骨肉瘤MG63细胞中VEGF-C/VEGFR-3/iNOS信号转导对人脐静脉内皮细胞增殖的促进作用

doi:10.24920/003753

摘要/Abstract

摘要：

目的探讨血管内皮生长因子-C（vascular endothelial growth factor-C,VEGF-C）/血管内皮生长因子受体-3（vascular endothelial growth factor receptor-3,VEGFR-3）信号转导对人骨肉瘤MG63细胞中一氧化氮（nitric oxide,NO）的生成和诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）的表达以及对人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）增殖的影响。

方法MG63细胞分别以VEGF-C单独处理（VEGF-C组）,VEGF-C联合iNOS抑制剂氨基胍AG（AG组）以及VEGF-C联合VEGFR-3抑制剂MAZ-51（MAZ-51组）处理,将未处理的MG63细胞作为对照。采用硝酸还原酶比色法测定NO生成量,逆转录-聚合酶链反应（reverse transcription polymerase chain reaction,RT-PCR）和Western blot分别检测iNOS的mRNA和蛋白水平。为研究MG63细胞中VEGF-C/VEGFR-3/iNOS信号通路对HUVECs增殖的影响,实验设立如下6组：HUVECs组、HUVECs+MG63组、HUVECs+VEGF-C组、HUVECs+MG63+VEGF-C组、HUVECs+MG63+VEGF-C+AG组及HUVECs+MG63+VEGF-C+MAZ51组。用细胞计数试剂盒（Cell Counting Kit-8,CCK-8）、5-乙炔基-2'-脱氧尿嘧啶核苷（5 ethynyl 2' deoxyuridine,EdU）掺入法和增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）定量表达评估各组HUVECs的增殖情况。

结果VEGF-C促进了MG63细胞中iNOS在基因（LSD-t=4.152,P<0.01）和蛋白水平的表达（LSD-t=3.486,P<0.01）,增加了NO释放（LSD-t=3.774,P<0.01）。经AG或MAZ51处理均可降低上述作用（mRNA：LSD-t=9.183,P<0.001;LSD-t=8.639,P<0.001;protein：LSD-t=5.170,P<0.001;LSD-t=7.255,P<0.001;NO：LSD-t=10.326,P<0.001;LSD-t=10.540,P<0.001）。HUVECs与MG63细胞和/或VEGF-C共同培养可显著促进HUVECs的增殖（EdU：LSD-t=5.374,P<0.001;LSD-t=2.984,P<0.05;LSD-t=8.526,P<0.001;PCNA： LSD-t=9.267,P<0.001;LSD-t=5.515,P<0.001;LSD-t=14.873,P<0.001）。经AG（EdU：LSD-t=10.770,P<0.001;PCNA：LSD-t=19.940,P<0.001）或MAZ51（EdU：LSD-t=6.950,P<0.001;PCNA：LSD-t=14.001,P<0.001）处理后,MG63细胞和VEGF-C对HUVECs增殖的诱导作用减弱。

结论VEGF-C激活VEGFR-3可促进在人骨肉瘤MG63细胞iNOS的表达和NO的产生,进而诱导HUVECs的增殖。

关键词: 骨肉瘤, 血管内皮生长因子-C, 血管内皮生长因子受体-3, 诱导型一氧化氮合酶, 肿瘤血管生成

Abstract:

Objective To assess the effects of vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor-3 (VEGFR-3) signaling on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in human osteosarcoma MG63 cells and the subsequent impact on the proliferation of human umbilical vein endothelial cells (HUVECs).

MethodsMG63 cells were treated with VEGF-C alone (VEGF-C group), VEGF-C + iNOS inhibitor aminoguanidine (AG; AG group), and VEGF-C + VEGFR-3 inhibitor MAZ51 (MAZ51 group); untreated MG63 cells were used as controls. NO production was evaluated by a colorimetric method involving nitrate reductase. Meanwhile, mRNA and protein levels of iNOS were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. To explore the effect of VEGF-C/VEGFR-3/iNOS signaling of MG63 cells on proliferation of HUVECs, we set up six groups: HUVECs, HUVECs+MG63, HUVECs+VEGF-C, HUVECs+MG63+VEGF-C, HUVECs+MG63+VEGF-C+AG, and HUVECs+MG63+VEGF-C+MAZ51 groups. The proliferation of HUVEC cells was assessed by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay, and proliferating cell nuclear antigen (PCNA) expression quantitation.

