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阿魏酸對人類肺癌細胞之抗腫瘤作用
                                                                                                                    Anti-tumor effect of ferulic acid on human lung cancer H1299 cells
                                                                                                                                                                                                           長榮大學生物科技學系
                                                                                                                                                                                                               陳宥安
                                                                                                                                                                                                        實習單位:高雄醫學大學生物科技系
                                                                                                                                                                                                           指導老師: 邱建智老師

                               Abstract
                                由過去研究發現,阿魏酸Ferulic acid (FA) 在藥理活性上具抗發炎、抑制血栓形成、抗自由基、抑制腫瘤細
                             胞生長及調節人體免疫功能的作用等等的活性。本論文主要以人類非小細胞肺癌細胞株作為研究對象,探討
                             FA引起H1299細胞生長及轉移的抑制能力,並藉由蛋白質表現的分析,探討其對肺癌細胞的作用機制。研究
                             結果顯示,FA轉移能力及抑制活性,與FA處理濃度和時間有關,在高濃度FA有明顯的生長抑制活性、轉移
                             能力也下降。利用PI的染色結果,FA能夠引起H1299細胞細胞週期停滯於G2/M期及誘導H1299細胞內ROS表
                             現下降,及不會導致細胞內粒線體膜電位下降。以明膠蛋白酵素電泳法 zymography assay進行肺癌細胞外培
                             養基的蛋白質分析,顯示FA處理後的 MMP-9及MMP-2 活性有明顯降低的趨勢。此外,以Migration assay及
                             Invasion assay進行FA對肺癌細胞作用的評估結果,也證實肺癌細胞在高濃度的轉移能力可被FA所抑制。綜合
                             上述結果,我們發現了FA萃取物與細胞轉移、移動能力有關,表示了天然植物所分離之物質FA在化學預防
___________________________________________________________________________
                             (chemoprevention)中有希望能成為癌症預防的新策略。

                             Experimental results                                                                                                                                                                                                                                                                                                                 FA




                                                                                                                                                                                                                      _________________________________
                                                                                                                                                                                                                      vehicle 0.03 0.06 0.15 0.3   0.6 (mM)
                                                                                                                                                                                                                                                                                                                                30


                                                                                                                                                                                                                                                                                                                                25
                                                                                                                                                                                                       Denuded zone




                                                                                                                                                                                                                                                                                                              Denude zone (%)
                                                                                                                                                                                                                                                                                                                                20
                                                                                                                                                                                                                                                                                                  0h
                                                                                                                                                                                                                                                                                                                                15


                                                                                                                                                                                                                                                                                                                                10

                                                                                                                                                                                                                                                                                                  14 h                          5


                                                                                                                                                                                                                                                                                                                                0
                                                                                                                                                                                                                                                                                                                                            con    0.03    0,06        0,15   0.3   0.6
                                                                                                                                                                                                                      Fig 4. NSCLC H1299 cells were incubated with a series of                                                                             FA dose (mM)

                                                                                                                                                                                                                      different concentration of FA for 14hr to observation cells
                                   Fig 1. Structure of Ferulic acid                                                                                                                                                   migration.

                                                                                                                                                                                                                        Vehicle       0.03         0.06                                    120
                                                                                                                                                                                                                                                                                                                                                   cell number

                                                             140                                                                                                                                                                                                                           100
                                                                                                                                                             24 hr
                                                                                                                                                                                                                                                                      invasion index (%)




                                                                                                                                                             48 hr
                                                             120                                                                                                                                                                                                                            80
                                    viable cell number (%)




                                                             100                                                                                                                                                                                                                            60

                                                              80                                                                                                                                                                                                                            40

                                                              60                                                                                                                                                                                                                            20

                                                              40                                                                                                                                                                                                                            0

                                                                                                                                                                                                                        0.15           0.3         0.6      (mM)
                                                                                                                                                                                                                                                                                                 con   0.03       0.06               0.15    0.3     0.6
                                                              20                                                                                                                                                                                                                  FA Dose (mM)

                                                                   0                                                                                                                                                  Fig 5. NSCLC H1299 cells were incubated with a series of different concentration
                                                                              Control 0.03          0.06     0.15                  0.3                 0.6
                                                                                               FA Dose (mM)
                                                                                                                                                                                                                      of FA for 16hr to observation cells invasion.
                                   Fig 2. NSCLC H1299 cells were incubated with a                                                                                                                                                                                                                                                                                      (mM)
                                   series of different concentration of FA for 24 and                                                                                                                                     Con            0.03          0.06          0.15                                         0.3                                       0.6
                                   48 hr to observation cell viability.

