Sulindac metabolites inhibit Sulindac metabolites inhibit epidermal growth factor receptor epidermal growth factor receptor

activation and expressionactivation and expression

Heather A Pangburn*1,2, Hanna Kraus3, Heather A Pangburn*1,2, Hanna Kraus3, Dennis J Ahnen2,3,4 and Pamela L Rice2,3,4Dennis J Ahnen2,3,4 and Pamela L Rice2,3,4

Journal of Carcinogenesis Journal of Carcinogenesis 2005, 2005, 44:16:16

AbstractAbstract Nonsteroidal anti-inflammatory drugs(NSAIDs)Nonsteroidal anti-inflammatory drugs(NSAIDs) 可以用來降低可以用來降低

colorectal cancaer (CRC)colorectal cancaer (CRC) 的死亡率，的死亡率， NSAIDsNSAIDs 可以誘導可以誘導 CRCCRC 細胞進行細胞進行apoptoticapoptotic ，並且可以抑制腫瘤細胞的生長。，並且可以抑制腫瘤細胞的生長。 NSAIDs sulindacNSAIDs sulindac 的代謝產物可的代謝產物可以 以 downregulate extracellular-signal regulated kinase ½(ERK1/2) downregulate extracellular-signal regulated kinase ½(ERK1/2) ，，而造成而造成 CRCCRC 細胞進行細胞進行 apoptosisapoptosis 。我們這個實驗的主要目的是要來證實我們的。我們這個實驗的主要目的是要來證實我們的假說， 假說， sulindac metabolitessulindac metabolites 可以抑制或促進可以抑制或促進 epidermal growth factor epidermal growth factor (EGF) receptor (EGFR)(EGF) receptor (EGFR) 表現，因而影響到表現，因而影響到 extracellular-signal regulated extracellular-signal regulated kinase ½(ERK1/2) kinase ½(ERK1/2) 的表現，而達到抑制的表現，而達到抑制 CRCCRC 細胞的生長的效果。細胞的生長的效果。

我們主要是利用我們主要是利用 HT29 human colon cancer cellHT29 human colon cancer cell 來進行實驗，利用在細胞來進行實驗，利用在細胞的培養基中加入的培養基中加入 EGFEGF 、 、 sulindac sulfide sulindac sulfide 或 或 sulindac sulfonesulindac sulfone 來培養，並利來培養，並利用用 western immunoblotting western immunoblotting 觀察細胞中的 觀察細胞中的 phosphorylated phosphorylated EGFGEGFG 、、 total EGFGtotal EGFG 、 、 phosphorylated ERK1/2phosphorylated ERK1/2 、、 total total ERK1/2ERK1/2 、、 activated caspase-3activated caspase-3 和和 α-tubulinα-tubulin 的表現量，和 的表現量，和 nuclear nuclear morphologymorphology 來觀察 來觀察 apoptotic cells apoptotic cells 和存活的細胞數量來判斷 和存活的細胞數量來判斷 sulindacsulindac 對對CRCCRC 細胞生長的影響情形。細胞生長的影響情形。

IntroductionIntroduction 在美國在美國 CRCCRC 是最常見是最常見 cancercancer 第二死因，每年約有第二死因，每年約有 104950104950 個案例，個案例，死亡率約死亡率約 56.2956.29 。在美國人的一生中平均約有。在美國人的一生中平均約有 66 ％的機會會得到％的機會會得到 CRCCRC ，，所以作者想要研究所以作者想要研究 NSAIDsNSAIDs 這類藥物對於這類藥物對於 CRCCRC 的影響，看是否可以降的影響，看是否可以降

