透過 caspase 8 抑制劑 Z-IETD-fmk 了解外在路徑對於 triazinone

從前面結果（Fig. 9，Fig. 12）中得知，triazinone triepoxide TATT 對於 HT-29 細胞中 caspase 8 的表現有時間與劑量上的影響，因此為 檢測 caspase 8 在 triazinone triepoxide TATT 引發之細胞凋亡中的重要 性，實驗首先使用 Z-IETD-fmk 進行西點墨法偵測 caspase 8 下游 caspase 3 的表現，結果（Fig. 15）發現，單獨使用 20 M Z-IETD-fmk 時，caspase 3 沒有變化，單獨使用 4 M triazinone triepoxide TATT 時，

caspase 3 則被活化（p < 0.05）。兩者合併處理時，caspase 3 再度受到 抑制，猜測 triazinone triepoxide TATT 造成 HT-29 細胞凋亡部分原因 來自於外在路徑。接著進一步使用相同實驗條件，以 hoechst 33258 染色進行倒立螢光顯微鏡觀察凋亡小體的發生情況，凋亡小體呈現較 亮的藍色，觀察到相同的結果（Fig. 16）。為了將凋亡的結果予以量 化，實驗再以相同條件處理細胞，並藉由 annexin V 染色進行流式細 胞儀分析，結果（Fig. 17）發現單獨使用 Z-IETD-fmk 時，細胞膜外 翻比例與控制組沒有差異（p > 0.05），單獨使用 triazinone triepoxide TATT 則與控制組有顯著差異（p < 0.05），兩者合併處理後細胞膜外 翻情況受到抑制，與控制組沒有差異（p > 0.05）。藉由三種實驗方法

凋亡。

Fig. 15 Inhibitory effect of caspase 8 inhibitor Z-IETD-fmk on triazinone triepoxide TATT-induced the levels of cleavage-caspase 3 protein in HT-29 cells by western blot. Cells (1 × 10⁵) were pretreated with and without 20 M Z-IETD-fmk for 4 h prior to exposure to 4 M of triazinone triepoxide TATT for 24 h. Whole cell lysates were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-caspase 3 antibody was served as a probe. -actin was used as a loading control. Data of quantification of cleavage-caspase 3 protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments. Triazinone triepoxide TATT

(4 M) Triazinone triepoxide TATT

(4 M)

Fig. 16 Effect of triazinone triepoxide TATT and the inhibitory effect of 20 M Z-IETD-fmk on the formation of apoptotic bodies. Cells (1

× 10⁵) pretreated without or with 20 M Z-IETD-fmk for 4 h prior to exposure to 4 M of triazinone triepoxide TATT for 24 h were stained with hoechst 33258 and analyzed by fluorescence microscopy. The arrowhead indicates the formation of apoptotic bodies. Results are representative of three independent experiments.

Triazinone

Fig. 17 Inhibitory effect of caspase 8 inhibitor Z-IETD-fmk on

triazinone triepoxide TATT-induced apoptosis in HT-29 cells. Cells (1

× 10⁵) were pretreated without or with 20 M Z-IETD-fmk for 4 h prior to exposure to 4 M of triazinone triepoxide TATT for 24 h. Cells were then stained with annexin V and analysed by flow cytometry. The percentages in the figure indicate the proportion of apoptotic cells with externalization of phosphatidylserine. Data are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

128

10¹ 10² 10³

Fluorescence intensity

Relative cell number

z-IETD-fmk

Triazinone triepoxide TATT (4 M)

Relative cell number

z-IETD-fmk

Triazinone triepoxide TATT

(4 M)

第八節 透過抑制劑 cyclosporine A 了解內在路徑對於

triazinone triepoxide TATT 造成 HT-29 細胞凋亡的重要性

為瞭解內在路徑的調控對於 triazinone triepoxide TATT 造成細胞 凋亡的重要性，實驗使用抑制劑 transition pore inhibitor（cyclosporine A）與 triazinone triepoxide TATT 處理細胞。為確認 cyclosporine A 的 有效性，細胞以 rhodamine 123 進行染色，再以共軛焦顯微鏡觀察活 細胞粒線體膜電位的改變。結果發現（Fig. 18），單獨 4 M triazinone triepoxide TATT 處理細胞，粒線體膜電位明顯改變，具顯著差異（p <

