胰腺癌内质网应激微环境活化核转录因子-κB通路对内质网分子伴侣葡萄糖调节蛋白78的调控作用

Nuclear factor-κB signaling pathway is activated by endoplasmic reticulum stress microenvironment in pancreatic cancer and regulates endoplasmic reticulum chaperon glucose-regulated protein 78

目的探讨内质网应激(ERS)肿瘤微环境对胰腺癌中核转录因子(NF)-κB信号通路的激活效应,以及NF-κB对ERS分子伴侣葡萄糖调节蛋白78(GRP78)的调控作用。方法采用Western印迹法检测经ERS诱导剂毒胡萝卜素(Tg)处理后人胰腺癌细胞系PANC-1细胞中ERS分子伴侣GRP78和NF-κB信号通路相关分子的表达情况,以及RNA干扰NF-κB/P65表达后GRP78的表达情况。通过反复Tg处理PANC-1细胞,建立ERS诱导细胞系PANC-ERS,采用细胞计数试剂盒(CCK)-8比色法检测其增殖能力,应用Transwell迁移试验检测其迁移能力。同时,采用Western印迹法检测PANC-ERS细胞中GRP78和NF-κB信号通路相关分子的表达情况。结果 Tg 2.5、5.0μg/mL处理组GRP78条带的光密度值分别为555 855.3±28 310.3、945 831.0±20 643.2,均显著高于未处理组的352 606.7±15 843.8(P值均<0.05),较未处理组分别上调了(1.57±0.05)和(2.64±0.13)倍。Tg 2.5、5.0μg/mL处理组NF-κB/P65条带的光密度值分别为1 470 315.0±18 036.6、2 231 275.3±218 250.8,均显著高于未处理组的1 061 648.3±61 723.7(P值均<0.05),较未处理组分别上调了(1.38±0.05)和(2.07±0.16)倍。Tg 2.5、5.0μg/mL处理组的IKBα条带的光密度值分别为228 153.3±325 929.1、106 070.0±6 550.5,均显著低于未处理组的318 308.3±20 897.7(P值均<0.05),较未处理组分别下调了(72.2±13.9)%和(32.9±2.1)%。shNF-κB/P65干扰后NF-κB/P65和GRP78的光密度值分别为714 109.7±12 774.6和467 384.3±10 745.5,均显著低于shSCR干扰后的1 448 839.3±15 362.5和894 971.3±6 772.0(P值均<0.05),下调幅度分别为(47.6±0.5)%和(50.4±2.3)%。培养2、3、4d的PANC-ERS细胞的光密度值分别为0.557±0.066、0.883±0.082、1.375±0.125,分别显著高于PANC-1细胞的0.445±0.059、0.686±0.069、0.948±0.114(P值均<0.05)。Transwell迁移实验显示,光学显微镜下见穿过小室的PANC-ERS细胞数为171.2±9.4,显著多于PANC-1细胞的127.7±8.9(P<0.05)。正常培养基培养3代后的PANC-ERS细胞中GRP78和NF-κB/P65条带的光密度值分别为1 147 799.3±47 206.4和1 387 829.0±34 749.2,均显著高于PANC-1细胞中的656 958.0±12 639.2和903 576.0±7 672.9(P值均<0.05),较PANC-1细胞分别上调(1.76±0.06)和(1.55±0.02)倍;PANC-ERS细胞与PANC-1细胞中NF-κB的抑制蛋白IKBα的光密度值分别为345 592.3±13 767.2和319 723.3±1 035.6,差异无统计学意义(P>0.05)。结论体外模拟胰腺癌ERS微环境,NF-κB信号通路可被激活,GRP78表达上调。ERS微环境下GRP78表达上调可能与NF-κB信号通路激活有关。慢性的ERS刺激可增高胰腺癌细胞表型的恶性程度,胰腺癌的高度恶性可能与其ERS微环境相关。

