當細胞受到刺激活化磷脂?A2（phospholipase A2）時，細胞膜的磷脂質將會被分解成花生四烯酸（arachidonic acid），花生四烯酸再經由環氧合酵素（cyclooxygenase，COX）分解成各種前列腺素，前列腺素在每一種組織細胞皆能產生，目前已有許多報告指出前列腺素和細胞凋亡（apoptosis）有關，但其作用機制仍不清楚。細胞凋亡是一種受刺激而引起的細胞死亡模式，目的是為了清除多餘、突變、損傷或老化的細胞，粒線體對於細胞凋亡扮演很重要的角色，許多物質會作用在粒線體上引發粒線體膜電位（mitochondrial transmembrane potential，△ψm）下降，釋放cytochrome c等物質活化caspases造成細胞核內DNA被分解成片段，使細胞發生凋亡。本研究探討人類肺腫瘤細胞（A549）在加入NS-398（COX-2抑制劑）所引發的細胞凋亡是否經由前列腺素調控粒線體所引起的，本實驗以不同劑量NS-398處理A549細胞觀察細胞凋亡情形，並於處理48小時後偵測PGF2α、PGI2及TXA2在細胞外、細胞質中及粒線體內的量，結果發現在加入100 μM NS-398處理48小時後，每毫升所含的細胞數目和控制組相比減少53%，細胞凋亡比率約為控制組的7.5倍，證明NS-398的確會抑制細胞的增殖，並且會促使細胞發生凋亡。A549培養48小時後細胞外控制組的PGF2α、TXB2及6-keto- PGF1α濃度分別為細胞質中的6267、44及140倍，加入100 μM NS-398處理48小時後細胞外的PGF2α、TXB2及6-keto- PGF1α濃度分別為細胞質的3887、77及61倍，可見前列腺素會經由擴散作用及前列腺素運輸蛋白（prostaglandin transporter；PGT）被運送至細胞外，而控制組粒線體內的PGF2α、TXB2及6-keto- PGF1α濃度分別為細胞質的3187、4.6及0.9倍，加入100 μM NS-398處理48小時後粒線體內的PGF2α、TXB2及6-keto- PGF1α濃度分別為細胞質中的3793、5.7及1.4倍，由此可推測粒線體上具有類似PGT等構造，可將前列腺素運輸至粒線體中，在加入100 μM NS-398處理48小時後粒線體內PGF2α及TXB2量分別為控制組的2.8倍及2.9倍，這代表隨著細胞凋亡比率升高，粒線體內PGF2α及TXB2相對含量也明顯增加，所以PGF2α等前列腺素在細胞質產生後，可能會經由PGT或其他管道運送至細胞外及粒線體。△ψm下降是細胞發生凋亡的一項重要指標，本實驗A549細胞經100 μM NS-398處理後，由△ψm下降引發的細胞凋亡為66%，故推測PGF2α等前列腺素在細胞受到NS-398刺激後會經PGT等構造進入粒線體中引起
△ψm下降，使細胞發生凋亡。加入PGT抑制劑BrCG後，實驗發現100 μM NS-398+
3 μM BrCG組及100 μM NS-398+10 μM BrCG組細胞數分別為100 μM NS-398組的144%及193%，可見BrCG可恢復NS-398所抑制的細胞增殖。100 μM NS-398+3 μM BrCG組細胞凋亡比例僅為100 μM NS-398組的71%，100 μM NS-398+10 μM BrCG組更只有100 μM NS-398組的48%，可見BrCG的加入的確可抑制NS-398所促進的細胞凋亡比率。100 μM NS-398+3 μM BrCG及100 μM NS-398+10 μM BrCG處理後在粒線體內的PGF2α濃度分別較100 μM NS-398組減少為60%及35%，以上實驗顯示當PGT受到抑制時，隨著進入粒線體內的PGF2α濃度降低，細胞凋亡的比例也隨之降低。本實驗推論當人類肺腫瘤細胞株A549受到NS-398這種細胞凋亡刺激時，PGF2α等前列腺素會經由PGT運送至粒線體內，迫使△ψm瓦解後使得PTP打開，引發細胞進行凋亡。

