLC). Chemotherapy, one of the most commonly used therapies, has shown good clinical outcome, but the 5-year survival rate of late-stage patients is still lower than 10%. Besides, the cytotoxicity and side effects have limited extensive use of chemotherapeutic agents. Therefore, combination therapy can be a potential approach to reduce the doses and toxicity of chemotherapeutic drugs while sensitizing anticancer activities. Among a number of new compounds synthesized by our colleagues, DRH-9 showed lower PDE5 inhibition and the highest sensitizing activities when combined with vinorelbine, a chemotherapeutic drug for metastatic NSCLC patients. Interestingly, DRH-9 alone did not affect cell survival, but profoundly potentiated vinorelbine-induced anticancer effects in sulforhodamine B assay with decreasing GI50 from 5.16±0.31 nM to 2.41±0.11 nM against NSCLC cell line NCI-H460. The synergistic effect was further substantiated by using both colony formation assay and flow cytometric analysis of CFSE staining. Moreover, the results of cell cycle distribution analysis detected by flow cytometry and apoptosis assay detected using a cell death ELISA kit showed that DRH-9 dramatically sensitized vinorelbine-induced apoptotic cell death. Although DRH-9 did not directly interact with tubulins, it amplified vinorelbine’s effect on microtubule destabilization and exaggerated the formation of mitotic cells with abnormal spindles. Furthermore, the combinatory treatment of DRH-9 and vinorelbine significantly increased the cell populations arrested in mitotic phase and the expression levels of related proteins, including cyclin B1, mitosis-related protein phosphorylation and Bcl-2 phosphorylation, which activated downstream intrinsic apoptotic pathways and eventually led to much more apoptotic cells. In conclusion, the data suggest that DRH-9 is a potential chemosensitizer displaying low PDE5 inhibiting activity but high sensitization capability on vinorelbine-induced anticancer effect against NSCLC.