Huang Zhi-qiang, Buchsbaum Donald J, Raisch Kevin P, Bonner James A, Bland Kirby I, Vickers Selwyn M
Division of General Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294-0016, USA.
J Surg Res. 2003 May 15;111(2):274-83. doi: 10.1016/s0022-4804(03)00076-3.
Pancreatic cancer remains a devastating disease, with 95% of all patients diagnosed with the disease dying within 2 years. The combined therapy using Erbitux, gemcitabine, and radiation caused complete tumor regression using a nude mouse model inoculated with pancreatic MiaPaCa-2 cells but only a delay in tumor growth with BxPC-3. We investigated the effect of prolonged Erbitux treatment to the sensitivity to gemcitabine and/or radiation and the epidermal growth factor receptor (EGFR) signal transduction pathway.
MiaPaCa-2 and BxPC-3 cells were cultured with or without Erbitux for 6 weeks. Cells were then treated with gemcitabine and/or radiation, harvested 48 h after treatment, and counted. Differences in EGFR expression after exposure to Erbitux were analyzed by FACS. Internalization rates of EGFR induced by Erbitux on these cell lines were determined using 125I-EGF binding assay after removal of Erbitux by acidic wash. Cell lysates were harvested after cells were stimulated with EGF, FGF, or IGF-1 respectively, and EGFR was immunoprecipitated using Erbitux. Samples were separated using SDS-PAGE and transferred to PVDF membrane. The membranes were probed with antibody against human growth factor receptor binding protein (Grb2) to detect the association of this Ras-MAPK upstream adaptor protein to EGFR. Cell lysates were also separated with SDS-PAGE and probed with rabbit anti-human PARP after samples were transferred to PVDF membrane. Expression of BAX and Bcl-(XL) were probed in the cells treated with or without Erbitux.
Proliferation assays indicated that prolonged exposure to Erbitux increased the sensitivities of MiaPaCa-2 to gemcitabine and radiation therapy (41 +/- 16% vs 52 +/- 9% for gemcitation, 28 +/- 9 vs 39 +/- 9% for combination; P = 0.015) but not for BxPC-3. FACS analysis showed that the expressed EGFR level decreased by about 42% on MiaPaCa 2 whereas no loss was seen on BxPC-3. Expression of BAX was upregulated on MiaPaCa-2. Poly (ADP-ribose) polymerase cleavage indicated the killing was mediated by apoptosis. Immunoblots showed that Grb2 was co-immunoprecipitated with EGFR after EGF stimulation. Incubation with Erbitux blocked Grb2 binding in MiaPaCa-2 but not BxPC 3. FGF transactivated EGFR down stream Ras-MAPK in the presence or absence of Erbitux. Internalization of EGFR induced by Erbitux did not differ between MiaPaCa-2 and BxPC-3.
胰腺癌仍然是一种极具毁灭性的疾病，所有确诊患者中有95%在2年内死亡。使用爱必妥、吉西他滨和放疗的联合疗法，在接种了胰腺MiaPaCa - 2细胞的裸鼠模型中导致肿瘤完全消退，但在BxPC - 3模型中仅使肿瘤生长延迟。我们研究了延长爱必妥治疗对吉西他滨和/或放疗敏感性以及表皮生长因子受体（EGFR）信号转导通路的影响。
将MiaPaCa - 2和BxPC - 3细胞在有或没有爱必妥的情况下培养6周。然后用吉西他滨和/或放疗处理细胞，处理后48小时收获细胞并计数。通过流式细胞术分析暴露于爱必妥后EGFR表达的差异。在通过酸性洗涤去除爱必妥后，使用125I - EGF结合试验测定爱必妥诱导的这些细胞系上EGFR的内化率。在用EGF、FGF或IGF - 1分别刺激细胞后收获细胞裂解物，并用爱必妥免疫沉淀EGFR。样品用SDS - PAGE分离并转移到PVDF膜上。用抗人生长因子受体结合蛋白（Grb2）抗体检测该膜，以检测这种Ras - MAPK上游衔接蛋白与EGFR的结合。样品转移到PVDF膜后，细胞裂解物也用SDS - PAGE分离，并用兔抗人PARP检测。在有或没有爱必妥处理的细胞中检测BAX和Bcl - (XL)的表达。
增殖试验表明，长时间暴露于爱必妥可增加MiaPaCa - 2对吉西他滨和放疗的敏感性（吉西他滨：41±16%对52±9%，联合治疗：28±9对39±9%；P = 0.015），但对BxPC - 3无此作用。流式细胞术分析表明，MiaPaCa - 2上表达的EGFR水平下降约42%，而BxPC - 3上未见下降。MiaPaCa - 2上BAX表达上调。聚（ADP - 核糖）聚合酶裂解表明细胞死亡是由凋亡介导的。免疫印迹显示，EGF刺激后Grb2与EGFR共免疫沉淀。用爱必妥孵育可阻断MiaPaCa - 2中Grb2的结合，但不影响BxPC - 3。在有或没有爱必妥的情况下，FGF均可激活EGFR下游的Ras - MAPK。爱必妥诱导的EGFR内化在MiaPaCa - 2和BxPC - 3之间无差异。
1）在爱必妥存在下，EGF诱导的BxPC - 3中Grb2与EGFR的结合表明存在Ras - MAPK激活替代途径，这可能与肿瘤对治疗的抗性有关；2）FGF对EGFR下游Ras - MAPK途径的反式激活导致对治疗的抗性；3）EGFR的下调可能增加对治疗的反应。