Prostaglandins are derived from arachidonic acid liberated from phospholipids in the cell membrane by the action of PLA2 enzymes. Prostaglandins are synthesized via the cyclooxygenase（COX）pathway in a variety of cells in response to various physiological and pathophysiological stimuli. Prostaglandins are quickly transported outside the cells and act as autocrine or paracrine mediators in the vicinity of their sites of production to maintain local homeostasis. In every second, several millions of human body cells undergo apoptosis. All cells require at least one pathway for apoptosis. Mitochondria play a central role in the regulation of apoptosis. Numerous stimuli trigger an increase in mitochondrial membrane permeability, and the permeability transition pores（PTP）are then opened. Opening of the pores has dramatic consequences on mitochondrial function, including mitochondrial transmembrane potential（△ψm）collapse, and efflux of cytochrome c and apoptosis-inducing factors from the mitochondria. These products are necessary for nuclear apoptosis to occur. Selective COX-2 inhibitors such as NS-398 have been reported to induce apoptosis in a variety of cancer cell lines. Apoptosis in A549 cells, which was confirmed by triple-combination-labeling staining and detection of chromatin condensation, appeared within 48 h after NS-398 treatment. Incubation of the cell with 100 μmol/L NS-398 induced apoptosis（54%）, and inhibited cell proliferation （53%）. To determine whether prostaglandins are relevant to apoptosis, PGF2α, PGI2 and TXA2 concentrations of culture medium, cytosol and mitochondria were extracted and assayed by ELISA. In this study, PGF2α, 6-keto-PGF1α and TXB2 concentrations of the control medium were 6267-fold, 44-fold and 140-fold higher than those of the cytosol, respectively. After NS-398 treatment（100 μmol/L for 48 h）, PGF2α, 6-keto-PGF1α and TXB2 concentrations of culture medium were 3887-fold, 77-fold and 61-fold higher than that of the cytosol, respectively. It indicates that prostaglandins can efflux from cells by use of simple diffusion and prostaglandin transporter（PGT）. PGF2α, 6-keto-PGF1α and TXB2 concentrations of the mitochondrial matrix of control group were 3187-fold, 4.6-fold and 0.9-fold higher than those of the cytosol, respectively. PGF2α, 6-keto-PGF1α and TXB2 concentrations in the mitochondrial matrix in 100 μmol/L NS-398 treatment group were 3793-fold, 5.7-fold and 1.4-fold higher than those of the cytosol, respectively. Surprisingly, PGF2α was transported to mitochondria with high efficiency, whereas 6-keto-PGF1α was not transported to mitochondria. TXB2 was transported to mitochondria with low efficiency. Thus it is possible that PGT is also present in the membrane of the mitochondria. NS-398 treatment enhanced PGF2α and TXB2 concentrations inside mitochondria compared with the control by 2.77-fold and 2.87-fold, respectively. Consistent with apoptosis, prostaglandins of mitochondria were markedly increased in A549 cells. It is therefore suggested that prostaglandins are transported to mitochondria in order to induce apoptosis. To support this hypothesis, PGT inhibitor, BrCG, was used to treat the cell. The results showed that apoptosis was inhibited in A549 cells by NS-398 treatment （48%）in response to BrCG. BrCG can recover cell proliferation ability that was inhibited by NS-398 treatment. Incubation with 10 μmol/L BrCG reduced PGF2α concentration in mitochondrial matrix by a 35% level. These results suggest that the proapoptotic effect of NS-398 in A549 cells was achieved by promoting the uptake of prostaglandins into mitochondria through the PGT.

Andrews P, Krygier S and Daniel D. Dihydrotestosterone（DHT）modulates the ability of NSAIDs to induce apoptosis of prostate cancer cells. Cancer Chemother Pharmacol 2002；49：179-86.

Bennett A and Stockley HL. The contribution of prostaglandins in the muscle of human isolated small intestine to neurogenic responses. Br J Pharmacol 1977 ；61：573-8.

Bito LZ. Accumulation and apparent active transport of prostaglandins by some rabbit tissues in vitro. J Physiol（London）1972；221：371-87.

Burleson FG, Chambers TM and Wiedbrauk DL. Virology：a laboratory manual, Academic Press Inc, California, 1992, pp. 19-24.

Cao Y and Prescott SM. Many actions of cyclooxygenase-2 in cellular dynamics and in cancer. J Cell Physiol 2002；190：279-86.

Chan BS, Satriano JA, Pucci M and Schuster VL. Mechanism of prostaglandin E2 transport across the plasma membrane of HeLa cells and Xenopus Oocytes expressing the prostaglandin transporter "PGT". J Biol Chem 1998；273：6689-97.

Coceani F. Prostaglandins and the central nervous system. Arch Intern Med 1974 ；133：119-29.

