肿瘤相关巨噬细胞通过PI3K/Akt途径促进胰腺癌浸润迁移的研究

doi:10.3877/cma.j.issn.1674-0793.2015.03.006

目的

研究PI3K/Akt信号通路对胰腺癌细胞PANC-1浸润迁移能力的影响,进一步探讨肿瘤相关巨噬细胞促进胰腺癌发生发展的分子机制。

方法

通过密度梯度离心法从健康成人外周血中分离单个核细胞,用IL-4体外诱导选择性激活的巨噬细胞(M2)。采用实时荧光定量PCR和Western blotting法检测胰腺癌PANC-1细胞PI3K、Akt mRNA和蛋白表达水平的变化,利用Transwell侵袭实验与划痕实验观察细胞浸润迁移能力的变化。

结果

体外模拟胰腺癌微环境,将胰腺癌PANC-1细胞与不同激活状态的巨噬细胞共培养,证明M2可显著上调胰腺癌PANC-1细胞PI3K、Akt的mRNA和蛋白水平,共培养20 h后可明显促进胰腺癌PANC-1细胞的浸润与迁移能力。

结论

肿瘤相关巨噬细胞可通过PI3K/Akt信号通路促进胰腺癌细胞的浸润与迁移。

关键词: 胰腺癌, 肿瘤相关巨噬细胞, PI3K, Akt, 浸润迁移

Objective

To explore the influence of PI3K/Akt signaling pathway on invasive and migratory ability of pancreatic cancer cell line PANC-1, and further investigate the molecule mechanism of tumor-associated macrophages’promotion in the occurrence and development of pancreatic cancer.

Methods

Mononuclear cells were isolated from peripheral blood of healthy adults by density gradient centrifugation, and treated with IL-4 to obtain M2 in vitro. Expression of PI3K and Akt was evaluated by quantitative Real-time polymerase chain reaction and Western blotting. The ability of cellular invasion and migration was assessed by transwell chamber assay and wound healing assay.

Results

To simulate pancreatic cancer microenvironment, we co-cultured pancreatic cancer PANC-1 cells and Ua or M2. The mRNA and protein levels of PI3K and Akt were remarkably increased analyzing by qRT-PCR and western blotting. In addition, transwell chamber assays and wound healing assays revealed that alternatively activated macrophages significantly promoted the invasion and migration of PANC-1 cells in 20 h.

Conclusion

Tumor-associated macrophages may promote invasion and migration of pancreatic cancer cells via PI3K/Akt signaling pathway.

Key words: Pancreatic cancer, Tumor-associated macrophages, PI3K, Akt, Invasion and migration

表1 PCR特异性引物序列

名称	引物序列
PI3K	5′-ATAGGCAAGTCGAGGCAATGGA-3′
?	5′-TGAGCAGGGTTTAGAGGAGACAGAA-3′
?	5′- CTTCTTTGCCGGTATCGTGTGG-3′
Akt	5′- TGTCATCTTGGTCAGGTGGTGTG-3′
?	5′-AATCTGGCACCACACCTTCTAC-3'
β-actin	5′- ATAGCACAGCCTGGATAGCAAC-3′

表1 PCR特异性引物序列

名称	引物序列
PI3K	5′-ATAGGCAAGTCGAGGCAATGGA-3′
?	5′-TGAGCAGGGTTTAGAGGAGACAGAA-3′
?	5′- CTTCTTTGCCGGTATCGTGTGG-3′
Akt	5′- TGTCATCTTGGTCAGGTGGTGTG-3′
?	5′-AATCTGGCACCACACCTTCTAC-3'
β-actin	5′- ATAGCACAGCCTGGATAGCAAC-3′

