薑黃素與 Sorafenib 合併使用對肝癌細胞的影響

在前節實驗探討 Yao-ram-2-7 與薑黃素合併使用毒殺 Hep 3B 的 效果，結果顯示合併使用對生長抑制有加乘的效用，可能與薑黃素導 致 G2/M 期停滯有關，具有使癌細胞分裂減少的潛力。為比較臨床抗 癌用藥 Sorafenib 與薑黃素合併使用對細胞是否具有影響，實驗利用 細胞毒殺試驗方法測試 Sorafenib（0-10 M）、薑黃素（30 M）及兩 者合併使用下對細胞生長抑制比率，並計算出 q 值。

結果發現（Fig. 12）在單獨處理情況下，薑黃素比 Sorafenib 各 濃度效果好，有顯著差異（p < 0.05）。薑黃素與 Sorafenib 合併使用，

效果比單獨使用 Sorafenib 佳（p < 0.05），但沒有比單獨使用薑黃素 好，不具顯著差異（p > 0.05）。以 q 值來看合併效用，Sorafenib 各濃 度與薑黃素合併使用後 q 值均為拮抗。為進一步了解合併使用對細胞 凋亡或細胞週期的影響，選用 Sorafenib 劑量 10 M 及薑黃素 30 M，

處理 Hep 3B 細胞 48 小時，利用 PI 染劑經流式細胞儀分析並計算細 胞週期各分期的 q 值。凋亡 Sub-G1 期如圖所示（Fig. 13），單獨處理 薑黃素或 Sorafenib 引起的 Sub-G1 百分比間無顯著差異（p > 0.05）；

薑黃素和 Sorafenib 合併使用 Sub-G1 百分比也與單獨處理無顯著差異

（p > 0.05）且數值較低，顯示薑黃素和 Sorafenib 合併使用引起的凋

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亡效用是相互拮抗（Table 5）。分析細胞週期並計算 q 值（Table 5），

在 G0/G1 期的百分比發現單獨使用 Sorafenib 較薑黃素及合併使用高

（p < 0.05）。S 期則是 Sorafenib 較薑黃素及合併使用低（p < 0.05）。

在 G2/M 期發現 Sorafenib 與控制組比較無統計差異（p > 0.05），但在 合併使用兩者與單獨使用薑黃素具有 G2/M 期停滯的趨勢。細胞週期 各期在 q 值結果均為拮抗。

實驗以 western blotting 方式觀察 caspase 3 的表現，使用 Sorafenib、

薑黃素及兩者合併使用處理細胞，如同前述實驗條件，收取 whole cell lysates。結果如 Fig. 14 所示，細胞經 Sorafenib 處理，引起

cleavage-caspase 3 的表現量多於控制組，具有顯著差異（p < 0.05）， 此結果也可證實 PI 染色實驗（Fig. 5），Sorafenib 藉由活化 caspase 3 蛋白誘發細胞凋亡。比較薑黃素、Sorafenib 及合併使用這三組，

cleavage-caspase 3 的表現量均多於控制組（p < 0.05），但彼此之間並 無顯著差異（p > 0.05），結果可能因接受藥物處理的這三組引起的 Sub-G1 百分比相當有關（Fig. 13）。在第三節實驗已確認 Sorafenib 處理 Hep 3B 細胞會產生些微的酸性囊狀胞器（Fig. 7），為確認自噬 體上 LC3-II 蛋白的表現及 Sorafenib 合併使用薑黃素後是否對細胞自 噬現象產生影響，實驗利用 western blotting 觀察 LC3-II 蛋白的表現 量。結果發現（Fig. 15），單獨處理 Sorafenib 與控制組相比其 LC3-II

75

蛋白表現無顯著差異（p > 0.05），也證實前面實驗結果（Fig. 7），10 M 的 Sorafenib 引起細胞自噬與控制組並無顯著差異（p > 0.05）。在 Sorafenib 與薑黃素合併處理後，LC3-II 蛋白表現量比單一 Sorafenib 處理顯著增加（p < 0.05）；但比單獨處理薑黃素顯著減少（p < 0.05），

此結果可能與合併使用產生拮抗有關。

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Fig. 12 Effect of Sorafenib with or without curcumin on Hep 3B cell viability determined by MTT assay and the interaction of Sorafenib and curcumin determined by the value of q.

Hep 3B cells (8×10³

cells/well) were treated with various concentrations (0, 2.5, 5, 10 M) of Sorafenib with or without 30 M curcumin for 48 h. Percentage of inhibition on Hep 3B cells was measured by MTT assay. The value of q indicates synergism when greater than 1.15, antagonism when smaller than 0.85, and additivity when located between 0.85 and 1.15. 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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Fig. 13 The effect of curcumin on Sorafenib-induced apoptosis.

