1. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression Heather A Pangburn*1,2, Hanna Kraus3, Dennis J Ahnen2,3,4 and Pamela L Rice2,3,4 Journal of Carcinogenesis 2005, 4:16

2. Abstract Nonsteroidal anti-inflammatory drugs(NSAIDs) 可以用來降低 colorectal cancaer (CRC) 的死亡率， NSAIDs 可以誘導 CRC 細胞進行 apoptotic ，並且可以抑制腫瘤細胞的生長。 NSAIDs sulindac 的代謝產物 可以 downregulate extracellular-signal regulated kinase ½ (ERK1/2) ，而造成 CRC 細胞進行 apoptosis 。我們這個實驗的主要目的 是要來證實我們的假說， sulindac metabolites 可以抑制或促進 epidermal growth factor (EGF) receptor (EGFR) 表現，因而影響到 extracellular-signal regulated kinase ½ (ERK1/2) 的表現，而達到抑 制 CRC 細胞的生長的效果。 Nonsteroidal anti-inflammatory drugs(NSAIDs) 可以用來降低 colorectal cancaer (CRC) 的死亡率， NSAIDs 可以誘導 CRC 細胞進行 apoptotic ，並且可以抑制腫瘤細胞的生長。 NSAIDs sulindac 的代謝產物 可以 downregulate extracellular-signal regulated kinase ½ (ERK1/2) ，而造成 CRC 細胞進行 apoptosis 。我們這個實驗的主要目的 是要來證實我們的假說， sulindac metabolites 可以抑制或促進 epidermal growth factor (EGF) receptor (EGFR) 表現，因而影響到 extracellular-signal regulated kinase ½ (ERK1/2) 的表現，而達到抑 制 CRC 細胞的生長的效果。 我們主要是利用 HT29 human colon cancer cell 來進行實驗，利用 在細胞的培養基中加入 EGF 、 sulindac sulfide 或 sulindac sulfone 來培 養，並利用 western immunoblotting 觀察細胞中的 phosphorylated EGFG 、 total EGFG 、 phosphorylated ERK1/2 、 total ERK1/2 、 activated caspase-3 和 α-tubulin 的表現量，和 nuclear morphology 來 觀察 apoptotic cells 和存活的細胞數量來判斷 sulindac 對 CRC 細胞生長 的影響情形。 我們主要是利用 HT29 human colon cancer cell 來進行實驗，利用 在細胞的培養基中加入 EGF 、 sulindac sulfide 或 sulindac sulfone 來培 養，並利用 western immunoblotting 觀察細胞中的 phosphorylated EGFG 、 total EGFG 、 phosphorylated ERK1/2 、 total ERK1/2 、 activated caspase-3 和 α-tubulin 的表現量，和 nuclear morphology 來 觀察 apoptotic cells 和存活的細胞數量來判斷 sulindac 對 CRC 細胞生長 的影響情形。

3. Introduction 在美國 CRC 是最常見 cancer 第二死因，每年約有 104950 個案例， 死亡率約 56.29 。在美國人的一生中平均約有 6 ％的機會會得到 CRC ， 所以作者想要研究 NSAIDs 這類藥物對於 CRC 的影響，看是否可以降低 CRC 的死亡率。 Sulindac 在肝臟可以快速的代謝成兩種產物 sulindac sulfide 和 sulindac sulfone ，我們將對此兩種藥物對於 CRC 細胞的 影響加以研究，其中 sulindac sulfone 並不屬於 NSAIDs 這類藥物，但 他對於 CRC 細胞的生長還是有影響，我們將再接下來的實驗中為大家 證實。剛開始認為 NSAIDs sulindac sulfide 會抑制 CRC 細胞生長主要 是因為他可以抑制 cycloxygenase-1 (cox-1) 或 cox-2 的活性，因而使 細胞進行 apoptosis 但在我們後來的實驗中發現，最主要是因為使 EGFR 的表現量下降，才會使細胞進行 apoptosis 。所以我們設計此實 驗來證實 sulindac sulfide 和 sulindac sulfone 會影響 EGFR 的表現量， 進而使 ERK1/2 的表現量下降，而使細胞進行 apoptosis 。 在美國 CRC 是最常見 cancer 第二死因，每年約有 104950 個案例， 死亡率約 56.29 。在美國人的一生中平均約有 6 ％的機會會得到 CRC ， 所以作者想要研究 NSAIDs 這類藥物對於 CRC 的影響，看是否可以降低 CRC 的死亡率。 Sulindac 在肝臟可以快速的代謝成兩種產物 sulindac sulfide 和 sulindac sulfone ，我們將對此兩種藥物對於 CRC 細胞的 影響加以研究，其中 sulindac sulfone 並不屬於 NSAIDs 這類藥物，但 他對於 CRC 細胞的生長還是有影響，我們將再接下來的實驗中為大家 證實。剛開始認為 NSAIDs sulindac sulfide 會抑制 CRC 細胞生長主要 是因為他可以抑制 cycloxygenase-1 (cox-1) 或 cox-2 的活性，因而使 細胞進行 apoptosis 但在我們後來的實驗中發現，最主要是因為使 EGFR 的表現量下降，才會使細胞進行 apoptosis 。所以我們設計此實 驗來證實 sulindac sulfide 和 sulindac sulfone 會影響 EGFR 的表現量， 進而使 ERK1/2 的表現量下降，而使細胞進行 apoptosis 。

