由本研究結果得知，台灣山豆根根部粗抽物（EFR）對人類肝癌細胞（Hep3B）
的抗癌分子機轉有兩個方向，一是EFR 會誘導 Hep3B 細胞細胞週期停滯在 S 期 並使得細胞凋亡。其主要機轉是抑制細胞週期調控分子cyclin A、cyclin E、CDK2 及 cyclin A、cyclin B1、CDK1 的蛋白表現，使得細胞週期停滯在 S 期；另一方 面也活化了Fas/ Fas L 進而促使 caspase-8 的活化，並啟動 caspase-3 與 Bid 的裂 解更影響了粒線體膜電位的下降，造成cytochrome c 的釋出、ROS 的產生；也 因內質網釋出鈣離子並使得GADD153 增加，caspase-12 的活化進而造成細胞的 凋亡。其主要的週期停滯與細胞凋亡的路徑如（Figure 54）。
另外，在抑制癌細胞轉移的部分，在有關的致癌發生（癌化作用）到生長因 子受體中最重要的路徑有PI3K （phosphatidylinositol 3-kinase）/Akt 和 Ras/MAPK
（Mitogen -activated protein kinase） pathways，這在細胞增生（cell proliferation）、 分化（differentiation）、轉型（transformation）及存活上扮演重要的角色。由實驗 結果得知會經由抑制 protein kinase C（PKC）的活性進而抑制下游的 MAPK pathway 訊 息 傳 遞 路 徑 之 蛋 白 的 活 化 ， 更 而 抑 制 金 屬 蛋 白 酶 -9 （ matrix metalloproteinases-9；MMP-9）以及 MMP-1，-2，-10 的表現。另外會抑制一些 轉錄因子（transcription factor）像是 NF-κB 和 AP-1 的表現以及抑制像是細胞貼 附分子、趨化物質（chemokine）、TNF、COX-2 及 MMP-9 基因的表現。綜合各 個結果得知細胞因藥物影響而降低血管新生、細胞移動、侵襲之能力。並以西方 墨點法（Western Blot），分析細胞轉移機轉的相關蛋白分子影響。最後以（Figure 55）預估 EFR 對 Hep3B 細胞抑制轉移機轉的表現。
許多的中草藥成分中同時具有治療以及預防癌症之功能，而 EFR 在體外試 驗中可以抑制癌細胞的增生，造成細胞凋亡及抑制癌細胞的轉移，若依此分子檢 測模型，了解藥物造成癌細胞的蛋白毒性之相關機制，不僅未來可針對開發台灣 固有種植物成為新藥物的研發，尚可提供抗癌藥物治療癌症之另一可行方向。
B A
圖十六、台灣山豆根之生藥組織圖
Figure 17. The viable cells were determined by flow cytometry ; Each point is mean ± SD of three experiments. *p <0.05.
Figure 18. The apoptotic cells were determined by flow cytometry ; Each point is mean ± SD of three experiments. *p <0.05.
Figure 19. Morphology change of Hep3B cells after treatment with different concentrations of EFR（0, 50, 75, 100, 125, 150 μg/ml）for 24 h. The cells were photographed under a phase contrast microscope 200X.
Figure 20. Morphology change of Hep3B cells after treatment with different concentration of EFR（0, 50, 75, 100, 125, 150 μg/ml）for 48 h. The cells were photographed under a phase contrast microscope 200X.
Figure 21. Morphology change of Hep3B cells after treatment with different concentration of EFR（0, 50, 75, 100, 125, 150 μg/ml）for 72 h. The cells were photographed under a phase contrast microscope 200X.
Figure 23. The effects of EFR on cell cycle and apoptosis in human hepatocellular carcinoma (Hep3B) cells. Panel A: representative profile after 24 h incubation ; panel B: percentage of cells in different phases after 24 h incubation ; panel C: percentage of apoptotic cells. Data represents mean ± SD of three experiments. *p<0.05
B
A
C
Figure 24. The effects of EFR on cell cycle and apoptosis in human hepatocellular carcinoma (Hep3B) cells. Panel A: representative profile after 48 h incubation;
B C
A
Figure 25. The effects of EFR on cell cycle and apoptosis in human hepatocellular carcinoma (Hep3B) cells. Panel A: representative profile after 72 h incubation; panel B: percentage of cells in different phases after 72 h incubation ; panel C: percentage of apoptotic cells. Data represents mean ± SD of three experiments. *p<0.05
B C
A
Figure 26. The Hep3B cells were incubated with various concentrations of EFR for 24 h and apoptosis were determined by DAPI staining and fluorescence microscopy as described in Materials and Methods.
Figure 28. The Hep3B cells were incubated with various concentrations of EFR for 24 h and apoptosis were determined by Comet assay and fluorescence microscopy as described in Materials and Methods.
