背景:TGF-β基因是吉西他滨(GEM)抗性基因;然而,其调节胰腺癌GEM耐药的机制尚不清楚.
方法:用GEM处理PANC-1细胞系,然后用TGF-β刺激。随后,我们构建了抗GEM的胰腺癌细胞系,在这些细胞系中敲除TGF-β,并检测耐药癌细胞增殖和凋亡的变化。此外,测定KLF-4、GFI-1和ZEB-1的蛋白表达水平。通过将GEM抗性PANC-1细胞皮下注射到小鼠腋下,构建了裸鼠异种移植瘤模型。肿瘤被切除了,解剖,6周后称重。定量肿瘤组织中KLF-4、GFI-1和ZEB-1的蛋白质水平。此外,使用流式细胞术确定肿瘤组织中M2巨噬细胞的百分比。
结果:GEM处理后,胰腺癌细胞中TGF-β的蛋白水平明显下降。KLF-4蛋白表达下调,而TGF-β刺激后GFI-1和ZEB-1的表达上调。TGF-β敲低GEM耐药胰腺癌细胞凋亡增加,增殖减少,此外,沉默TGF-β可促进Caspase3和cleavedcaspase3的表达。此外,KLF-4蛋白表达上调,而GFI-1和ZEB-1的表达下调。Further,TGF-β敲除后,移植瘤的体积和重量降低。KLF-4蛋白表达上调,而GFI-1和ZEB-1在肿瘤组织中表达下调。此外,TGF-β敲除后,肿瘤组织中M2巨噬细胞的百分比降低。
结论:TGF-β的敲低抑制上皮-间质转化,抑制耐药癌细胞的增殖并促进其凋亡,并减少巨噬细胞向M2表型的极化,因此改善胰腺癌的GEM耐药性。
BACKGROUND: The TGF-β gene is a gemcitabine (GEM) resistance gene; however, the mechanism by which it regulates GEM resistance in pancreatic cancer remains unclear.
METHODS: The PANC-1 cell line was treated with GEM and then stimulated with TGF-β. Subsequently, we constructed GEM-resistant pancreatic cancer cell lines, knocked down TGF-β in these cell lines, and detected changes in the proliferation and apoptosis of drug-resistant cancer cells. In addition, the protein expression levels of KLF-4, GFI-1, and ZEB-1 were determined. The xenograft tumor models of nude mice were constructed by subcutaneously injecting GEM-resistant PANC-1 cells into mouse axilla. The tumors were removed, dissected, and weighed after 6 weeks. The protein levels of KLF-4, GFI-1, and ZEB-1 in tumor tissues were quantified. In addition, the percentage of M2 macrophages in tumor tissues was determined using flow cytometry.
RESULTS: The protein levels of TGF-β in pancreatic cancer cells were significantly decreased after GEM treatment. The protein expression of KLF-4 was downregulated, whereas the expressions of GFI-1 and ZEB-1 were upregulated after TGF-β stimulation. Apoptosis increased and proliferation decreased after TGF-β knockdown in GEM-resistant pancreatic cancer cells, moreover, silencing TGF-β promoted the expression of Caspase 3 and Cleaved caspase 3. In addition, the protein expression of KLF-4 was upregulated, whereas the expressions of GFI-1 and ZEB-1 were downregulated. Further, the volume and weight of the transplanted tumor decreased after TGF-β knockdown. The protein expression of KLF-4 was upregulated, whereas the expressions of GFI-1 and ZEB-1 were downregulated in tumor tissues. In addition, the percentage of M2 macrophages decreased in tumor tissues after TGF-β knockdown.
CONCLUSIONS: The knockdown of TGF-β inhibits epithelial-to-mesenchymal transition, suppresses the proliferation and promotes the apoptosis of drug-resistant cancer cells, and decreases the macrophage polarization to the M2 phenotype, consequently ameliorating GEM resistance in pancreatic cancer.