4. The EGFR/MAPK/ERK1/2 signaling pathway. We have previously shown that sulindac metabolites inhibit both MEK1/2 and ERK1/2 activation. The goal of this study was to determine if this inhibition is due to downregulation of the EGFR.

5. Material and method Morphologic quantitation of apoptotic cell death 利用 ethidium bromide/acridine orange double-dye morphological assay 來觀察有多少細胞存活和多少細胞進行 apoptosis ，來做數量的統計。 利用 ethidium bromide/acridine orange double-dye morphological assay 來觀察有多少細胞存活和多少細胞進行 apoptosis ，來做數量的統計。 western immunoblotting 1. 將細胞從 plates 上取出，利用冰的 phosphate buffer saline (PBS) 洗一次。 2.2400g 離心 5min 去上清液，再重複上述步驟兩次，注意在所有的過程中都要 盡量保持低溫。 3. 在將離心所得的 pellet 放置到 extraction buffer 30min on ice ，每十分鐘取 出來 vortex 一下。 4. 離心 18000g 4 ℃ 10min 取上清液，在利用 Lowry 的方法來测蛋白質濃度 ( 約 50μg)

6. 5. 利用 sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS PAGE) 來分離蛋白質，再利用 electrotansferred overnight onto immobilon-P polyvinylidene difluoride membranes 上。 6. 在將 membranes 浸泡在含有 Tris-neutral saline with 1 ％ dry milk and 0.05 ％ Tween 20 30min 來做 blooking 。 7. 取出並放入含有 phosphorylated EGFG 、 total EGFG 、 phosphorylated ERK1/2 、 total ERK1/2 、 activated caspase-3 和 α-tubulin primary antibody 的溶液 37 ℃下作用 1 小時 8. 取出之後放到含有 secondary antibody 溶液中作用 1 小時。 9. 放置含有 chemiluminescent substrate 中作用一分鐘 10. 在將所得到的結果拿去测光密度即可知道各種不同的蛋白之含量。

7. EGF induces EGFR and ERK1/2 phosphorylation. HT29 human colon cancer cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 48 h before treatment with vehicle (water), 10, or 100 ng/ml EGF. Cells were harvested 10 min after EGF treatment and lysates prepared for (A) Immunoblotting with antibodies raised against pEGFR(pY1068), total EGFR, ERK1/2, and total ERK1/2; α-tubulin immunoblots of the same lysates served as loading controls. Thegraphs show the densitometry results of the pEGFR bands (B) and pERK1/2 bands (C) normalized for the loading controls.

8. Dose response of sulindac sulfide inhibition of EGFR. HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.1% DMSO), 40, 80, 120, or 160 μM sulindac sulfide, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C). Results shown in figure are representative of 3 separate experiments.

9. Dose response of sulindac sulfone inhibition of EGFR. HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.2% DMSO), 200, 400, 600, or 800 μM sulindac sulfone, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C). Results shown in figure are representative of 3 separate experiments.

10. Dose response and time course of sulindac sulfide inhibition of EGFR. HT29 cells were grown to confluence in medium containing 10% FBS and treated with vehicle (0.1% DMSO), 160, or 180 μM sulindac sulfide for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls.(A) 1 h, 12 h, and 24 h immunoblot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C).

11. Dose response and time course of sulindac sulfone inhibition of EGFR. HT29 cells were grown to confluence in medium containing 10% FBS followed by treatment with vehicle (0.2% DMSO), 400, or 600 μM sulindac sulfone for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h Western blot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C).

12. Effect of the caspase inhibitor, ZVAD, on apoptosis and inhibition of EGFR. HT29 colon cancer cells were grown to confluence in medium containing 10% FBS followed by pretreatment with or without 25 μM zvad for 1 h. Cells were then treated with vehicle (0.2% DMSO) or 600 μM sulfone for 48 h. Cells were harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, and cleaved caspase 3; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show morphological apoptosis results (A) 48 h Western immunoblot results (B) and densitometry of the pEGFR bands (C) and total EGFR bands (D).

13. Result 1.sulindac sulfide 和 sulindac sulfone 都可以使 EGFR 的活性降低， 而使 CRC 細胞進行 apoptosis ，但 sulindac sulfone 的效果比較沒有 這麼好。 2.EGFR 被抑制並不是因為 caspase-3 被活化的原因。 3.sulindac 是直接使 EGFR 的表現量下降。而跟 COX 沒有關係。