Cullen L, Kelly L, Connor SO and Fitzgerald DJ. Selective cyclooxygenase-2 inhibition by nimesulide in man. J Pharmacol Exp Therap 1998；28：578-82.

DeWitt DL and Smith WL. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci USA 1988；85：1412-6.

Endo S, Nomura T, Chan BS, Lu R, Pucci ML, Bao Y and Schuster VL. Expression of PGT in MDCK cell monolayers：polarized apical localization and induction of active PG transport. Am J Physiol Renal Physiol 2002；282：F618-22.

Evan GI and Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature 2001；411：342-8.

Ferri KF, Jacotot E, Blanco J, Este JA, Zamzami A, Susin SA, Brothers G, Reed JC, Penninger JM and Kroemer G. Apoptosis control in syncytia induced by the HIV-1-envelope glycoprotein complex. Role of mitochondria and caspases. J Exp Med 2000；192：1081-92.

Fitzgerald GA and Patrono C. The coxibs. Selective inhibitors of cyclooxygenase-2. N Engl J Med 2001；345：433-42.

Flower RJ. The development of COX2 inhibitors. Nat Rev Drug Discov 2003；2：171-91.

Foglieni C, Meoni C and Davalli AM. Fluorescent dyes for cell viability：an application on prefixed conditions. Histochem Cell Biol 2001；115：223-9.

Fuller KM and Arriaga EA. Advances in the analysis of single mitochondria. Curr Opin Biotechnol 2003；14：35-41.

Gerber JG, Anderson RJ, Schrier RW and Nies AS. Prostaglandins and the regulation of renal circulation and function. Adv Prostaglandin Thromboxane Leukot Res 1982；10：227-54.

Grimm S, Bauer MK, Baeurle PA and Schulze-Osthoff K. Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis. J Cell Biol 1996；134：13-23.

Gupta S. Molecular steps of death receptor and mitochondrial pathways of apoptosis. Life Sci 2001；69：2957-64.

Hanahan D and Weinberg RA. The hallmarks of cancer. Cell 2000；100：57-70.

Hawk ET, Viner JL, Dannenberg A and DuBois RN. COX-2 in cancer-a player that's defining the rules. J Natl Cancer Inst 2002；94：545-6.

Hayflick L. Mortality and immortality at the cellular level. A review. Biochemistry 1997；62：1180-90.

Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilent L, DuBois G, Lazebnik Y, Zervos AS, Fernandes-Alnemri T and Alnemri ES. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts IAP-caspase interaction. J Biol Chem 2001；17：17.

Hirsh PD, Hillis LD, Campbell WB, Firth BG and Willerson JT. Release of prostaglandins and thromboxane into the coronary circulation in patients with ischemic heart disease. N Engl J Med 1981 ；304：685-91.

Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM and Chen CS. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 2000；275：11397-403.

Hu KQ, Yu CH, Mineyama Y, McCracken JD, Hillebrand DJ and Hasan M. Inhibited proliferation of cyclooxygenase-2 expressing human hepatoma cells by NS-398, a selective COX-2 inhibitor. Int J Oncol 2003；22：757-63.
Hussain T, Gupta S and Mukhtar H. Cyclooxygenase-2 and prostate carcinogenesis. Cancer Lett 2003；191：125-35.

Jacobson MD, Burne JF and Raff MC. Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J 1994；13：1899-910.

Johnson M, Carey F and McMillan RM. Alternative pathways of arachidonate metabolism: prostaglandins, thromboxane and leukotrienes. Essays Biochem 1983；19：40-141.

Kamijo T, Sato T, Nagatomi Y and Kitamura T. Induction of apoptosis by cyclooxygenase-2 inhibitors in prostate cancer cell lines. Int J Urol 2001；8：S35-9.

Kanai N, Lu R, Satriano JA, Bao Y, Wilkoff AW and Schuster VL. Identification and characterization of a prostaglandin transporter. Science 1995；268：866-9.

Katori M and Majima M. Cyclooxygenase-2：its rich diversity of roles and possible application of its selective inhibitors. Inflamm Res 2000；49：367-92.

Katz RL and Katz GJ. Prostaglandins--basic and clinical considerations. Anesthesiology 1974 ；40：471-93.

Kawamura T, Horie S, Maruyama T, Akira T, Imagawa T and Nakamura N. Prostaglandin E1 transported into cells blocks the apoptotic signals induced by nerve growth factor deprivation. J Neurochem 1999；72：1907-14.

