目的 探討三磷酸腺苷結(jié)合轉(zhuǎn)運(yùn)體G2(ABCG2)與胰腺癌采用吉西他濱化療抵抗的相互關(guān)系。方法 觀察胰腺癌手術(shù)患者的癌和癌旁組織免疫組化及胰腺癌細(xì)胞系中ABCG2的表達(dá),流式細(xì)胞術(shù)測定胰腺癌細(xì)胞系在吉西他濱刺激0、3、12、24、48、72 h后的ABCG2膜表達(dá)結(jié)果,選擇高表達(dá)水平的PANC-1細(xì)胞株進(jìn)行相關(guān)機(jī)制試驗(yàn)。觀察吉西他濱作用0、15、30、45、120、180 min后P-AKT表達(dá)水平,及用PI3K/AKT抑制劑LY294002或AKT siRNA阻斷AKT表達(dá)后ABCG2表達(dá)水平和生存率變化。免疫熒光共聚焦技術(shù)觀察在對(duì)照、吉西他濱、LY294002和吉西他濱+LY294002 4種實(shí)驗(yàn)條件下細(xì)胞膜膜表面的ABCG2蛋白表達(dá)變化。結(jié)果 ABCG2蛋白表達(dá)量在胰腺癌組織中較癌旁組織明顯增高,ABCG2+細(xì)胞在不同病理分化程度的胰腺癌細(xì)胞中均有表達(dá)。胰腺癌細(xì)胞系在吉西他濱刺激后總ABCG2水平無明顯變化,而ABCG2+細(xì)胞數(shù)有明顯增加。PANC-1在吉西他濱刺激后細(xì)胞總ABCG2蛋白表達(dá)無明顯變化;p-AKT蛋白表達(dá)水平呈現(xiàn)增加趨勢;ABCG2+細(xì)胞數(shù)明顯增加,并呈時(shí)間相關(guān)性。LY294002或LY294002+吉西他濱處理細(xì)胞后ABCG2總蛋白表達(dá)無明顯變化,siRNA-AKT2基因敲除或LY294002處理后,腫瘤細(xì)胞對(duì)吉西他濱的敏感性明顯增加。免疫熒光共聚焦表明吉西他濱調(diào)節(jié)胰腺癌PI3K/AKT信號(hào)分子,介導(dǎo)ABCG2質(zhì)膜移位。結(jié)論 吉西他濱活化PI3K/AKT信號(hào)分子,促進(jìn)ABCG2質(zhì)膜移位,抑制胰腺癌化療敏感性。

Objective To investigate the relationship between ATP binding cassette transporter G2 (ABCG2) and gemcitabine chemotherapy resistance in pancreatic cancer. Methods The patient's pancreatic cancer surgery and adjacent tissues were observed by immunohistochemistry and pancreatic cancer cell lines' ABCG2 expression. Pancreatic cancer cell lines ABCG2 membrane expression results were observed by flow cytometry after gemcitabine stimulation (0, 3, 12, 24, 48, and 72 h). Then high expression levels of PANC-1 cell line were selected for mechanisms tests. P-AKT expression levels after gemcitabine action (0, 15, 30, 45, 120, and 180 min), and ABCG2 expression levels and the change of survival rate after blocking AKT expression with the PI3K/AKT inhibitor LY294002 or AKT siRNA were observed. Immunofluorescence confocal was used to observe ABCG2 protein in membrane surface expressions under four experimental conditions (control, gemcitabine, LY294002, gemcitabine + LY294002). Results Gemcitabine enhanced the expression of ABCG2's protein in cellular surface and ABCG2+ cells, which induced chemotherapy-resistance. Furthermore it was found that gemcitabine could regulate ABCG2 translocation of cytoplasm-membrane via activation of PI3K/Akt signaling pathway to mediate pancreatic cancer cells' chemoresistance. Conclusion Gemcitabine can activate PI3K/AKT signaling molecules, promote the shift of the ABCG2 plasma membrane, and restrain pancreatic cancer chemotherapy sensitivity.

天津市應(yīng)用基礎(chǔ)與前沿技術(shù)研究計(jì)劃項(xiàng)目(13JCZDJC31300)