Objective To explore the role of endoplasmic reticulum stress （ERS） micrcenvironment for activating nuclear factor （NF）-κB signaling pathway and the regulation of NF-κB for glucose-regulated protein 78 （GRP78, molecular chaperones of ERS）. Methods The pancreatic cancer cell line, P,NNC-1, was treated with endeplasrnic reticulum stress inducer thepsigargin （Tg）, and then the changes of GRP78 and the molecules in NF-κB signaling pathway were measured by Western blotting. GRP78 protein was also detected with NF-κB/P65 down-regulation by RNA interference. A ceil line named PANC-ERS was established through treating PANC-1 with Tg periodically and repeatedly. CCK-8 proliferation assay and transwell migration assay were performed to evaluate its proliferation and migration activity. Meanwhile, Western blot was also conducted to detect the protein levels of GRP78 and the molecules in NF-κB signaling pathway. Results The optical density values of the binds of GRP78 treated with 2.5 μg/mL and 5.0 μg/mL Tg were 555 855.3 ± 28 310.3 and 945 831.0 ± 20 643.2, which were Significantly higher than that in untreated group （352 606.7± 15 843.8, all P〈0.05）. The up- regulation of optical density values of the binds of GRP78 treated with 2.5 μg/mL and 5.0 μg/ml- Tg were （ 1.57 ± 0.05） and （2.64 ± 0. 13） folds as compared with untreated binds. The optical density values of the binds of NF-κB/P65 treated with 2.5 pg/mL and 5.0 μg/rnL Tg were 1 470 315.0 ± 18 036.6 and 2 231 275.3 ± 218 250.8, respectively, which were significantly higher than those in untreated group （1 061 648.3±61 723.7, both P 0.05）. The up-regulation of optical density values of the binds of NF-κB/P65 treated with 2. 5 μg/mL and 5.0 pg/mL Tg were （ 1.38 ± 0.05） and （2.07 ± 0.16） folds as compared with untreated binds. The optical density values of the binds of IκBcx treated with 2.5 μg/mL and 5.0 μg/mL Tg were 228 153.3 ± 325 929.1 and 106 070.0 ±6 550.5, respectively, which were significantly lower than those in untreated group （318 308.3 ± 20 897.7, both P〈0.05）. The down-regulation of optical density values of the binds of IκBcc treated with 2.5 pg/mL and 5.0 μg/mL Tg were （72.2± 13.9）% and （32.9± 2.1 ）% as compared with untreated binds. The optical density values of the binds of NF-κB/P65 and GRP78 by shNF-κB/P65 interference were 714 109.7 ± 12 774.6 and 467 384.3 ± 10 745.5, respectively, which were significantly lower than that interfered by shSCR （1 448 839.3 ± 15 362.5 and 894 971.3± 6 772.0, both P〈0.05） ; NF-κB/P65 and GRP78 were correspondingly down-regulated to （47.6 ± 0.5） % and （50.4 ± 2.3） %. The optical density values of PANC-ERS were 0. 557 ± 0. 066, 0. 883 ± 0. 082, and 1. 375±0. 125 on day 2, 3 and 4 of culture, respectively, which were significantly higher than that of PANC-1 （0. 445 ± 0. 059, 0. 686 ± 0. 069, 0. 948 ± 0.114, all P〈 0.05）. Transwell migration assay showed the number of PANC-ERS cells migrating to the other side of the chamber was 171.2 ± 9.4, which was much more than PANC-1 （127.7 ± 8.9, P〈0.05）. The optical density values of the binds of GRP78 and NF-κB/P65 were 1 147 799.3~47 206.4 and 1 387 829.0 ± 34 749.2 in PANC-ERS cells cultured with normal medium for three days, which were both significantly higher than those in PANC-1 cells （656 958.0± 12 639.2 and 903 576.0± 7 672.9, both P〈0. 05） ; GRP78 and NF-κB/P65 were up-regulated by （ 1.76 ± 0.06） and （ 1.55 ± 0.02） folds in PANC-ERS cells, respectively. The optical density values of the binds of IκBcc in PANC-ERS and PANC-1 were 345 592.3 ± 13 767.2 and 319 723.3 ± 1 035.6, and there was no significant difference between them （P〉0.05）. Conclusion NF-κB signaling pathway can be activated in a stimulated endoplasmic reticulum stress microenvironment in vitro with GRP78 increase. Up-regulation of GRP78 in endoplasmic reticulum stress may correlate with NF-κB signaling pathway activation. Chronic stimulation of endoplasmic reticulum stress can result in more malignant phenotype of pancreatic cancer. The malignancy of pancreatic cancer may be related to endoplasmic reticulum stress microenvironment. （Shanghai Med J, 2014, 37= 686-690）