Figure 29. DNA fragmentation of Hep3B cells treated with EFR examined by DNA gel electrophoresis. Panel A: The Hep3B cells were incubated with various concentrations of EFR for 48 h or Panel B: treated with 125 μg/ml of EFR for various times. DNA was extracted before undergoing DNA gel electrophoresis and then photographed by fluorescence microscopy as described in Materials and Methods.
Figure 30. Flow cytometric analysis of ROS in human Hep3B cells treated with EFR for 2 hours. The percentage of cells that were stained by DCFH-DA dye for ROS.
*differences between EFR and control, p<0.05.
Figure 31. Human Hep3B cells ( 2x105 cells/ml ) were treated with 125 μg/ml of EFR ( control : 0 μg EFR ) for 0-24 hours to detect the changes of ROS. The percentage of cells that were stained by DCFH-DA dye for ROS. *differences between EFR and control, p<0.05.
Figure 32. Flow cytometric analysis of ΔΨm in human Hep3B cells treated with EFR for 24 hours. The percentage of cells that were stained by DiOC6 dye for ΔΨm.
*differences between EFR and control, p<0.05.
Figure 33. The Hep3B cells (2x105 cells/ml) were treated with 125 μg/ml of EFR (control: 0 μg EFR ) for 0-48 hours to detect the changes of ΔΨm.The percentage of cells that were stained by DiOC6 dye for ΔΨm. *differences between EFR and control, p<0.05.
Figure 34. Flow cytometric analysis of Ca2+ in human Hep3B cells treated with EFR for 24 hours. The percentage of cells that were stained by by Indo-1/AM dye for Ca2+.
*differences between EFR and control, p<0.05.
Figure 35. The Hep3B cells (2x105 cells/ml) were treated with 125 μg/ml of EFR (control: 0 μg EFR ) for 0-48 hours to detect the changes of Ca2+. the percentage of cells that were stained by Indo-1/AM dye for Ca2+. *differences between EFR and control, p<0.05.
Figure 36. Flow cytometric assays of the effects of EFR on caspase-3 activity. The
B
A
Figure 37. Effects of EFR on the levels of Cdc25a, cyclin A, cyclin B1, cyclinE, CDK1, CDK2, E2F-1, P-pRb, p21, p27 in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 38. Effects of EFR on the levels of Bad, Bcl-xl, Bcl-xs in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 39. Effects of EFR on the levels of cytochrome c, AIF, Endo G, cytosolic cytochrome c in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 40. Effects of EFR on the levels of caspase-3, 7, 9, PARP in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours
Figure 41. Effects of EFR on the levels of Fas, FasL, caspase-8, Bid in Hep3B cells.
The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 42. Effects of EFR on the levels of GRP78, GADD153, Caspase-12 in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 43. Effects of EFR on the invasion of Hep3B cells in vitro. Cells that penetrated through the matrigel to the lower surface of the filter were stained with crystal violet and shown under a light microscope at 200X.
Figure 44. Quantification of Hep3B cells in the lower chambers, which was done by counting at 200X. Columns, mean from three independent experiments. p＜0.01, significant difference between EFR-treated groups and the control as analyzed by
Figure 45. Effects of EFR on the migration of Hep3B cells in vitro. The filters with a reconstituted basement, type collagen were stained with 2% crystal violet, and Ⅰ migrated cells adherent to the underside of the filter were observed under a light microscope at 200X.
Figure 46. Quantification of Hep3B cells in the lower chambers, which was done by counting at 200X. Columns, mean from three independent experiments. p＜0.01, significant difference between EFR-treated groups and the control as analyzed by Student’s t-test.
Figure 47. In vitro wound closure. Confluent monolayers of cells were wounded with a scratch, rinsed to remove debris, and incubated in the absence or presence of EFR (50 and 75 μg/ml) as indicated for 24 and 48 hours. Photographs indicate relative wound closure as monitored by visual examination using a Nikon phase-contrast microscope. Fields shown are representative of the width of quadruplicate wounds made in triplicate cultures.
Figure 49. Immunofluorescence showing ROCK-1 localization. Hep3B cells were treated with or without EFR (125 μg/ml) and ROCK-1 localization was represented as green fluorescence. DAPI staining indicated nuclear localization.
Figure 50. Effects of EFR on the levels of JNK-p, c-jun, c-jun-p, PKC, Raf-1, Ras, SOS-1, GRB2, VEGF, HIF-1α in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 51. Effects of EFR on the levels of ERK1/2, JNK1/2, p38-p, p38, MEKK3, MKK7, PI3K, AKT Thr348, AKT Ser473 in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 53. Effects of EFR on the levels of MMP-1, -2, -9, -10, RhoA, ROCK-1 and FAK in Hep3B cells. The Hep3B cells (2×105/ml) were treated with 125 μg/ml EFR for 6, 12, 24 and 48 hours then cytosolic total protein was prepared and the individual protein levels were estimated by Western blotting.
Figure 54. Proposed model of EFR mechanism of action for apoptosis in Hep3B cells.
Figure 55. The proposed scheme of mechanism of EFR action on Hep3B cells.
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