以A549肺癌細胞株為模式篩選對非小細胞肺癌有治療潛力之中藥複方

Abstract

肺癌長期以來都是全世界致死率最高的癌症疾病。根據型態上和病理上的特徵分類之結果，絕大多數的肺癌病例屬於非小細胞肺癌。雖然現今已經發展出許多針對癌症的治療方法，卻依然沒有可以確實治癒癌症的方法。我們利用非小細胞肺癌細胞株A549為模式，篩檢十五種臨床上常用的中藥複方，期能找出能有效治療肺癌的替代性藥物。在細胞毒性的初步篩檢中，我們挑出三種可以有效抑制A549細胞生長卻不影響正常肺部組織細胞MRC5的中藥複方，分別是三號、五號及十四號藥方。在進一步的分析中發現，此三種複方的處理會造成A549細胞週期停滯進而抑制細胞的生長，但不會顯著地造成細胞死亡，同時我們也發現在經由三種複方的處理後，cyclin D3、cyclin B1、CDK4和CDK6這些細胞週期調控蛋白的表現，有顯著下降的現象，得到與細胞週期停滯現象一致之結果。此外，我們也發現三號及五號複方能有效地減少具有癌幹細胞特性的側群細胞(side population)的比率，而這個現象似乎與間葉表皮轉換(Mesenchymal to epithelial transition)的機制無關。我們也試著藉由檢測血管內皮新生因子A (VEGF-A)表現量去應證這些在中醫理論中具有活血化瘀效用的藥方是否對於癌症細胞分泌促血管增生因子的行為有調節的效用。本實驗的結果提供了分子生物學上的證據去解釋這些複方在臨床上可能的治病機轉以及中醫理論在分子生物學上的意義，更重要的是，我們發現三個中藥複方具有可以被用來抑制非小細胞肺癌細胞之潛力。Lung cancer has long been one of the most deadly diseases all over the world. According to the morphological and pathological classification, the majority of lung cancers are belonged to non-small cell lung cancer (NSCLC). Although there are a lot of therapeutic approaches toward cancers, none of the treatments can substantially cure patients suffering from cancers till now. Here we use non-small cell lung carcinoma cell line A549 to screen 15 different traditional Chinese herbal medicine (CHM) formulae to explore the possibility of alternative medicine. In this study, we identified three potential formulae could substantially decreased the survival of A549 cells and not affect normal lung tissue cells MRC5. Flow cytometry analysis showed that treatment of the three formulae induced cell cycle arrest in G1 (formulae 3, 5, and 14) and G2 phase (only formula 14) without causing significant cell death. These results were also confirmed by western blot analysis showed decreased expression of G1/S and G2/M promoting cell cycle machinery including cyclin D3, cyclin B1, CDK4, and CDK6. In addition, formulae 3 and 5 were also demonstrated to be able to reduce the stem-like cancer cells side population (SP) and the mesenchymal to epithelial transition (MET) process is not involved in the diminishing of SP cells. We also analyzed the expression of VEGF-A to examine whether the angiogenic effect of A549 cancer cells would be influenced by these CHM formulae. This study provides direct evidences to explain the possible formula working mechanism toward patients and also gives molecular biological evidences demonstrated the perspective of TCM theory. Most importantly, we identified three potential formulae which could be used in NSCLC diseases.