| 摘要: | 桑黃 (Phellinus linteus) 為東方人(尤其是中國、日本、南韓)的傳統中藥，目前已有許多相關的研究證實其在抗菌、抗癌、抗纖維化、抗氧化及抗發炎等方面具有療效。而在桑黃所含的多種活性成分之中，主要的成分 Hispolon 本身則具有相當強的抗氧化活性。
血管新生是癌症與許多種人類疾病快速發展所必需要關鍵的一個步驟，若是藥物能夠減緩血管新生的過程則也就能夠減輕疾病的嚴重程度。然而， Hispolon 對於血管新生是否具有抑制的能力或是直接對於前列腺癌細胞 PC-3 是否具有誘發凋亡的能力，目前並無相關文獻研究報導，因此本篇論文旨在希望能夠探討並了解其相關的機制。首先利用 MTT 試驗發現 Hispolon 對於抑制前列腺癌細胞 PC-3 的存活率具有劑量以及時間的依存效應，然後進一步利用 DAPI 染色法、 DNA 片斷化電泳分析以及 Annexin V-FITC/PI 螢光染色分析，證實了 Hispolon 的確能夠誘導 PC-3 走向凋亡而減少其存活率。另外也利用流式細胞儀分析，觀察 Hispolon 對細胞週期的影響，發現不同濃度的 Hispolon 也會使腫瘤細胞週期停頓在 G2 phase 。
而在研究血管新生試驗方面，由體外細胞實驗發現 Hispolon 能夠有效的抑制人類臍靜脈內皮細胞的轉移、侵襲及類血管管柱分化。另外在利用共培養系統研究後也發現 Hispolon 能夠抑制 HUVEC 經由人類腦部神經膠質瘤細胞 U87MG 所誘導的類血管管柱分化以及減緩由 VEGF 或是 MMP 所促進的 HUVEC 的轉移以及侵襲能力。而在動物離體試驗 (ex vivo and in vivo) 方面，利用大鼠主動脈環試驗、雞胚胎尿囊膜試驗、以及 Matrigel plug 試驗證實了 Hispolon 在動物活體實驗中的血管新生情形也具有抑制的能力。實驗最後，利用西方墨點法探討 TIMP-1 , PAI-1 , PI3K , AKT , P44/42 , phospho-P44/42 , RAS ,NF-κB 和 COX-2 等與血管內皮細胞生長因子的相關的訊息傳導蛋白表現。
綜合以上的實驗結果，本篇論文認為 Hispolon 能夠透過促使前列腺癌細胞凋亡而降低其存活率，並且也能經由血管內皮細胞生長因子的訊息傳導路徑來降低腫瘤誘導的血管新生，因此， Hispolon 應具有可開發成為抗血管新生藥物的潛力，期望未來能夠將其真正開發為藥物，為對抗癌症貢獻一份力量。
Phellinus linteus (PL) , is a traditional medicinal plant of oriental people (especially in China, Japan,and North Korea ) , was demonstrated to exhibit anti-bacterial , anti-tumour , anti-fibrotic , anti-oxidant and anti-inflammation functions in several studies. Hispolon is a major component of PL with great antioxidant activity.
Whether Hispolon induces prostate tumor cell apoptosis or inhibits angiogenesis,which is crucial for cancer and other human diseases, remains unknown. First, we investigated how hispolon induce apoptosis in the prostate tumor cell lines PC-3. In the MTT assay, treatment of Hispolon inhibited the growth of PC-3 in the dose- and time- dependent manners. Next ,Hispolon inducing PC-3 cells apoptosis was confirmed by nuclear condensation (DAPI staining), DNA fragmentation assay and Annexin V-FITC/PI staining . We also examined the Hispolon induced cell cycle arrest in the G2 phase by flow cytometric analysis.
In vitro angiogenesis study, we found that Hispolon effectively inhibited human umbilical vein endothelial cell migration, invasion, and tube formation. In a coculture study, Hispolon completely prevented U87MG cell–mediated capillary formation of HUVEC. This inhibitor also prevented that VEGF and MMP induced migration ability of HUVEC when cultured alone or cocultured with U87MG cells. In vivo angiogenesis study, we use the rat aortic ring assay, chicken chorioallantoic membrane assay (CAM) and Matrigel plug assay to evaluate anti-angiogenic effects. Finally, Western blot showed that Hispolon treatment suppressed the protein expression of TIMP-1 , PAI-1 , PI3K , AKT , P44/42 , phospho-P44/42 , RAS , NF-κB and COX-2, which were involved in endothelial cell survival, proliferation, migration and angiogeneis.
Our results indicated that Hispolon exerted an anti-tumor activity associated with decreased proliferation of tumor cells and induced the apoptosis of prostate tumor cell, and inhibited the anti-angiogenic activity by VEGF signaling pathway . |