图1 不同激活状态的巨噬细胞光学显微镜下形态（400×）

图2 人外周血单个核细胞分离流式鉴定结果

图3 M2促进胰腺癌PANC-1细胞PI3K、Akt的mRNA转录和蛋白表达

图4 选择性巨噬细胞促进胰腺癌PANC-1细胞的横向迁移

图5 选择性巨噬细胞促进胰腺癌PANC-1细胞的纵向浸润

[1]	Stathis A, Moore MJ. Advanced pancreatic carcinoma: current treatment and future challenges[J]. Nat Rev Clin Oncol, 2010, 7(3): 163-172.
[2]	Seufferlein T, Bachet JB, Van Cutsem E, et al. Pancreatic adenocarcinoma: ESMO-ESDO Clinical Practice Guidelines for diagnosis, treatment and follow-up[J]. Ann Oncol, 2012, 23(7): 33-40.
[3]	Ghaneh P, Costello E, Neoptolemos JP. Biology and management of pancreatic cancer[J]. Postgrad Med J, 2008, 84(995): 478-497.
[4]	Coffelt SB, Hughes R, Lewis CE. Tumor-associated macrophages: effectors of angiogenesis and tumor progression[J]. Biochim Biophys Acta, 2009, 1796(1): 11-18.
[5]	李奕，蔡真.肿瘤相关巨噬细胞与肿瘤生长和治疗的研究进展[J]. 中国肿瘤生物治疗杂志, 2011, 18(4): 441-447.
[6]	Laoui D, Movahedi K, Van Overmeire E, et al. Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions[J]. Int J Dev Biol, 2011, 55(7-9): 861-867.
[7]	Kedra B, Popiela T, Sierzega M, et al. Prognostic factors of longterm survival after resective procedures for pancreatic cancer[J]. Hepatogastroenterology, 2001, 48(42): 1762-1766.
[8]	Tezel E, Kaneko T, Sugimoto H, et al. Clinical significance of intraportal endovascular ultrasonography for the diagnosis of extrapancreatic nerve plexus invasion by pancreatic carcinoma[J]. Pancreatology, 2004, 4(2): 76-81.
[9]	Allavena P, Sica A, Solinas G, et al. The inflammatory micro-environment in tumor progression: the role of tumor-associated macr- ophages[J]. Crit Rev Oncol Hematol, 2008, 66(1): 1-9.
[10]	Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis[J]. Nat Rev Cancer, 2004, 4(1): 71-78.
[11]	Su S, Liu Q, Chen J, et al. A positive feedback loop between mesenchymal-like cancer cells and macrophages is essential to breast cancer metastasis[J]. Cancer Cell, 2014, 25(5): 605-620.
[12]	Ioannides CG, Whiteside TL. T cell recognition of human tumors:implications for molecular immunotherapy of cancer[J]. Clin Immunol Immunopathol, 1993, 66(2): 91-106.
[13]	Liou GY, Doppler H, Necela B, et al. Mutant KRAS-induced expression of ICAM-1 in pancreatic acinar cells causes attraction of macrophages to expedite the formation of precancerous lesions[J]. Cancer Discov, 2015, 5(1): 52-63.
[14]	李康，郭强，王翠妮,等. M1和M2型巨噬细胞表型的比较分析[J].现代免疫学, 2008, 28(3): 177-183.
[15]	Wang N, Liang H, Zen K. Molecular mechanisms that influence the macrophage m1-m2 polarization balance[J]. Front Immunol, 2014, 5(2): 614-615.
[16]	曹春雨，王清，王艳林.肿瘤相关巨噬细胞极化的研究进展[J].生命科学, 2013, 25(11): 1105-1108.
[17]	Lawrence T. Macrophages and NF-kappaB in cancer[J]. Curr Top Microbiol Immunol, 2011, 349(3): 171-184.
[18]	Mantovani A, Sozzani S, Locati M, et al. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes[J]. Trends Immunol, 2002, 23(11): 549-555.
[19]	李晓曦，郭宁，曹雪涛.肿瘤相关巨噬细胞促进肿瘤生长与转移的研究现状[J].中国肿瘤生物治疗杂志, 2008, 15(1): 78-81.
[20]	Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macrophages in tumour progression: implications for new anti-cancer therapies[J]. J Pathol, 2002, 196(3): 254-265.
[21]	耿晓强. M2型肿瘤相关巨噬细胞在肿瘤中作用的研究进展及肿瘤治疗的新思路[J].中国实用内科杂志, 2014, 34(1): 169-171.
[22]	Chen J. Regulation of tumor initiation and metastatic progression by Eph receptor tyrosine kinases[J]. Adv Cancer Res, 2012, 114(2): 1-20.
[23]	Saglam O, Garrett CR, Boulware D, et al. Activation of the serine/threonine protein kinase AKT during the progression of colorectal neoplasia[J]. Clin Colorectal Cancer, 2007, 6(9): 652-656.
[24]	Schlieman MG, Fahy BN, Ramsamooj R, et al. Incidence, mechanism and prognostic value of activated AKT in pancreas cancer[J]. Br J Cancer, 2003, 89(11): 2110-2115.
[25]	Asano T, Yao Y, Zhu J, et al. The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kappaB and c-Myc in pancreatic cancer cells[J]. Oncogene, 2004, 23(53): 8571-8580.
[26]	Stephan S, Datta K, Wang E, et al. Effect of rapamycin alone and in combination with antiangiogenesis therapy in an orthotopic model of human pancreatic cancer[J]. Clin Cancer Res, 2004, 10(20): 6993-7000.

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Tumor-associated macrophages promoting invasion and migration of pancreatic cancer via PI3K/Akt signaling pathway

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Tumor-associated macrophages promoting invasion and migration of pancreatic cancer via PI3K/Akt signaling pathway