Hep 3B cells (1×10⁵ cells/well) were treated with 10 M Sorafenib in the presence and absence of 30 M cucumin for the 48h. After treatment, Hep 3B cells were stained with PI (2 g/ml) for 30 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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Table 5 The percentages of cells at different phases of cell cycle in Fig. 13 and the value of q

Time (h) 48

Sorafenib (10 M)

－ － ＋ ＋

Curcumin (30 M)

－ ＋ － ＋

% q

Sub-G1 1.3 ± 0.3

^b

13.5 ± 3.7

^a

10.9 ± 3.2

^a

8.4 ± 0.6

^ab

0.41 ± 0.07

G0/G1 38.0 ± 1.8

^a

23.1 ± 2.0

^b

39.1 ± 5.6

^a

23.6 ± 1.4

^b

0.45 ± 0.02

S 24.8 ± 2.2

^a

20.7 ± 0.2

^ab

11.7 ± 1.1

^c

20.4 ± 0.7

^b

0.68 ± 0.01 G2/M 36.0 ± 2.4

^b

42.5 ± 3.2

^ab

38.2 ± 3.1

^b

47.3 ± 0.5

^a

0.74 ± 0.04

The percentages of cells at different phases of cell cycle in Fig. 13 were quantified by WinMDI 2.8 software. The value of q indicates synergism when greater than 1.15, antagonism when smaller than 0.85, and additivity when located between 0.85 and 1.15. Data are presented as means ± SEM. Means in the same cell cycle without a common letter differ, p < 0.05.

Results are representative of three independent experiments.

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Fig. 14 The change of caspase 3 protein expression in

Sorafenib-treated Hep 3B cells in the presence and absence of curcumin. Hep 3B cells (1×10

⁶ cells/dish) were treated with 10 M of Sorafenib with or without 30 M curcumin for 48 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. Quantification of cleavage-caspase 3

protein expressions from independent experiments 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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Fig. 15 The change of LC3 protein expression in Sorafenib-treated Hep 3B cells in the presence and absence of curcumin. Hep 3B cells

(1×10⁶ cells/dish) were treated with 10 M of Sorafenib with or without 30 M curcumin for 48 h. Whole cell lysates were resolved in

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

Anti-LC3 antibody was served as a probe. -actin was used as a loading control. Quantification of LC3-II protein expressions from independent experiments are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

81 第六節 驗證連接網路資料庫比對結果

Yao-ram-2-7 處理 Hep 3B 細胞後分析細胞內基因的表現變化，經 連接網路資料庫比對，發現 Yao-ram-2-7 影響細胞基因表現與資料庫 中藥物 AR-A014418 可能有類似的效用。因此，實驗利用 AR-A014418 處理 Hep 3B 細胞，與前述檢測之 Yao-ram-2-7 的結果比對。

實驗先以 MTT assay 檢測 AR-A014418 是否對 Hep 3B 具有抑制 作用。使用 AR-A014418 劑量由 0-40 M，作用 Hep 3B 細胞時間為 24、48 及 72 小時。實驗結果發現（Fig. 16）AR-A014418 隨劑量增 加及作用時間延長，細胞生長抑制率有增加趨勢，但與 Yao-ram-2-7 比較，AR-A014418 在最高劑量 40M 的抑制率趨勢與 Yao-ram-2-7 最高劑量 20 M 較為相似。從 MTT assay 結果得知細胞生長抑制率 可能是因細胞死亡或細胞分裂減少，所以實驗下一步利用 PI 染劑以 流式細胞儀分析凋亡產生及細胞週期的變化。結果如圖所示（Fig. 17），

Hep 3B 經 AR-A014418 處理，凋亡的細胞落在 Sub-G1 期的百分比僅 有在時間點 48 小時最高劑量 40 M 與 20 M 和 72 小時的 20 M 具 有顯著差異（p < 0.05），在所有時間點劑量 0-20 M 均無統計差異（p

> 0.05），在 40 M 下的三個時間點也無統計差異（p > 0.05）。在不同 時間劑量下，針對細胞週期各期的百分比進行統計分析（Table 6），

82

在 G0/G1 期均沒有顯著差異（p > 0.05）。S 期在時間點 24 及 48 小時 沒有顯著差異（p > 0.05），到 72 小時劑量 40 M 時 S 期的百分比顯 著降低（p < 0.05）。G2/M 期隨時間延長及劑量增高比例有增加的趨 勢，且在時間點 72 小時劑量 40 M 與 20 M 比較下具有顯著差異（p

< 0.05），此結果也推知 AR-A014418 對細胞生長抑制是透過 S 期的減 少及 G2/M 期的停滯，使細胞分裂減少。另外，我們透過 western blotting 方式觀察 caspase 3 蛋白表現也發現（Fig. 18）細胞經