Kennedy I, Coleman RA, Humphrey PPA, Levy GP and Lumley P. Studies on the characterization of prostanoid receptors：a propesed classification. Prostaglandins 1982；24：667-89.

Konturek SJ, Tasler J, Kwiecien N, Cieszkowski M and Obtulowicz W. Mechanisms of the inhibitory action of prostaglandins on meal-induced gastric secretion. Digestion 1978 ；17：281-90.

Kosaka T, Miyata A, Ihara H, Hara S, Sugimoto T, Takeda O, Takahashi E and Tanabe T. Characterization of the human gene (PTGS2) encoding prostaglandin-endoperoxide synthase 2. Eur J Biochem 1994 ；221：889-97.

Kraemer SA, Meade EA and DeWitt DL. Prostaglandin endoperoxides synthase gene structure：identification of the transcriptional start site and 5'-flanking regulatory sequences. Arch Biochem Biophys 1992；293：391-400.

Kroemer G, Dallaporta B and Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 1998；60：619-42.

Leahy KM, Ornberg RL, Wang Y, Zweifel BS, Koki AT and Masferrer JL. Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. Cancer Res 2002；62：625-31.

Lecomte M, Laneuville O, Ji C, DeWitt DL and Smith WL. Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. J Biol Chem 1994 ；269：13207-15.

Levine RA, Kohen KR, Schwartzel EH Jr and Ramsay CE. Prostaglandin E2-histamine in interactions on cAMP, cGMP, and acid production in isolated fundic glands. Am J Physiol 1982；242：G21-6.

Li M, Wu X and Xu XC. Induction of apoptosis by cyclooxygenase-2 inhibitor NS398 through a cytochrome c-dependent pathway in esophageal cancer cells. Int J Cancer 2001；93：218-23.
Lipsky PE and Isakson PC. Outcome of specific COX-2 inhibition in rheumatoid arthritis. J Rheumatol 1997；24：9-14.

Liu X, Kim CN, Yang J, Jemmerson R and Wang X. Induction of apoptotic program in cell- free extracts：Requirement for dATP and cytochrome c. Cell 1996；86：147-57.

Liu XH, Yao S, Kirschenbaum A and Levine AC. NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells. Cancer Res 1998；58：4245-9.

Lu R, Kanai N, Bao Y and Schuster VL. Cloning, in vitro expression, and tissue distribution of a human prostaglandin transporter cDNA（hPGT）. J Clin Invest 1996；98：1142-9.

Merlie JP, Fagan D, Mudd J and Needleman P. Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase（cyclooxygenase）. J Biol Chem 1988；263：3550-3.

Milton AS and Wendlandt S. The effect of different environmental temperatures on the hyperpyrexia produced by the intraventricular injection of pyrogen, 5-hydroxytryptamine and prostaglandin E 1 in the conscious cat. J Physiol (Paris) 1971 ；63：340-2.

Montelione GT, Wuthrich K, Burgess AW, Nice EC, Wagner G, Gibson KD and Scheraga HA. Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints. Biochemistry 1992 14；31：236-49.

Morita I. Distinct functions of COX-1 and COX-2. Prostaglandins Other Lipid Mediat 2002；68-69：165-75.
Nakata H, Uemura Y, Kobayashi M, Harada R and Taguchi H. Cyclooxygenase-2 inhibitor NS-398 suppresses cell growth and constitutive production of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells. Cancer Sci 2003；94：173-80.

Newmeyer DD and Ferguson-Miller S. Mitochondria：releasing power for life and unleashing the machineries of death. Cell 2003；112：481-90.

Ogino K, Hatanake K, Kawamura M, Katori M and Harada Y. Evaluation of pharmacological profile of meloxicam as an anti-inflammatory agent, with particular reference to its relative selectivity for cyclooxygenase-2 over cyclooxygenase-1. Pharmacol 1997；55：44-53.

Oida H, Hirata M, Sugimoto Y, Ushikubi F, Onishi H, Mizuno N, Ichikawa A and Narumiya S. Expression of messenger RNA for the prostaglandin D receptor in the leptomeninges of the mouse brain. FEBS Lett. 1997；417：53-6

Padmanabhan K, Padmanabhan KP, Tulinsky A, Park CH, Bode W, Huber R, Blankenship DT, Cardin AD and Kisiel W. Structure of human des(1-45) factor Xa at 2.2 A resolution. J Mol Biol 1993 ；232：947-66.