低低 CRCCRC 的死亡率。 的死亡率。 SulindacSulindac 在肝臟可以快速的代謝成兩種產物 在肝臟可以快速的代謝成兩種產物 sulindac sulfide sulindac sulfide 和 和 sulindac sulfonesulindac sulfone ，我們將對此兩種藥物對於，我們將對此兩種藥物對於CRC CRC 細胞的影響加以研究，其中細胞的影響加以研究，其中 sulindac sulfonesulindac sulfone 並不屬於並不屬於 NSAIDsNSAIDs這類藥物，但他對於這類藥物，但他對於 CRC CRC 細胞的生長還是有影響，我們將再接下來的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。剛開始認為實驗中為大家證實。剛開始認為 NSAIDs sulindac sulfideNSAIDs sulindac sulfide 會抑制會抑制CRCCRC 細胞生長主要是因為他可以抑制細胞生長主要是因為他可以抑制 cycloxygenase-1 (cox-1)cycloxygenase-1 (cox-1) 或或cox-2cox-2 的活性，因而使細胞進行的活性，因而使細胞進行 apoptosisapoptosis 但在我們後來的實驗中發現，但在我們後來的實驗中發現，最主要是因為使最主要是因為使 EGFREGFR 的表現量下降，才會使細胞進行的表現量下降，才會使細胞進行 apoptosisapoptosis 。。所以我們設計此實驗來證實所以我們設計此實驗來證實 sulindac sulfide sulindac sulfide 和 和 sulindac sulfonesulindac sulfone 會會影響影響 EGFREGFR 的表現量，進而使的表現量，進而使 ERK1/2ERK1/2 的表現量下降，而使細胞進行的表現量下降，而使細胞進行apoptosisapoptosis 。。

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.

Material and methodMaterial and methodMorphologic quantitation of apoptotic cell deathMorphologic quantitation of apoptotic cell death 利用 利用 ethidium bromide/acridine orange double-dye morphological ethidium bromide/acridine orange double-dye morphological

assayassay 來觀察有多少細胞存活和多少細胞進行來觀察有多少細胞存活和多少細胞進行 apoptosisapoptosis ，來做數量的統計。，來做數量的統計。 western immunoblottingwestern immunoblotting1.1. 將細胞從將細胞從 platesplates 上取出，利用冰的上取出，利用冰的 phosphate buffer saline (PBS)phosphate buffer saline (PBS) 洗一次。洗一次。2.2400g2.2400g 離心 離心 5min5min 去上清液，再重複上述步驟兩次，注意在所有的過程中都去上清液，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。要盡量保持低溫。3.3. 在將離心所得的在將離心所得的 pelletpellet 放置到放置到 extraction buffer 30min on iceextraction buffer 30min on ice ，每十分鐘，每十分鐘取出來取出來 vortexvortex 一下。一下。4.4. 離心離心 18000g 4℃ 10min 18000g 4℃ 10min 取上清液，在利用取上清液，在利用 LowryLowry 的方法來测蛋白質濃度的方法來测蛋白質濃度(( 約約 50μg)50μg)

5.5. 利用利用 sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS PAGE)PAGE) 來分離蛋白質，再利用來分離蛋白質，再利用 electrotansferred overnight onto electrotansferred overnight onto immobilon-P polyvinylidene difluoride membranesimmobilon-P polyvinylidene difluoride membranes 上。上。

6.6. 在將在將 membranesmembranes 浸泡在含有浸泡在含有 Tris-neutral saline with 1Tris-neutral saline with 1 ％ ％ dry milk and dry milk and 0.050.05 ％ ％ Tween 20 30minTween 20 30min 來做 來做 blookingblooking 。。

7.7. 取出並放入含有取出並放入含有 phosphorylated EGFGphosphorylated EGFG 、、 total EGFGtotal EGFG 、 、 phosphorylated phosphorylated ERK1/2ERK1/2 、、 total ERK1/2total ERK1/2 、、 activated caspase-3activated caspase-3 和和 α-tubulin primary α-tubulin primary antibodyantibody 的溶液的溶液 37℃37℃ 下作用下作用 11 小時小時

8.8. 取出之後放到含有取出之後放到含有 secondary antibodysecondary antibody 溶液中作用溶液中作用 11 小時。小時。9.9. 放置含有 放置含有 chemiluminescent substratechemiluminescent substrate 中作用一分鐘中作用一分鐘10.10. 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。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。

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.

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 performedon 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.

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 performedon 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.

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).

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).

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).

ResultResult1.sulindac sulfide 1.sulindac sulfide 和 和 sulindac sulfonesulindac sulfone 都可以使都可以使 EGFREGFR 的活性降低，的活性降低，而使而使 CRCCRC 細胞進行細胞進行 apoptosisapoptosis ，但，但 sulindac sulfonesulindac sulfone 的效果比較的效果比較沒有這麼好。沒有這麼好。2.EGFR2.EGFR 被抑制並不是因為被抑制並不是因為 caspase-3caspase-3 被活化的原因。被活化的原因。3.sulindac3.sulindac 是直接使是直接使 EGFREGFR 的表現量下降。而跟的表現量下降。而跟 COXCOX 沒有關係。沒有關係。