                                    _________________                                         FA
                                                              0           0.03 0.06 0.15 0.3 0.6 (mM)
                                                                                                                                                                                MMP-2
                                                                                                                                                                                MMP-9

                             120                                                                                                                 120


                             100                                                                                                                 100                                                                    Fig 6. NSCLC H1299 cells were incubated with a series of different concentration
                                                                                                                    Fold relative activity (%)
Flod relative activity (%)




                             80                                                                                                                  80
                                                                                                                                                                                                                        of FA for 16hr to observation cell cycle distribution through flow cytometric
                             60                                                                                                                  60
                                                                                                                                                                                                                        analysis.
                             40                                                                                                                  40


                             20


                              0
                                                                                                                                                 20


                                                                                                                                                  0
                                                                                                                                                                                                                       Discussion
                                                             con       0.03     0.06   0.15   0.3      0.6                                               con         0.03    0.06   0.15   0.3   0.6
                                                                               FA Dose (mM)                                                                                 FA Dose (mM)
                                                                                                                                                                                                                           1、The inhibitory effect of FA on migration of lung cancer cells,
                                   Fig 3. NSCLC H1299 cells were incubated with a                                                                                                                                            suggesting that FA could be a chemoprevention agent in the
                                   series of different concentration of FA for 24hr after                                                                                                                                    future。
                                   take supernatant to observation matrix metallopro-
                                   teinases (MMP-2 and MMP-9) activity.

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陳宥安

  • 1. 阿魏酸對人類肺癌細胞之抗腫瘤作用 Anti-tumor effect of ferulic acid on human lung cancer H1299 cells 長榮大學生物科技學系 陳宥安 實習單位:高雄醫學大學生物科技系 指導老師: 邱建智老師 Abstract 由過去研究發現,阿魏酸Ferulic acid (FA) 在藥理活性上具抗發炎、抑制血栓形成、抗自由基、抑制腫瘤細 胞生長及調節人體免疫功能的作用等等的活性。本論文主要以人類非小細胞肺癌細胞株作為研究對象,探討 FA引起H1299細胞生長及轉移的抑制能力,並藉由蛋白質表現的分析,探討其對肺癌細胞的作用機制。研究 結果顯示,FA轉移能力及抑制活性,與FA處理濃度和時間有關,在高濃度FA有明顯的生長抑制活性、轉移 能力也下降。利用PI的染色結果,FA能夠引起H1299細胞細胞週期停滯於G2/M期及誘導H1299細胞內ROS表 現下降,及不會導致細胞內粒線體膜電位下降。以明膠蛋白酵素電泳法 zymography assay進行肺癌細胞外培 養基的蛋白質分析,顯示FA處理後的 MMP-9及MMP-2 活性有明顯降低的趨勢。此外,以Migration assay及 Invasion assay進行FA對肺癌細胞作用的評估結果,也證實肺癌細胞在高濃度的轉移能力可被FA所抑制。綜合 上述結果,我們發現了FA萃取物與細胞轉移、移動能力有關,表示了天然植物所分離之物質FA在化學預防 ___________________________________________________________________________ (chemoprevention)中有希望能成為癌症預防的新策略。 Experimental results FA _________________________________ vehicle 0.03 0.06 0.15 0.3 0.6 (mM) 30 25 Denuded zone Denude zone (%) 20 0h 15 10 14 h 5 0 con 0.03 0,06 0,15 0.3 0.6 Fig 4. NSCLC H1299 cells were incubated with a series of FA dose (mM) different concentration of FA for 14hr to observation cells Fig 1. Structure of Ferulic acid migration. Vehicle 0.03 0.06 120 cell number 140 100 24 hr invasion index (%) 48 hr 120 80 viable cell number (%) 100 60 80 40 60 20 40 0 0.15 0.3 0.6 (mM) con 0.03 0.06 0.15 0.3 0.6 20 FA Dose (mM) 0 Fig 5. NSCLC H1299 cells were incubated with a series of different concentration Control 0.03 0.06 0.15 0.3 0.6 FA Dose (mM) of FA for 16hr to observation cells invasion. Fig 2. NSCLC H1299 cells were incubated with a (mM) series of different concentration of FA for 24 and Con 0.03 0.06 0.15 0.3 0.6 48 hr to observation cell viability. _________________ FA 0 0.03 0.06 0.15 0.3 0.6 (mM) MMP-2 MMP-9 120 120 100 100 Fig 6. NSCLC H1299 cells were incubated with a series of different concentration Fold relative activity (%) Flod relative activity (%) 80 80 of FA for 16hr to observation cell cycle distribution through flow cytometric 60 60 analysis. 40 40 20 0 20 0 Discussion con 0.03 0.06 0.15 0.3 0.6 con 0.03 0.06 0.15 0.3 0.6 FA Dose (mM) FA Dose (mM) 1、The inhibitory effect of FA on migration of lung cancer cells, Fig 3. NSCLC H1299 cells were incubated with a suggesting that FA could be a chemoprevention agent in the series of different concentration of FA for 24hr after future。 take supernatant to observation matrix metallopro- teinases (MMP-2 and MMP-9) activity.