AR-A014418 劑量 40 M 處理 72 小時，引起 cleavage-caspase 3 蛋白 表現與控制組沒有顯著差異（p > 0.05），此結果也證實 PI 染色實驗

（Fig. 17）。

為確認 AR-A014418 是否同 Yao-ram-2-7 具有誘發細胞自噬現象，

實驗利用 AO 染劑與自噬小體染色結合，經流式細胞儀分析。實驗使 用 AR-A014418 劑量由 0-40 M，作用 Hep 3B 細胞時間為 24、48 及 72 小時。結果發現（Fig. 19）在 24 及 48 小時各劑量沒有顯著差異，

但在 72 小時劑量 40 M 產生的自噬比例較劑量 20 M 多，也比 48 小時劑量 40 M 的自噬百分比多，具有顯著差異（p < 0.05）。另外，

透過 western blotting 方式觀察 LC3-II 蛋白表現也發現（Fig. 20）細胞 經 AR-A014418 劑量 40 M 處理 72 小時，引起 LC3-II 蛋白表現量顯 著多於控制組（p < 0.05）。以上結果也證實 AO 染色實驗（Fig. 19），

83

AR-A014418 如同 Yao-ram-2-7（Fig. 6）具有誘發細胞自噬現象。

AR-A014418 為一種選擇性的 GSK-3 蛋白抑制劑，為比對

Yao-ram-2-7 是否具有相似功能，實驗使用 Yao-ram-2-7 劑量 10 M 處 理細胞 48 小時，收取 whole cell lysates，以 western blotting 方式觀察 細胞內 phospho-GSK-3及 GSK-3的表現。實驗結果發現（Fig. 21）， Yao-ram-2-7 處理 Hep 3B 細胞後沒有改變 phospho-GSK-3蛋白表現 量但 phospho-GSK-3蛋白表現減少，具有顯著差異（p < 0.05）。在總 GSK-3蛋白表現雖無統計差異，但有減少趨勢（Fig. 22）。以上結果 顯示 Yao-ram-2-7 似 AR-A014418，具有抑制 phospho-GSK-3蛋白表 現的潛力。

 

84







Fig. 16 Effect of Yao-ram-2-7 or AR-A014418 on Hep 3B cell viability determined by MTT assay.

Hep 3B cells (8×10³ cells/well) were treated with 0-20 M Yao-ram-2-7 or 0-40 M AR-A014418 for 24, 48 and 72 h. Percentage of inhibition on Hep 3B cells was measured by MTT assay. Data are presented as means ± SEM. Results are

representative of three independent experiments.

 

85



Fig. 17 Effect of AR-A014418 on apoptosis of Hep 3B cells. Hep 3B

cells were synchronized at the beginning of G0 phase by starvation

(DMEM supplemented with 1% FBS for 24 h). To determine the effect of AR-A014418, Hep 3B cells were treated with different dosages (0-40M) of AR-A014418 for the indicated time periods. Hep 3B cells were stained with PI (2 g/ml) for 30 min, and 10000 cells were examined by flow cytometry. The percentages indicate the proportion of apoptotic cells.

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

experiments.

86

Table 6 The percentage of cells at different phases of cell cycle in Fig. 17

Time (h) 24

The percentages of cells at different phases of cell cycle in Fig. 17 were quantified by WinMDI 2.8 software. Data are presented as means ± SEM. Means in the same cell cycle without a common letter differ, p < 0.05. Results are representative of three independent experiments.

87

Fig. 18 The change of caspase 3 protein expression in

AR-A014418-treated Hep 3B cell. Hep 3B cells (1×10

⁶ cells/dish) were treated with 40 M of AR-A014418 for 72 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. Quantification of cleavage-caspase 3 protein expressions from independent experiments are presented as means ± SEM.

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

88

Fig. 19 Effect of AR-A014418 on autophagy of Hep 3B cells.

Hep 3B cells (1×10⁵ cells/well) were exposed to 5 concentrations (2.5-40

M) of AR-A014418 for 24, 48, and 72 h. After treatment, the cells

were stained with AO (1.5 g/ml) for 15 min before flow cytometry.

The percentages in the figure indicate the proportion of cells (upper two quadrants) with AVOs staining. Data are presented as means ± SEM.

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

89

Fig. 20 The change of LC3 protein expression in

AR-A014418-treated Hep 3B cell. Hep 3B cells (1×10

⁶ cells/dish) were treated with 40 M of AR-A014418 for 72 h. Whole cell lysates were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-LC3 antibody was served as a probe. -actin was used as a loading control. Quantification of LC3-II protein expressions from independent experiments are presented as means ± SEM. Means without a common letter differ, p < 0.05. Results are representative of three independent experiments.

