| 摘要: | 近年來，乳癌的發生率及死亡率逐年攀升，高居女性十大癌症發生率第一位，死亡率則排名第四位。放射線治療是乳癌綜合治療中不可或缺之治療方式；但是，腫瘤細胞對放射線產生之抗性的確為乳癌治療失敗最常見之原因。傳統的輻射增敏劑因有較大的副作用，故限制其臨床應用。因此，本實驗希望能從中草藥中尋找高效低毒的輻射增敏劑，以期提高乳癌放射線治療的效果。
本實驗之目的係探討放射線配合中藥五味子萃取物對MCF-7乳癌細胞株之輻射增敏的效果及作用機轉。實驗材料與方法以MTT評估五味子萃取物之毒性，以細胞群落生成探討MCF-7細胞存活曲線，利用流式細胞儀之PI染色法、Annexin V-FITC/PI雙染法、粒線體膜電位變化等進行細胞凋亡分析，以及Hoechst 33258細胞核染色檢視細胞凋亡後期之DNA斷裂，再以西方墨點法檢測AIF及凋亡相關蛋白之表現。
本實驗結果發現五味子萃取物對MCF-7之抑制率與濃度及作用時間呈正相關性。放射線併用五味子萃取物對MCF-7細胞群落生成比例均有減少的趨勢，表示五味子萃取物具輻射增敏性而達到抑制腫瘤生長之效果。此外，放射線併用五味子萃取物誘導MCF-7停滯在G2/M期。JC-1染色發現放射線併用五味子萃取物降低粒線體膜電位（ΔΨm），經由Annexin V-FITC/PI 雙染法得知放射線併用五味子萃取物之apoptotic cells亦明顯增加，且以Hoechst螢光染色偵測DNA斷裂，而五味子萃取物會增加放射線所造成的凋亡蛋白Bcl-2與Cytosolic AIF的表現，並調降抗凋亡蛋白Bcl-2及Mitochondrial AIF的表現。此均證實MCF-7細胞走向凋亡。
本實驗結論，中藥五味子萃取物對MCF-7細胞株具增強「輻射誘導細胞凋亡路徑（Radiation-induced apoptotic pathway）」之作用，具有進一步研究與乳癌輻射增敏劑開發臨床應用之潛力。
Purpose:
We investigate the radiosensitive mechanism of Schisandra chinensis induced apoptosis in MCF-7 cell line, hormone sensitive breast cancer cells with caspase 3-deficiency.
Materials & Methods:
In our study, the crude sample was extracted from Schisandra chinensis (Trucz.) Baill seed in a serial process. The cell viability of human breast cancer MCF-7 cells was determined by MTT colorimetric assay. We used clonogenic assay to count cell survival fraction of MCF-7 cells after treating S. chinensis with radiation (RT) at different condition. Moreover, we analyzed MCF-7 cell-cycle distribution, mitochondrial membrane protential (ΔΨm), and proportion of apoptosis by using flow cytometry. DNA damage of MCF-7 cells was detected by Hoechst 33258 nucleus staining. Finally, we measured expression of apoptosis-related proteins, such as Bax, Bcl-2, and AIF, by using western blotting assay.
Results:
The MCF-7 survival curve decent was proportional to doses of extract of S. chinensis, and IC50 were 196.33 μg/ml for 24 h, 118.63 μg/ml for 48 h and 57.13 μg/ml for 72 h, respectively. The clonogenic assay of MCF-7 cells treated with RT combining S. chinensis showed that survival curve was more radiosensitive than RT alone. The cell-cycle arrest in G2/M phase in RT 15Gy and S. chinensis 40 μg/ml was 48.36% ± 3.11 vs. 42.24 ± 4.43% in RT 15Gy alone (p < 0.05). Loss of ΔΨm were 23.23 ± 3.90% and 27.15 ± 8.96% in RT combining S. chinensis 20 μg/ml and 40 μg/ml, respectively, compared with 15.08 ± 3.71% in RT 15Gy alone. The percentage of apoptotic cells evaluated by the AnnexinV/PI assay were 27.10 ± 0.53% and 25.30 ± 1.60% in RT combining S. chinensis 20 μg/ml and 40 μg/ml, respectively, vs. 19.83 ± 0.8% in RT 15 Gy alone (p < 0.01). Loss of ΔΨm were 23.23 ± 3.90% and 27.15 ± 8.96% in RT combining S. chinensis 20 μg/ml and 40 μg/ml, respectively, compared with 15.08 ± 3.71% in RT 15Gy alone. The indices of DNA staining detected by Hoechst 33258 nuclear stain calculated 28/250 in IR alone vs. 53/250 in RT and S. chinensis (p < 0.01). The expression of apoptosis-related proteins showed that Bax and cytosolic AIF up-regulated, while Bcl-2 and mitochondrial AIF down-regulated in RT combining S. chinensis.
Conclusion:
The radiosensitization of extract of Schisandra chinensis may relate with cell apoptosis through cell cycle arrest at G2/M phase, depolarization of ΔΨm, and regulation of Bax, Bcl-2 and caspase-independent AIF pathway in MCF-7 human breast cancer cells. |
