| Abstract: | 研究目的: 過去研究證實宵楠植物樹皮萃取物具有抑癌作用，但其抗癌機轉不甚清楚。而防己根部萃取物具有利尿及減肥作用，然而卻引來腎衰竭及致癌的問題。因此本研究旨在探討宵楠萃取物與防己根部萃取物化學成份(馬兜鈴酸與粉防己鹼)在泌尿系統中的藥理作用。
材料及方法:本實驗採用宵楠的葉部萃取液(Florin leaf extracts)及防己根部萃取物化學成份:馬兜鈴酸(AA)與粉防己鹼(TET)為材料進行膀胱癌細胞株及正常腎臟細胞株及C3HeN小鼠實驗。在體外細胞株以MTT 試驗檢測細胞存活率、流式細胞儀進行細胞週期分析、免疫螢光分析細胞β-微管蛋白的表現、西方點墨法分析β-微管蛋白的表現、細胞調控週期蛋白激?及凋亡蛋白ADP核糖聚合?(PARP)與Bcl-2 及Bax 蛋白的表現及酵素免疫分析蛋白?-3、-8、-9的活性和Annexin V-FITC雙螢光染色、DAPI螢光染色及DNA片段電泳進行凋亡分析。在體內動物實驗則進行血液生化分析、H.E.組織病理染色及TUNEL凋亡分析。
結果: Florin 萃取物作用於膀胱癌細胞株試驗證實其具有抑制膀胱癌細胞株生長的效果(48小時，IC50為~9-17µg/ml)，且經由減少調控細胞週期的蛋白激?Cyclin B1、 Cdc2 激?及P-Cdc2 激?磷酸化而使細胞停滯在G2/M 期(>90%)。此外，亦發現可經由活化caspase-3、-8、-9而導致細胞凋亡。相對馬兜鈴酸與粉防己鹼對腎臟細胞株的影響，從MTT試驗、DAPI 染色、DNA片段電泳、Annexin V-FITC雙螢光染色及蛋白?-3活性分析，證實粉防己鹼具有較強的毒性促使腎臟細胞株凋亡。從動物實驗中亦證實，連續三個月腹腔注射不同濃度AA 和TET (分別為1, 10, 50 mg/kg)，發現AA 10mg/kg 造成小鼠腎毒性、血中的尿素氮、肌酸酐增加及增加腎小管損傷。相對的粉防己鹼TET須要50 mg/kg，才會造成腎臟遠端小管水腫樣變性。
結論與建議: 本研究結果證實Florin可促使膀胱癌細胞經由粒線體內在路徑與接受體外在路徑導致細胞凋亡，具有很好的抗癌潛力。而馬兜鈴酸(AA)的毒性遠超過於粉防己鹼(TET)，推測可能因為不同的血漿蛋白結合作用或是不同的代謝機轉導致，未來仍須進一步驗證。
Objective: Calocedrus formosana (Florin) bark acetone/ethylacetate extracts are known to exert an antitumor effect on some human cancer cell lines, but the mechanism is yet to be defined. In this research, we investigated anti-bladder cancer cell activity in the leaf methanol extracts of Florin and the mechanism by which the extracts exert such activity. We also assessed the nephrotoxicity of aristolochic acid (AA) and tetrandrine (TET), the major bioactive components in the root extracts of Aristolochiae fangchi and Stephaniae tetrandra which have been used as diuretics and slimming agents.
Materials and Methods: Florin leaf methanol extracts, and AA and TET were used for indentifying anti-bladder cancer activity and toxicity studies, respectively. Human bladder cancer cell lines, and normal renal cells (MDCK cells) and C3HeN mice were used in these studies. Methods included MTT test, flow cytometry, SDS-PAGE/Western blot, ELISA assay, immunofluorescence analyses, HE stain and TUNEL assay.
Results: Florin extracts inhibited growth of bladder cancer cells by inducing G2/M phase arrest, attenuating β-tubulin polymerization and causing apoptosis as determined by MTT assay, flow cytometry, immunofluorescence staining, Western blot analysis of apoptotic proteins, respectively. In the nephrotoxicity assessment, although TET was more potent than AA in inhibiting MDCK cell growth in vitro as determined by apoptosis assay, mice treated with AA (10 mg/kg) by i.p. for 3 months showed higher nephrotoxicity, elevated blood urea nitrogen and increased renal tubular injuries when compared with those in mice treated with TET.
Conclusions and suggestions: Florin extracts potently and specifically inhibit growth of human bladder cancer cells. AA but not TET exhibits a significant nephrotoxicity in mice under the experimental conditions. These suggest that Florin extracts may be an effective anti-bladder cancer agent and that the levels of AA in Chinese herbs should be an important concern for its nephrotoxicity. |