ResultsVEGF-C treatment enhanced iNOS expression at both gene and protein levels (mRNA: LSD-t=4.152, P<0.01; protein: LSD-t=3.486, P<0.01) and increased NO release of MG63 cells (LSD-t=3.774, P<0.01); treatment with either AG or MAZ51 decreased these effects (mRNA: LSD-t=9.183, P<0.001; LSD-t=8.639, P<0.001; protein: LSD-t=5.170, P<0.001; LSD-t=7.255, P<0.001; NO production:LSD-t=10.326, P<0.001; LSD-t=10.540, P<0.001). Interestingly, co-incubation of HUVECs with MG63 cells and/or VEGF-C significantly promoted HUVEC proliferation (EdU: LSD-t=5.374, P<0.001; LSD-t=2.984, P<0.05; LSD-t=8.526, P<0.001; PCNA: LSD-t=9.267, P<0.001; LSD-t=5.515, P<0.001; LSD-t=14.873, P<0.001).The proliferation effects of HUVEC induced by MG63 cells and VEGF-C attenuated by the treatment of AG (EdU: LSD-t=10.770, P<0.001; PCNA: LSD-t=19.940, P<0.001) or MAZ51 (EdU: LSD-t=6.950, P<0.001; PCNA: LSD-t=14.001, P<0.001).

ConclusionIn human osteosarcoma MG63 cells, activation of VEGFR-3 by VEGF-C promotes iNOS expression and NO production, which subsequently induces HUVEC proliferation.

Key words: osteosarcoma, VEGF-C, VEGFR-3, iNOS, tumor angiogenesis

基金资助: 山西省自然科学基金(201801D121220)

Jie Lv,Jie Yuan,Chaojian Xu,Jiaqi Hao,Yichuan Qin,Xiaoqiang Wang,Yongfeng Wang. VEGF-C/VEGFR-3/iNOS Signaling in Osteosarcoma MG63 Cells Mediates Stimulatory Effects on Human Umbilical Vein Endothelial Cell Proliferation[J].Chinese Medical Sciences Journal, 2021, 36(1): 35-42.

图/表 2

参考文献 29.

