- 學位論文
探討靈芝三萜類T-612之抗癌分子機制
Anticancer Molecular Mechanism of Ganoderma Triterpene T-612
摘要
靈芝是傳統中草藥之一，先前研究發現，靈芝三萜類T-612是一個具有抗癌活性的小分子，但詳細作用機制尚未清楚。拓樸異構酶II (Topoisomerase, Topo II)為解開DNA超螺旋結構的酵素，在DNA複製、轉錄及基因重組時扮演重要角色，許多化療藥物能夠藉由抑制Topo II而達到抑制癌細胞生長的效果。乳癌是全球女性罹患率極高的癌症，在臨床上，雌性素、黃體素受體以及HER2受體均為陰性的三陰性乳癌 (triple negative breast cancer, TNBC)為較惡性、治療效果有限及復發率高的乳癌亞型，在臨床上只能使用化療藥物治療，因此發展治療TNBC的藥物是乳癌治療上重要的課題之一。本篇研究目的是探討T-612抑制Topo II活性的機制與T-612對於TNBC細胞株MDA-MB-231之抗癌應用。In vitro試管實驗結果顯示，T-612能有效抑制Topo II活性 (IC50: 1.2 μM)，且抑制的方式為不可逆的，另外，將T-612與Topo II預先反應之後發現，Topo II的活性更顯著受到抑制，推斷T-612能透過直接與Topo II結合而抑制Topo II活性；在in vitro 及in vivo的系統中，T-612皆能造成DNA-Topo II complex累積。T-612處理能夠造成細胞中DNA斷裂、DNA損傷相關基因表現量上升、細胞週期停滯於G2/M時期與細胞凋亡發生，顯示T-612不論是在in vitro或是in vivo實驗，皆有Topo II poison的特性。此外，T-612處理也可造成細胞內活性氧化物 (ROS)大量累積、引發細胞自噬。與乳癌常見化療藥物Doxorubicin合併處理後，T-612可增強Doxorubicin所造成的細胞毒性，細胞中DNA-Topo II complex與ROS累積量增加、細胞週期停滯現象更加顯著，並誘發細胞凋亡。綜合以上結果得知，T-612造成DNA-Topo II complex累積，為一個Topo II poison的藥物；與Doxorubicin合併使用，能藉由增加DNA斷裂與細胞週期停滯，進而造成細胞凋亡，顯示T-612能夠有效增強Doxorubicin的抗癌效果，具有作為Doxorubucin治療TNBC佐劑之潛力。
並列摘要
Ganoderma (Lingzhi) has been a notable Chinese traditional medicinal herb for centuries. T-612, a triterpene from Ganoderma tsugae, has been previously found to possess anticancer activity, but the mechanism remains unclear. Breast cancer is the most life threatening malignant disease in women worldwide. In clinical, the breast cancer cells which do not overexpress estrogen receptor, progesterone receptor, and HER-2 genes, called triple negative breast cancer (TNBC) subtype, should be treated by chemotherapy. In this study, we studied anticancer activity of T-612 in MDA-MB-231 cells (a TNBC cell line), and characterized its inhibitory activity on topoisomerase II (Topo II), that is an enzyme essential for DNA replication and transcription, and especially crucial for cancer cell proliferation. T-612 was found to irreversibly inhibit Topo II activity with an IC50 of 1.2 μM. Both in vitro and in vivo, T-612 treatment caused Topo II poison, accumulation of the Topo II-DNA complex. In addition, T-612 caused DNA breaks, G2/M arrest, DNA damage-related gene expression and apoptosis, which are responses caused by Topo II poison. T-612 also caused reactive oxygen species (ROS) accumulation and induced autophagy. We further investigated whether T-612 is able to enhance the efficacy of Doxorubicin (another Topo II inhibitor) through further Topo II inhibition. The combination of T-612 and Doxorubicin enhanced DNA-Topo II complex formation and ROS accumulation in MDA-MB-231 cells. This combined treatment displayed synergistic cytotoxicity through higher level of G2/M arrest and apoptosis compared to Doxorubicin alone. The synergistic effect of T-612 and Doxorubicin in MDA-MB-231 cells indicated that the T-612 might be a potential anticancer adjuvant for treatment of TNBC.