抑制細胞自噬能促進 JA 毒殺 Hep 3B 細胞及細胞凋亡，而

細胞自噬與細胞凋亡之間有許多蛋白分子會交互影響，彼此可能 會促進或抑制，在之前的研究已發現 JA 會促進細胞凋亡進行(Su, et al., 2006)。因此，為釐清 JA 所誘發的細胞自噬與細胞凋亡之間的關 係，利用細胞自噬抑制劑 BAF 來抑制自噬小體與溶酶體結合，觀察 細胞凋亡下游分子 caspase 3 的表現量並觀察細胞凋亡的比例是否有 受影響。實驗也使用細胞凋亡抑制劑 pan-caspase 蛋白抑制劑

zVAD-fmk 抑制細胞凋亡蛋白質表現，並觀察細胞自噬相關蛋白質的 表現量，以確認細胞凋亡與細胞自噬之間的相關性。

實驗先以細胞毒殺能力試驗檢測有使用與未使用細胞自噬抑制 劑 BAF 前處理對於 JA 毒殺細胞的能力，實驗結果發現（Fig. 7），JA 濃度在 0.5 M 的濃度下有加入抑制劑前處理的組別細胞死亡的比例 明顯增加（p＜0.05），但在 JA 1 及 2 M 的濃度下，加入抑制劑前處 理的組別細胞死亡的比例並無明顯增加（p＞0.05）。為確認細胞凋亡 情形，利用 PI 染劑經流式細胞儀檢測細胞凋亡 Sub-G1 的比例，依前 節實驗（Fig. 1）時間與劑量的自噬比例，選用細胞自噬較明顯的時 間點 48 小時及劑量 JA 1 及 2 M 的濃度，如（Fig. 8）所示，單獨使 用抑制劑的組別，Sub-G1 比例沒有明顯差異。加入 JA 後，Sub-G1

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比例隨著劑量增加而上升，並且在加入細胞自噬抑制劑 BAF 前處理 的組別，其 Sub-G1 的比例較未加入 BAF 前處理的組別高（p＞0.05），

顯示抑制了細胞自噬有增加細胞凋亡比例的趨勢，因此利用西方墨點 法來確認細胞凋亡相關蛋白質的表現量，實驗結果發現（Fig. 9），細 胞凋亡下游分子 caspase3 活化型的表現量，也是在有加入細胞自噬抑 制劑前處理的組別中，表現量較多（p＜0.05），與 Sub-G1 細胞凋亡 比例的結果一致，證實抑制 JA 所誘發的細胞自噬能增加細胞凋亡的 比例。

從前面結果（Fig. 7、8、9）得知抑制了 JA 誘發的細胞自噬後會 促進細胞凋亡的進行，為檢測細胞凋亡對於細胞自噬的影響，實驗使 用細胞凋亡抑制劑 zVAD-fmk 抑制 caspase 蛋白活性，利用西方墨點 法檢測 caspase 3、LC3、p62 及 LAMP2a 的表現，來觀察抑制細胞凋 亡後 JA 對於細胞自噬的影響。結果（Fig. 10）顯示單獨使用 50M zVAD-fmk 預處理的組別，caspase 3 沒有變化；單獨使用 1 M 及 2 M 濃度的 JA，caspase 3 表現量上升（p＜0.05）；而合併處理後，活化型 caspase 3 表現受到抑制，且有顯著差異（p＜0.05）。細胞自噬相關蛋 白部分，單獨使用 50M zVAD-fmk 預處理的組別，LC3、p62 及 LAMP2a 表現量沒有變化，但加入 JA 後，有使用與未使用 zVAD-fmk 預處理的組別相比，如結果（Fig. 11、12、13）所示，並不影響 LC3、

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p62 及 LAMP2a 的蛋白表現量（p＞0.05），顯示 JA 所誘發的細胞凋 亡並不影響細胞自噬。

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Fig. 7 BAF increased JA-induced cell growth inhibition in Hep 3B cells.

Hep 3B cells (1 × 10⁵ cell/well) were pretreated with or without 25 nM Bafilomycin A1 (BAF) for 2 h. The cells were then treated with 0.5, 1

M and 2 M JA for 48 h. Growth inhibition was evaluated by MTT

assay. Data are presented as mean ± SEMs. Means without a common letter differ, p＜0.05. Results are representative of three independent experiments.

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Fig. 8 BAF increased JA-induced sub G1 percentage in Hep 3B cells.

Hep 3B cells (1 × 10⁵ cells/well) were pretreated with or without 25 nM Bafilomycin A1 (BAF) for 2 h. The cells were then treated with 1 M or 2 M JA for 48 h. The cells were stained with PI (40 g/ml) for 30 min before flow cytometry. The percentages in the figure indicate the

proportion of apoptotic cells. Data are presented as mean ± SEMs. Means without a common letter differ, p＜0.05. Results are representative of three independent experiments.

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Fig. 9 BAF increased JA-induced cleavage caspase 3 expression in Hep 3B cells.

Hep 3B cells (1 × 10⁶ cells/dish) were pretreated with or without 25 nM BAF for 2 h. The cells were then treated with 1 M or 2 M JA for 48 h.

Whole cell lysates were prepared and subjected to Western blot analysis using anti-caspase 3 antibody. -actin antibody was used as an internal control. Quantification of cleavage-caspase 3 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. 10 zVAD-fmk decreased JA-induced cleavage caspase 3 expression in Hep 3B cells.

Hep 3B cells (1 × 10⁶ cells/dish) were pretreated with or without 50 M zVAD-fmk for 2 h. The cells were then treated with 1 M or 2 M JA for 48 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-caspase 3 antibody. -actin antibody was used as an internal control. Quantification of cleavage-caspase 3 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. 11 zVAD-fmk didn’t affect JA-induced LC3-II expression in Hep 3B cells.

Hep 3B cells (1 × 10⁶ cells/dish) were pretreated with or without 50 M zVAD-fmk for 2 h. The cells were then treated with 1 M or 2 M JA for 48 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-LC3 antibody. -actin antibody was used as an

internal control. Quantification of LC3-II 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. 12 zVAD-fmk didn’t affect JA-induced p62 expression in Hep 3B cells.

Hep 3B cells (1 × 10⁶ cells/dish) were pretreated with or without 50 M zVAD-fmk for 2 h. The cells were then treated with 1 M or 2 M JA for 48 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-p62 antibody. -actin antibody was used as an internal control. Quantification of p62 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. 13 zVAD-fmk didn’t affect JA-induced LAMP2a expression in Hep 3B cells.

Hep 3B cells (1 × 10⁶ cells/dish) were pretreated with or without 50 M zVAD-fmk for 2 h. The cells were then treated with 1 M or 2 M JA for 48 h. Whole cell lysates were prepared and subjected to Western blot analysis using anti-LAMP2a antibody. -actin antibody was used as an internal control. Quantification of LAMP2a protein expressions from three independent experiments are presented as means ± SEM. Means without a common letter differ, p < 0.05.

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第三節 JA 所誘發的細胞自噬與 MEK/ERK 路徑的活化有關