Doherty Glen A, Byrne Sinead M, Molloy Eamonn S, Malhotra Vikrum, Austin Sandra C, Kay Elaine W, Murray Frank E, Fitzgerald Desmond J
Molecular Medicine Group, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.
BMC Cancer. 2009 Jun 26;9:207. doi: 10.1186/1471-2407-9-207.
Prostaglandin E2 (PGE2) is the major product of Cyclooxygenase-2 (COX-2) in colorectal cancer (CRC). We aimed to assess PGE2 cell surface receptors (EP 1-4) to examine the mechanisms by which PGE2 regulates tumour progression.
Gene expression studies were performed by quantitative RT-PCR. Cell cycle was analysed by flow cytometry with cell proliferation quantified by BrdU incorporation measured by enzyme immunoassay. Immunohistochemistry was employed for expression studies on formalin fixed paraffin embedded tumour tissue.
EP4 was the most abundant subtype of PGE2 receptor in HT-29 and HCA7 cells (which show COX-2 dependent PGE2 generation) and was consistently the most abundant transcript in human colorectal tumours (n = 8) by qRT-PCR (ANOVA, p = 0.01). G0/G1 cell cycle arrest was observed in HT-29 cells treated with SC-236 5 microM (selective COX-2 inhibitor) for 24 hours (p = 0.02), an effect abrogated by co-incubation with PGE2 (1 microM). G0/G1 arrest was also seen with a specific EP4 receptor antagonist (EP4A, L-161982) (p = 0.01). Treatment of HT-29 cells with either SC-236 or EP4A caused reduction in intracellular cAMP (ANOVA, p = 0.01). Early induction in p21WAF1/CIP1 expression (by qRT-PCR) was seen with EP4A treatment (mean fold increase 4.4, p = 0.04) while other genes remained unchanged. Similar induction in p21WAF1/CIP1 was also seen with PD153025 (1 microM), an EGFR tyrosine kinase inhibitor, suggesting EGFR transactivation by EP4 as a potential mechanism. Additive inhibition of HCA7 proliferation was observed with the combination of SC-236 and neutralising antibody to amphiregulin (AR), a soluble EGFR ligand. Concordance in COX-2 and AR localisation in human colorectal tumours was noted.
COX-2 regulates cell cycle transition via EP4 receptor and altered p21WAF1/CIP1 expression. EGFR pathways appear important. Specific targeting of the EP4 receptor or downstream targets may offer a safer alternative to COX-2 inhibition in the chemoprevention of CRC.
前列腺素E2（PGE2）是结直肠癌（CRC）中环氧化酶-2（COX-2）的主要产物。我们旨在评估PGE2细胞表面受体（EP1 - 4），以研究PGE2调节肿瘤进展的机制。
通过定量逆转录聚合酶链反应（RT-PCR）进行基因表达研究。采用流式细胞术分析细胞周期，通过酶免疫测定法检测溴脱氧尿苷（BrdU）掺入量来量化细胞增殖。利用免疫组织化学对福尔马林固定石蜡包埋的肿瘤组织进行表达研究。
EP4是HT - 29和HCA7细胞（显示COX - 2依赖性PGE2生成）中PGE2受体最丰富的亚型，并且通过qRT-PCR在人结直肠癌肿瘤（n = 8）中始终是最丰富的转录本（方差分析，p = 0.01）。用5 microM的SC - 236（选择性COX - 2抑制剂）处理HT - 29细胞24小时后观察到G0/G1细胞周期停滞（p = 0.02），与PGE2（1 microM）共同孵育可消除该效应。用特异性EP4受体拮抗剂（EP4A，L - 161982）也观察到G0/G1停滞（p = 0.01）。用SC - 236或EP4A处理HT - 29细胞导致细胞内cAMP减少（方差分析，p = 0.01）。用EP4A处理后，p21WAF1/CIP1表达早期诱导（通过qRT-PCR）（平均增加倍数4.4，p = 0.04），而其他基因保持不变。用表皮生长因子受体（EGFR）酪氨酸激酶抑制剂PD153025（1 microM）处理也观察到p21WAF1/CIP1的类似诱导，提示EP4介导EGFR反式激活是一种潜在机制。观察到SC - 236与双调蛋白（AR，一种可溶性EGFR配体）中和抗体联合使用对HCA7增殖有相加抑制作用。注意到在人结直肠癌肿瘤中COX - 2和AR定位的一致性。
COX - 2通过EP4受体调节细胞周期转变并改变p21WAF1/CIP1表达。EGFR途径似乎很重要。在结直肠癌化学预防中，特异性靶向EP4受体或其下游靶点可能提供一种比COX - 2抑制更安全的替代方法。