0.05）。單獨使用 10 M cyclosporine A 處理細胞後，粒線體膜電位 沒有改變（p > 0.05）。當兩者合併處理時，粒線體膜電位與控制組相 比沒有改變（p > 0.05）。進一步以相同條件進行西方點墨法偵測 caspase 9 的表現，結果發現（Fig. 19）單獨使用 4 M 的 triazinone triepoxide TATT 時，caspase 9 的活化態增加。單獨只有 10 M

cyclosporine A 時，caspase 9 沒有被活化。當兩者合併處理細胞之後，

其 caspase 9 的活化態被抑制，並有顯著差異（p < 0.05）。為了量化 內在路徑所扮演的角色，實驗再以相同條件處理細胞，並藉由 annexin V 染劑染色，以流式細胞儀進行分析，檢測細胞產生細胞膜內層外翻 的比例。結果（Fig. 20）發現，單獨只有 triazinone triepoxide TATT 處理時，細胞膜外翻比例增加。單獨使用 10 M cyclosporine A 時，

細胞膜內層外翻比例沒有增加趨勢。而 triazinone triepoxide TATT 與 10 M cyclosporine A 合併處理之後，triazinone triepoxide TATT 引發 之細胞膜內層外翻比例降低（p < 0.05）。藉由三種實驗方法證實，

triazinone triepoxide TATT 也透過內在路徑促使 HT-29 發生細胞凋亡。

Fig. 18 Effect of triazinone triepoxide TATT and the inhibitory effect of 10 M CsA on the change of Δψm in HT-29 cells. Cells (1 × 10⁵) pretreated without or with 10 M cyclosporine A (CsA) for 4 h prior to exposure to 4 M of triazinone triepoxide TATT for 24 h were stained with rhodamine 123 and analyzed by confocal microscopy. Results are representative of three independent experiments.

Control CsA 10 M

10 M

Triazinone triepoxide TATT 4 M

Triazinone triepoxide

TATT+

CsA

Control CsA 10 M

10 M

Triazinone triepoxide TATT 4 M

Triazinone triepoxide

TATT+

CsA

Fig. 19 Inhibitory effect of cyclosporine A (CsA) on triazinone triepoxide TATT-induced the levels of cleavage-caspase 9 protein in HT-29 cells by western blot. Cells (1 × 10⁵) were pretreated with and without 10 M cyclosporine A (CsA) for 4 h prior to exposure to 4 M of triazinone triepoxide TATT for 24 h. Whole cell lysates were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-caspase 9 antibody was served as a probe. -actin was used as a loading control. Data of quantification of cleavage-caspase 9 protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments. Triazinone triepoxide TATT

(4 M) Triazinone triepoxide TATT

(4 M)

Fig. 20 Inhibitory effect of cyclosporine A (CsA) on triazinone triepoxide TATT-induced apoptosis in HT-29 cells. Cells (1 × 10⁵) were pretreated without or with 10 M cyclosporine A (CsA) for 4 h prior to exposure to 4 M of triazinone triepoxide TATT for 24 h. Cells were then stained with annexin V and analysed by flow cytometry. The percentages in the figure indicate the proportion of apoptotic cells with externalization of phosphatidylserine. Data are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

128

10¹ 10² 10³

Fluorescence intensity Relative cell number

CsA

(10 M)

-

-- +

Triazinone triepoxide TATT

(4 M)

- +

Relative cell number

CsA

(10 M)

-

-- +

Triazinone triepoxide TATT

(4 M)