90

Fig. 21 The change of phospho-GSK-3α/β protein expression in Yao-ram-2-7-treared Hep 3B cell. Hep 3B cells (1×10

⁶ cells/dish) were treated with 10 M of Yao-ram-2-7 for 48 h. Whole cell lysates were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-phospho-GSK-3α/β antibody was served as a probe.

-actin was used as a loading control. Quantification of phospho-GSK-3α

and phospho-GSK-3 protein expressions from independent experiments are presented as means ± SEM. Means in a row without a common letter differ, p < 0.05. Results are representative of three independent

experiments.

91

Fig. 22 The change of total GSK-3β protein expression in

Yao-ram-2-7-treated Hep 3B cell. Hep 3B cells (1×10

⁶ cells/dish) were treated with 10 M of Yao-ram-2-7 for 48 h. Whole cell lysates were resolved in SDS-PAGE gel and the proteins were transferred to PVDF membrane. Anti-GSK-3β antibody was served as a probe. -actin was used as a loading control. Quantification of total GSK-3β protein

expressions from independent experiments are presented as means ± SEM.

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

92

第五章 討論

本實驗中發現由 1,2,3-triazoles 及 2H-1,4-benzoxazin-3-(4H)-one 兩種化合物經結構修而得的 Yao-ram-2-7 與臨床肝癌標靶用藥

Sorafenib 毒殺人類肝癌細胞 Hep 3B 的效用相當，但對人類正常細胞 更具安全性。Yao-ram-2-7 在合併薑黃素處理下，對於毒殺肝癌細胞 具有加乘的效用。在比對基因資料庫 CMAP 後發現 Yao-ram-2-7 與 GSK-3 抑制劑 AR-A014418 的基因表現雷同，實驗也進一步證實 AR-A014418 似 Yao-ram-2-7 可誘發 Hep 3B 產生細胞自噬；

Yao-ram-2-7 似 AR-A014418 具抑制 phospho-GSK-3β 及 GSK-3β 蛋白 的表現。

癌細胞有 11 項特徵：（1）促進自我的生長、（2）對停止生長的 訊息敏感度降低、（3）抵抗細胞死亡程序、（4）使血管增生以供應 其生長之營養、（5）持續生長（6）侵犯附近之組織及轉移（7）處 在低度發炎的環境、（8）不正常的代謝途徑（9）不受免疫系統控制

（10）促進發炎反應（11）基因異常且不穩定(Colotta, Allavena, Sica, Garlanda, & Mantovani, 2009; Hanahan & Weinberg, 2011)，因此抗癌用 藥的研發常以誘發細胞凋亡及使癌細胞的細胞週期停滯為目標(Choi et al., 2011)。先前的研究指出 triazole 的衍生物在乳癌及胰臟癌具有 抑制癌細胞增殖與誘發凋亡的潛力(Sim, Lee, Ee, & Go, 2008; S. Wang

93

et al., 2010)，本研究透過 MTT assay 方式篩檢出具有抑制癌細胞增殖 效用的化合物，其中 Yao-ram-2-7 即為 triazole 的衍生物。實驗使用人 類肝癌細胞株 Hep 3B，Hep 3B 細胞特點為無 p53 基因，所以無法誘 發與 p53 蛋白相關的細胞凋亡及細胞週期停滯，且其 Raf-1/ERK 蛋白 持續的表現活化，對誘發凋亡的刺激表現出抗性，因此研究認為 Hep 3B 是較為惡性的癌細胞，可代表肝癌末期的情況(Cagnol & Chambard, 2010; Yip-Schneider et al., 2009)。實驗結果發現 Yao-ram-2-7 抑制癌細 胞 Hep 3B 生長與劑量及時間呈正相關，與臨床抗癌用藥 Sorafenib 比

et al., 2010)，本研究透過 MTT assay 方式篩檢出具有抑制癌細胞增殖 效用的化合物，其中 Yao-ram-2-7 即為 triazole 的衍生物。實驗使用人 類肝癌細胞株 Hep 3B，Hep 3B 細胞特點為無 p53 基因，所以無法誘 發與 p53 蛋白相關的細胞凋亡及細胞週期停滯，且其 Raf-1/ERK 蛋白 持續的表現活化，對誘發凋亡的刺激表現出抗性，因此研究認為 Hep 3B 是較為惡性的癌細胞，可代表肝癌末期的情況(Cagnol & Chambard, 2010; Yip-Schneider et al., 2009)。實驗結果發現 Yao-ram-2-7 抑制癌細 胞 Hep 3B 生長與劑量及時間呈正相關，與臨床抗癌用藥 Sorafenib 比

在文檔中 探討單獨使用新穎化學合成物Yao-ram-2-7或合併使用薑黃素對肝癌細胞的抑制並利用連接網路資料庫比對及證實Yao-ram-2-7之生理功能 (頁 88-0)