第四章 結果
第三節 JA 所誘發的細胞自噬與 MEK/ERK 路徑的活化有關
成之外，還有 Atg12-Atg5-Atg16L、Beclin 1 及 Class III PI3K 會一起 幫助延展雙層膜的構造(Ravikumar et al., 2010)，而細胞內也有許多路 徑會調控細胞自噬的進行，除了促進細胞自噬進行的 Class III PI3K 路徑，另一條 Class I PI3K/Akt/mTOR 路徑被抑制也會促進細胞自噬 進行(Petiot, Ogier-Denis, Blommaart, Meijer, & Codogno, 2000)。為檢 測 JA 所誘發的細胞自噬是否與此兩條路徑相關，實驗使用 1 M 濃 度的 JA 處理 Hep 3B 細胞，培養不同時間（0、24、48、72、96 小時）
後，收取 Whole cell lysates，利用西方墨點法分析 JA 對於促自噬蛋 白 Atg12-Atg5、Beclin 1 及 Class III PI3K 的表現及抑制自噬蛋白 Class I PI3K/Akt/mTOR 的表現量。
實驗結果發現， Atg12-Atg5 複合物的表現量在各個時間點下並 無明顯增加或下降表現量，同時 Atg5 的蛋白質表現量也沒有因 JA 處 理而有差異（p > 0.05）（Fig. 14）。在 Beclin 1 及 Class III PI3K 蛋白 質表現量方面，由（Fig. 15）則發現在時間點 24 小時 Beclin 1 的表 現量有些微增加（p > 0.05），至 96 小時其蛋白質表現量隨作用時間 降低，表現量並無明顯差異，在 Class III PI3K 蛋白質表現量可發現
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也無明顯的變化，在量化的結果發現 Beclin 1 與 Class III PI3K 蛋白質 表現量有隨時間點而減少的趨勢（p > 0.05）。
在檢測 JA 所誘發的細胞自噬與抑制 Class I PI3K/Akt/mTOR 路徑 方面則發現，phospho-mTOR 蛋白質的表現量（Fig. 16）在 24 小時有 增加的情形，但隨時間點增加表現量則遞減（p > 0.05），到 96 小時 表現量與 24 小時相比則有明顯減少（p < 0.05），但與 0 小時的表現 量相比則沒有明顯差異（p > 0.05）；mTOR 的上游分子 Akt 表現量如
（Fig. 17）所示，phospho-Akt 在 24 小時的表現量與 0 小時的表現量 相比有明顯增加（p < 0.05），其表現量隨作用時間增加有下降的趨勢，
但與 0 小時相比其表現量依然較多（p > 0.05），Class I PI3K/Akt/mTOR 路徑的上游分子 Class I PI3K，其活化型態的表現量與作用時間 0 小 時相比，24 至 96 小時的表現量較 0 小時多但無明顯變化（p > 0.05）
（Fig. 18）。
之前的研究也發現 Class I PI3K 的上游分子 Ras 蛋白不僅為 Class I PI3K 的上游分子也是 Raf/MEK/ERK 的上游分子(Peyssonnaux, Provot, Felder-Schmittbuhl, Calothy, & Eychene, 2000)，Raf/MEK/ERK 路徑與 Class I PI3K/Akt/mTOR 路徑作用相反，活化此路徑能促進細 胞自噬的進行。實驗結果發現，上游分子 Ras 蛋白質表現量在作用 0-72 小時之間隨時間點增加而上升（Fig. 19），到 72 小時的表現量明
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顯較 0 小時多（p < 0.05），但到 96 小時卻明顯降低（p < 0.05），下游 分子 MEK 及 ERK 的活化型態表現（Fig. 20、21），在作用時間點 0-72 小時，表現量隨時間增加而上升，在 72 小時的表現量明顯較 0 小時 多（p < 0.05），到 96 小時 MEK 的表現量降低（p < 0.05）。推論與 JA 誘發細胞自噬的相關路徑與 MEK/ERK 路徑的活化有關，與 Class I PI3K/Akt/mTOR 路徑被抑制或 Class III PI3K/Beclin 1 路徑活化無 關。
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Fig. 14 Expression of Atg12-Atg5 and Atg5 in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-Atg5 antibodies. -actin antibody was used as an internal control. Quantification of Atg12-Atg5 and Atg5 protein expressions from three independent experiments are presented as means ± SEM. Means without a common letter differ, p < 0.05.
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Fig. 15 Expression of Beclin 1and Class III PI3K in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-Beclin 1 and anti-Class III PI3K
antibodies. -actin antibody was used as an internal control.
Quantification of Beclin 1and Class III PI3K protein expressions from three independent experiments are presented as means ± SEM. Means without a common letter differ, p < 0.05.
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Fig. 16 Expression of total mTOR and phospho-mTOR in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-mTOR and anti-phoshpo-mTOR
antibodies. -actin antibody was used as an internal control.
Quantification of phospho-mTOR protein expressions from three
independent experiments are presented as means ± SEM. Means without a common letter differ, p < 0.05.
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Fig. 17 Expression of total Akt and phospho-Akt in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-Akt and anti-phoshpo-Akt antibodies.
-actin antibody was used as an internal control. Quantification of
phospho-Akt protein expressions from three independent experiments are presented as means ± SEM. Means without a common letter differ, p <
0.05.
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Fig. 18 Expression of total class I PI3K and phospho-class I PI3K in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-class I PI3K and anti-phoshpo-class I PI3K antibodies. -actin antibody was used as an internal control.
Quantification of phospho-class I PI3K protein expressions from three independent experiments are presented as means ± SEM. Means without a common letter differ, p < 0.05.
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Fig. 19 Expression of pan-Ras in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-pan-Ras antibodies. -actin antibody was used as an internal control. Quantification of pan-Ras protein expressions from three independent experiments are presented as means ± SEM.
Means without a common letter differ, p < 0.05.
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Fig. 20 Expression of total MEK and phospho-MEK in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-MEK and anti-phoshpo-MEK antibodies.
-actin antibody was used as an internal control. Quantification of
phospho-MEK protein expressions from three independent experiments are presented as means ± SEM. Means without a common letter differ, p
< 0.05.
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Fig. 21 Expression of total ERK and phospho-ERK in JA-treated Hep 3B cells.
Hep 3B cells (1 × 106 cells/dish) were treated with or without 1 M JA for 0, 24, 48, 72 and 96 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-ERK and anti-phoshpo-ERK antibodies.
-actin antibody was used as an internal control. Quantification of
phospho-ERK protein expressions from three independent experiments are presented as means ± SEM. Means without a common letter differ, p
< 0.05.
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第四節 JA 與臨床藥物 Sorafenib 合併使用對於肝癌細胞的影響