- +

第九節 Triazinone triepoxide TATT 造成 HT-29 細胞粒線體 膜電位改變的相關蛋白質

以 western blotting 分析與粒線體相關的蛋白質。結果（Fig. 21 ~ Fig. 32）發現，4 M triazinone triepoxide TATT 處理 HT-29 細胞 24 小時後，細胞質中的 cytochrome c、Smac、Bcl-2 與 Bcl-w 的表現與 控制組比較是增加的，其中 cytochrome c 與 Bcl-w 與控制組是有顯著 差異（p < 0.05）。Smac 與 Bcl-2 雖也有增加，但與控制組沒有顯著 差異。細胞質中的 Bax 與 Bid 的表現與控制組比較是減少的，並且均 具有顯著差異（p < 0.05）。另外粒線體中的 cytochrome c、Smac、

Bcl-2 與 Bcl-w 的表現與控制組比較是減少的，並且均有顯著差異（p

< 0.05）。在粒線體中的 Bax 與 tBid 的表現與控制組比較是增加的，

其中 Bax 具有顯著差異（p < 0.05）。粒線體內的 tBid 雖然減少，但 與控制組沒有顯著差異（p > 0.05）。

Fig. 21 Expression of cytosolic cytochrome c protein by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Cytosolic fractions were resolved in

SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-cytochrome c antibody was served as a probe. -actin was used as a loading control. Data of quantification of cytochrome c protein

expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲20

▲42

-actin Cyto. Cytochrome c^▲

▲ Cyto. Cytochrome c^▲

▲

Fig. 22 Expression of mitochondrial cytochrome c protein by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Mitochondrial fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-cytochrome c antibody was served as a probe. -actin was used as a loading control. Data of quantification of cytochrome c protein

expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲20

▲42

-actin Mito. Cytochrome c^▲

▲ Mito. Cytochrome c^▲

▲

Time (h) 0 24 KDa

1b 0.6±0.1a

Triazinone triepoxide TATT (4 M)

Fig. 23 Expression of cytosolic Smac protein by western blot analysis.

HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Cytosolic fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-Smac antibody was served as a probe. -actin was used as a loading control. Data of quantification of Smac protein expression are presented as means ± SEM.

Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲25

Fig. 24 Expression of mitochondrial Smac protein by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Mitochondrial fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-Smac antibody was served as a probe. -actin was used as a loading control. Data of quantification of Smac protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲25

Fig. 25 Expression of cytosolic Bax protein by western blot analysis.

HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Cytosolic fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-Bax antibody was served as a probe. -actin was used as a loading control. Data of

quantification of Bax protein expression are presented as means ± SEM.

Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲23

Fig. 26 Expression of mitochondrial Bax protein by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Mitochondrial fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-Bax antibody was served as a probe. -actin was used as a loading control. Data of quantification of Bax protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲23

Fig. 27 Expression of cytosolic Bid protein by western blot analysis.

HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Cytosolic fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-Bid antibody was served as a probe. -actin was used as a loading control. Data of

quantification of BID protein expression are presented as means ± SEM.

Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲22

Fig. 28 Expression of mitochondrial tBid protein by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Mitochondrial fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-tBid antibody was served as a probe. -actin was used as a loading control. Data of quantification of BID protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲15

Fig. 29 Expression of cytosolic Bcl-2 protein by western blot analysis.

HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Cytosolic fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-Bcl-2 antibody was served as a probe. -actin was used as a loading control. Data of quantification of Bcl-2 protein expression are presented as means ± SEM.

Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲26

Fig. 30 Expression of mitochondrial Bcl-2 protein by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Mitochondrial fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-Bcl-2 antibody was served as a probe. -actin was used as a loading control. Data of quantification of Bcl-2 protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲26

Fig. 31 Expression of cytosolic Bcl-w protein by western blot analysis.

HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Cytosolic fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-Bcl-w antibody was served as a probe. -actin was used as a loading control. Data of

quantification of Bcl-w protein expression are presented as means ± SEM.

Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲18

Fig. 32 Expression of mitochondrial Bcl-w protein by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Mitochondrial fractions were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-Bcl-w antibody was served as a probe. -actin was used as a loading control. Data of quantification of Bcl-w protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

▲18

第十節 Triazinone triepoxide TATT 造成 HT-29 細胞週期停 止的相關蛋白質

由 Fig. 8 及 Table 2 得知，triazinone triepoxide TATT 不只造成 HT-29 產生細胞凋亡，同時影響細胞週期。從細胞週期的變化發現，

triazinone triepoxide TATT 會使得 HT-29 細胞的 G2/M 期下降。由文獻 得知，許多蛋白質可調控細胞週期，包括 checkpoint kinase-2（Chk2）、 p53、cdc25 phosphatase isoforms-A（CDC25A）、checkpoint kinase-1

（Chk1）、cdc25 phosphatase isoforms-C（CDC25C）、mitogen-activated protein kinase-activated proteinkinase-2（MK2）、cdc25 phosphatase isoforms-B（CDC25B）、p21、Wee1、cyclin A/E、cyclin-dependent kinase 2（CDK2）、cyclin B1、cyclin-dependent kinase 1（CDK1）(Bucher &

Britten, 2008)。其中 cyclin B1、CDC25B 和 CDC25C 是參與細胞週期 G2/M 期相關的蛋白質，當細胞停止在 G2/M 期時，因 CDC25B 或 CDC25C 表現下降造成 cyclin B1/CDK 複合體的活性降低。因此實驗 藉由 western blotting 方法，檢驗 triazinone triepoxide TATT 造成 HT-29 細胞週期 G2/M 期的下降，是否與這些蛋白質有關。結果（Fig. 33 ~ Fig.

35）發現，triazinone triepoxide TATT 處理 24 小時後的 HT-29 細胞，

其 cyclin B1 的表現增加，而 CDC25B 和 CDC25C 的表現則減少，且 結果經統計後均有顯著差異（p < 0.05）。因此猜測 triazinone triepoxide

TATT 處理 24 小時後的 HT-29 細胞產生細胞週期 G2/M 期的比例減少 與可能與 CDC25B 和 CDC25C 的參與有關。

Fig. 33 Involvement of Cyclin B1 protein in G2/M arrest of HT-29 cells by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4

M of triazinone triepoxide TATT for 24 h. Whole cell lysates were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-Cyclin B1 antibody was served as a probe. -actin was used as a loading control. Data of quantification of cyclin B1 protein expression are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

Fig. 34 Expression of CDC25B protein in of HT-29 cells by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Whole cell lysates were resolved in

SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-CDC25B antibody was served as a probe. -actin was used as a loading control. Data of quantification of CDC25B protein expression are presented as means ± SEM. Means without a common letter differ, p <

0.05. Results are representative of three independent experiments.

▲62

Fig. 35 Expression of CDC25C protein in of HT-29 cells by western blot analysis. HT-29 cells (1 × 10⁶) were treated with 4 M of triazinone triepoxide TATT for 24 h. Whole cell lysates were resolved in

SDS-PAGE gel and the proteins were transferred to PVDF membrane.

Anti-CDC25C antibody was served as a probe. -actin was used as a loading control. Data of quantification of CDC25C protein expression are presented as means ± SEM. Means without a common letter differ, p <

0.05. Results are representative of three independent experiments.

▲55

第十一節 Triazinone triepoxide TATT 與薑黃素合併處理造 成 HT-29 細胞核內蛋白質的變化

薑黃素在許多研究中已被證明，在不同惡性腫瘤細胞系統中，透 過促進細胞色素c從粒線體釋放後再誘發第二粒線體衍生caspase的作

薑黃素在許多研究中已被證明，在不同惡性腫瘤細胞系統中，透 過促進細胞色素c從粒線體釋放後再誘發第二粒線體衍生caspase的作

在文檔中 植化素薑黃素藉由引發細胞凋亡促進新穎及傳統鉑金屬大腸直腸癌化療藥物之敏感性 (頁 115-0)