Piazza GA, Rahm ALK, Krutzsch M, Sperl G, Paranka NS, Gross PH, Brendel K, Burt RW, Alberts DS, Pamukcu R and Ahnen DJ. Antineoplastic drugs sulindac sulfide and sulfone inhibit cell growth by inducing apoptosis. Cancer Res 1995；55：3110-6.

Picot D, Loll PJ and Garavito RM. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature 1994 ；367：243-9.

Pucci ML, Bao Y, Chan B, Itoh S, Lu R, Copeland NG, Gilbert DJ, Jenkins NA and Schuster VL. Cloning of mouse prostaglandin transporter PGT cDNA：species-specific substrate affinities. Am J Physiol 1999；277：R734-41

Ravagnan L, Roumier T and Kroemer G. Mitochondria, the killer organelles and their weapons. J Cell Physiol 2002；192：131-7.

Robert A. Prostaglandins: effects on the gastrointestinal tract. Clin Physiol Biochem 1984；2：61-9.

Schuster VL. Prostaglandin transport. Prostaglandins Other Lipid Mediat 2002 ；68-69：633-47.

Schuster VL. Molecular mechanisms of prostaglandin transport. Annu Rev Physiol 1998；60：221-42.

Shay JW and Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer 1997；33：787-91.

Shivapurkar N, Reddy J, Chaudhary PM and Gazdar AF. Apoptosis and lung cancer：a review. J Cell Biochem 2003；88：885-98.

Skobe M and Fusenig NE. Tumorigenic conversion of immortal human keratinocytes through stromal cell activation. Proc Natl Acad Sci USA 1998；95：1050-5.

Smith WL, DeWitt DL and Garavito RM. Cyclooxygenases：structural, cellular, and molecular biology. Ann Rev Biochem 2000；69：145-82.

Song X, Lin HP, Johnson AJ, Tseng PH, Yang YT, Kulp SK and Chen CS. Cyclooxygenase-2, player or spectator in cyclooxygenase-2 inhibitor-induced apoptosis in prostate cancer cells. J Natl Cancer Inst 2002；94：585-91.

Steinbach G, Lynch PM, Philips RK, Wallace MH, Hawk E, Gordon GB, Wakabayashi N, Saunders B, Shen Y, Fujimura T, Su LK and Levin B. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med 2000；342：1946-52.

Subbaramaiah K and Dannenberg AJ. Cyclooxygenase-2：a molecular target for cancer prevention and treatment. Trends Pharmacol Sci 2003；24：96-102.

Toh H. Prostaglandin endoperoxide synthase contains an EGF-like domain. FEBS Lett. 1989 ；258：317-9.

Thor P, Konturek JW, Konturek SJ and Anderson JH. Role of prostaglandins in control of intestinal motility. Am J Physiol 1985 ；248：G353-9.

Turcotte LP. Mitochondria：biogenesis, structure, and function-symposium introduction. Med Sci Sports Exerc 2003；35：82-5.

Turini ME and DuBois RN. Cyclooxygenase-2：a therapeutic target. Annu Rev Med 2002；53：35-57.

Vane JR, Bakhle YS and Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 1998；38：97-120.

Vezza R, Rokach J and Fitzgerald GA. Prostaglandin F2α receptor-dependent regulation of prostaglandin transport. Mol Pharmacol 2001；59：1506-13.

Xu XM, Tang JL, Chen X, Wang LH and Wu KK. Involvement of two Spl elements in basal endothelial prostaglandin H synthase-1 promoter activity. J Biol Chem 1997；272：6943-50.

Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001；15：2922-33.

Willams CS, Mann M and Dubois RN. The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 1999；18：7908-16.

Williams PI, Hosie J and Scott DL. Etodolac therapy for osteoarthritis：a double-blind, placebo-controlled trial. Current Med Res Opinion 1989；11：463-70.

Yamazaki R, Kusunoki N, Matsuzaki T, Hashimoto S and Kawai S. Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells. FEBS Lett 2002；531：278-84.

Yokoyama C, Takai T and Tanabe T. Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence. FEBS Lett 1988；231：347-51.

Yusuf K, Smith SD, Levy R, Schaiff WT, Wyatt SM, Sadovsky Y and Nelson DM. Thromboxane A2 limits differentiation and enhances apoptosis of cultured human trophoblasts. Pediatr Res 2001；50：203-9.

Zhang X, Morham SG, Langenbach R and Young DA. Malignant transformation and antineoplastic actions of nonsteroidal anti-inflammatory drugs（NSAIDs）on cyclooxygenase-null embryo fibroblasts. J Exp Med 1999；190：451-9.