Triazinone triepoxide TATT引發HT-29細胞產生細胞週期停止及細

實驗使用Annexin V-FITC或PI兩種染劑染色檢測引發細胞凋亡 的時間與細胞比例。發生細胞凋亡初期PS由細胞膜內層翻向外側。

Annexin V-FITC是一種呈現綠色螢光的磷脂結合蛋白，對PS有高度親 和力，會與暴露於細胞外側的PS結合(Miller, 2004)。

實驗首先使用不同濃度（0、0.25、0.5、1、2 M）的triazinone triepoxide TATT處理HT-29細胞，培養12與24小時後收取細胞，首先 使用PI染色並以流式細胞儀偵測細胞，再以WinMDI 2.8分析軟體分析 細胞產生細胞凋亡的比例。實驗結果發現（Fig. 4），發生凋亡的細 胞於12小時隨著劑量的增加而有增加的趨勢。在24小時細胞凋亡增加 比例更為明顯。針對細胞週期各期的百分比進行統計分析（Table 1），

發現在12小時的條件下，G0/G1的比例隨劑量增加而減少，S期與G2/M 期的比例則沒有顯著差異（p > 0.05）。在24小時的條件下，G0/G1 期與S期比例隨劑量增加而減少，G2/M期比例則隨劑量增加而有增加

（p < 0.05）。在同樣濃度的triazinone triepoxide TATT處理HT-29之 後，其G2/M期的百分比也隨著處理的時間增長而有增加的趨勢。

實驗以細胞毒殺試驗方法測試發現（Fig. 5），4 M triazinone

對HT-29的抑制率比2 M高，因此實驗接著以4 M的triazinone triepoxide TATT或三種臨床抗癌用藥分別處理HT-29細胞12與24小 時，再以Annexin V-FITC染劑染色來檢測所引發之細胞膜內層外翻比 例。實驗結果（Fig. 6）得知，處理HT-29細胞12小時後，產生細胞膜 外翻的比例triazinone triepoxide TATT與cisplatin沒有顯著差異，但明 顯比carboplatin與oxaliplatin高。處理24小時之後細胞膜外翻的比例為 triazinone triepoxide TATT > cisplatin > oxaliplatin > carboplatin，並有 顯著差異（p < 0.05）。

Fig. 4 Triazinone triepoxide TATT induced apoptosis of HT-29 cells in a dose- and time-related manner. HT-29 cells were synchronized at the beginning of G0 phase by starvation (DMEM supplemented with 1% FBS for 24 h). To determine the effect of triazinone triepoxide TATT, HT-29 cells were treated with different dosages (0-2 M) of triazinone

triepoxide TATT for the indicated time periods. HT-29 cells were stained with PI (40 g/ml) for 1 h, and 10000 cells were examined by flow cytometry. Data are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

Fluorescence intensity

Relative cell number

200 400 200 400 200 400

0 0.25 0.5 1 (M)

128 128 128 128 128

200 400 200 400 200 400 200 400

200 400

1.4 ± 0.2 %

128 128 128 128 128

8.0 ± 0.6% 20.5 ± 5.9% 25.8 ± 5.6% 32.8 ± 4.6%

200 400 200 400 200 400

0 0.25 0.5 1 (M)

128 128 128 128 128

200 400 200 400 200 400 200 400

200 400

1.4 ± 0.2 %

128 128 128 128 128

8.0 ± 0.6% 20.5 ± 5.9% 25.8 ± 5.6% 32.8 ± 4.6%

Table 1 The percentages of cells at different phases of cell cycle in Fig.4-3

Time (h) 12

Triazinone triepoxide

TATT (M) 0 0.25 0.5 1 2

%

Sub-G1 1.4 ± 0.2^d 7.4 ± 1.7^d 8.0 ± 2.1^cd 14.4 ± 4.1^bcd 24.0 ± 2.2^ab G0/G1 55.7 ± 1.2^a 48.4 ± 1.6^b 47.6 ± 0.6^b 42.1 ± 1.0^c 34.7 ± 1.8^e

S 17.3 ± 1.7^c 14.2 ± 0.8^c 13.9 ± 0.2^c 15.6 ± 1.3^c 13.7 ± 0.8^c G2/M 14.5 ± 0.8^de 17.4 ± 2.9^cd 15.3 ± 1.6^de 15.4 ± 2.1^de 12.3 ± 0.7^e

Time (h) 24

Triazinone triepoxide

TATT (M) 0 0.25 0.5 1 2

%

Sub-G1 1.4 ± 0.2^d 8.0 ± 0.6^cd 20.5 ± 6.0^abc 25.9 ± 5.6^ab 32.9 ± 4.6^a G0/G1 53.9 ± 1.5^a 38.9 ± 1.6^d 26.1 ± 0.6^f 19.1 ± 0.3^g 14.4 ± 0.7^h S 24.6 ± 1.6^a 24.4 ± 0.3^a 22.0 ± 1.4^a 22.3 ± 1.7^a 19.0 ± 2.8^b G2/M 20.1 ± 0.4^c 28.8 ± 0.9^b 31.3 ± 0.9^ab 32.8 ± 4.2^a 33.8 ± 2.3^a The percentages of cells at different phases of cell cycle in Fig. 4-3 were quantified by WinMDI 2.8 software. Data are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

Fig. 5 Effect of triazinone triepoxide TATT, cisplatin, carboplatin and oxaliplatin with or without curcumin on HT-29 cell viability

determined by MTT assay. HT-29 cells (1.2 x 10³) were incubated in 96-well plates. The cells were treated with various concentrations (0, 2, 4

M) of triazinone triepoxide TATT, cisplatin (Cis), carboplatin (Car) or

oxaliplatin (Oxa) with or without 30 M curcumin for 24 h. Percentage of inhibition on HT-29 cells was measured by the metabolic dye-based MTT assay. Data are presented as mean ± SEM. Results are representative of three independent experiments.

0

Fig. 6 Triazinone triepoxide TATT exhibits stronger effects on inducing apoptosis of HT-29 at 12 h and 24 h post-treatment than cisplatin, carboplatin, and oxaliplatin. HT-29 cells (1 × 10⁵) were treated with 4M triazinone triepoxide TATT, cisplatin, carboplatin or oxaliplatin for the indicated time periods. After treatment, HT-29 cells were stained with annexin V-FITC (0.5 mg/ml) for 15 min, and 10000 cells were examined by flow cytometry. Data are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

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第三節 薑黃素使triazinone triepoxide TATT或臨床藥物引