目的观察TW-37干预对胰腺癌细胞增殖、凋亡、侵袭及血管生成的影响，探讨其可能机制。
方法应用TW-37干预胰腺癌细胞株BxPC3和HPAC，以转染NF-κB p65 cDNA(p65 cDNA)、靶向NF-κB p65的siRNA(siRNA-p65)细胞及未干预细胞作为对照组。采用MTT和ELISA法检测细胞增殖和细胞凋亡；应用Transwell小室检测细胞侵袭能力；采用体外人脐静脉内皮细胞(HUVECs)成管实验观察细胞培养上清对血管生成的影响；ELISA法检测细胞NF-κB活性；蛋白质印迹法检测细胞NF-κB靶向调节蛋白VEGF、MMP-9表达。
结果TW-37呈浓度和时间依赖性抑制BxPC3、HPAC细胞的增殖，诱导两株细胞的凋亡( A 405值1.29±0.21比0.09±0.01，1.07±0.18比0.08±0.01)，抑制细胞NF-κB活性及NF-κB p65、VEGF、MMP-9蛋白表达，差异均有统计学意义( P值均<0.05)。对照组、0.75 μmol/L TW-37干预组的穿膜细胞数分别为(46.7±5.24)、(10.3±1.26)个/200倍视野；HUVECs形成的血管数分别为(39.4±4.36)、(7.84±1.25)个/200倍视野，差异均有统计学意义( P值均为0.001)。转染p65 cDNA的两株细胞的NF-κB活性较对照组显著增加，用TW-37干预后NF-κB活性下降；转染siRNA-p65的两株细胞的NF-κB活性较对照组显著下降，用TW-37干预后NF-κB活性进一步下降，差异均有统计学意义( P<0.05或<0.01)。转染p65 cDNA对两株细胞凋亡无明显影响，用TW-37干预后凋亡略有变化；转染siRNA-p65的两株细胞凋亡显著增加，用TW-37干预后又进一步增加，差异均有统计学意义( P值均<0.01)。
结论TW-37通过NF-κB抑制胰腺癌细胞增殖、侵袭和血管生成，诱导细胞凋亡。
ObjectiveTo study the effect and mechanisms of TW-37 on cell proliferation, apoptosis, invasion and angiogenesis in pancreatic cancer cells in vitro and further explore the potential mechanism.
MethodsBxPC3 and HPAC cells were pretreated with TW-37 using untransfected or transfected with NF-κB p65 cDNA(p65 cDNA)or NF-κB p65 siRNA(siRNA-p65)cells as controls. Cell viability was determined by MTT assay. Cell apoptosis was assessed by enzyme-linked immunosorbent assay (ELISA). Cell invasion and angiogenesis was detected by Transwell and endothelial tube formation assay of HUVECs. ELISA assay was used to measure the activity of NF-κB, and its target proteins of MMP-9 and VEGF were detected by western blot.
ResultsTW-37 suppressed cell growth and induced apoptosis (A405: 1.29±0.21 vs 0.09±0.01, 1.07±0.18 vs 0.08±0.01), inhibited NF-κB activity and protein expression of NF-κB p65, VEGF and MMP-9(all P<0.05)in a dose- and time-dependent manner. The number of cells that invaded across the matrigel in the transwell chamber was (46.7±5.24) and (10.3±1.26)/×200 in BxPC3 control and 0.75 μmol/L TW-37 group ( P=0.001). The number of tube formation was (39.4±4.36) and (7.84±1.25)/×200, ( P=0.001). NF-κB activity was increased by p65 cDNA transfection, and decreased by TW-37 treatment in both of the two cell lines ( P<0.05). However, NF-κB activity was decreased by p65 siRNA transfection, and greatly decreased by TW-37 treatment in both two cell lines ( P<0.05 or P<0.01). Transfection of p65 cDNA did not significantly affect cell apoptosis. Transfection of p65 siRNA increased cell apoptosis, and greatly increased by TW-37 treatment in both two cell lines (all P<0.01).
ConclusionsTW-37 could inhibit the proliferation, invasion and angiogenesis in pancreatic cancer cells by regulating NF-κB signal pathway.
吴隆超,王琳娜,刘瑞东,等. TW-37调节NF-κB信号通路抑制体外胰腺癌细胞转移的实验研究[J]. 中华胰腺病杂志,2016,16(4)：237-242.
DOI:10.3760/cma.j.issn.1674-1935.2016.04.006版权归中华医学会所有。
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