1.	Messerschmitt PJ, Garcia RM, Abdul-Karim FW, et al.Osteosarcoma. J Am Acad Orthop Surg 2009; 17(8): 515-27. doi: 10.1097/BSD.0b013e31818d5fcb.
2.	Dai Z, Tang H, Pan Y, et al.Gene expression profiles and pathway enrichment analysis of human osteosarcoma cells exposed to sorafenib. FEBS Open Bio 2018; 8(5):860-7. doi: 10.1002/2211-5463.12428.
3.	Hameed M, Mandelker D. Tumor syndromes predisposing to osteosarcoma. Adv Anat Pathol 2018; 25(4):217-22. doi:10.1097/PAP.0000000000000190.
4.	Wang J, Cao L, Wu J, et al.Long non-coding RNA SNHG1 regulates NOB1 expression by sponging miR-326 and promotes tumorigenesis in osteosarcoma. Int J Oncol 2018; 52(1):77-88. doi: 10.3892/ijo.2017.4187.
5.	Nieto-Coronel MT, López-Vásquez AD, Marroquín-Flores D, et al.Central nervous system metastasis from osteosarcoma: Case report and literature review. Rep Pract Oncol Radiother 2018; 23(4):266-9. doi: 10.1016/j.rpor.2018.06.003. doi: 10.1016/j.rpor.2018.06.003 pmid: 30090025
6.	Harrison DJ, Geller DS, Gill JD, et al. Current and future therapeutic approaches for osteosarcoma. Expert Rev Anticancer Ther 2018; 18(1):39-50. doi:10.1080/14737140.2018.1413939.
7.	Briccoli A, Rocca M, Salone M, et al.Resection of recurrent pulmonary metastases in patients with osteosarcoma. Cancer 2005; 104(8):1721-25. doi:10.1002/cncr.21369.
8.	Hanahan D, Weinberg RA.Hallmarks of cancer: the next generation. Cell 2011; 144(5):646-74. doi:10.1016/j.cell.2011.02.013.
9.	Segaliny AI, Mohamadi A, Dizier B, et al.Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment. Int J Cancer 2015; 137(1): 73-85. doi:10.1002/ijc.29376.
10.	Yang X, Zhou W, Liu S.SPAG9 controls the cell motility, invasion and angiogenesis of human osteosarcoma cells. Exp Ther Med 2016; 11(2): 637-44. doi:10.3892/etm.2015.2932.
11.	Qu W, Wang Y, Wu Q, et al.Emodin inhibits HMGB1induced tumor angiogenesis in human osteosarcoma by regulating SIRT1. Int J Clin Exp Med 2015; 8(9): 15054-64.
12.	Kimura Y, Sumiyoshi M.Anti-tumor and anti-metastatic actions of wogonin isolated from Scutellaria baicalensis roots through anti-lymphangiogenesis. Phytomedicine 2013; 20(3-4): 328-36. doi: 10.1016/j.phymed.2012.10.016.
13.	Folkman J.What is the role of endothelial cells in angiogenesis? Lab Invest 1984; 51(6): 601-4.
14.	Dong Z, Nor JE.Transcriptional targeting of tumor endothelial cells for gene therapy. Adv Drug Deliv Rev 2009; 61(7-8): 542-53. doi:10.1016/j.addr.2009.02.006. doi: 10.1016/j.addr.2009.02.006 pmid: 19393703
15.	Kang JX, Liu A.The role of the tissue omega-6/omega-3 fatty acid ratio in regulating tumor angiogenesis. Cancer Metastasis Rev 2013; 32(1-2): 201-10. doi:10.1007/s10555-012-9401-9. pmid: 23090260
16.	Zhao J, Zhang ZR, Zhao N, et al.VEGF silencing inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via PI3K/AKT signaling pathway. Int J Clin Exp Med 2015; 8(8): 12411-7. doi:10.1007/s12013-015-0692-7. pmid: 26550152
17.	Abdeen A, Chou AJ, Healey JH, et al.Correlation between clinical outcome and growth factor pathway expression in osteogenic sarcoma. Cancer 2009; 115(22): 5243-50. doi: 10.1002/cncr.24562.
18.	Park HR, Min K, Kim HS, et al.Expression of vascular endothelial growth factor-C and its receptor in osteosarcomas. Pathol Res Pract 2008; 204(8): 575-82. doi: 10.1016/j.prp.2008.01.015.
19.	Chien MH, Ku CC, Johansson G, et al.Vascular endothelial growth factor-C (VEGF-C) promotes angiogenesis by induction of COX-2 in leukemic cells via the VEGF-R3/JNK/AP-1 pathway. Carcinogenesis 2009; 30(12): 2005-13. doi: 10.1093/carcin/bgp244.
20.	Curiel TJ, Cheng P, Mottram P, et al.Dendritic cell subsets differentially regulate angiogenesis in human ovarian cancer. Cancer Res 2004; 64(16): 5535-8. doi:10.1158/0008-5472.CAN-04-1272.
21.	Lahdenranta J, Hagendoorn J, Padera TP, et al.Endothelial nitric oxide synthase mediates lymphangiogenesis and lymphatic metastasis. Cancer Res 2009; 69(7): 2801-8. doi:10.1158/0008-5472.CAN-08-4051. pmid: 19318557
22.	Fukumura D, Kashiwagi S, Jain RK.The role of nitric oxide in tumour progression. Nat Rev Cancer 2006; 6(7): 521-34. doi:10.1038/nrc1910.
23.	Chen WL, Feng HJ, Li JS, et al.Expression and pathological relevance of inducible nitric oxide synthase in osteosarcoma of the jaws. Int J Oral Maxillofac Surg 2007; 36(6):541-4. doi:10.1016/j.ijom.2007.02.012.
24.	Cullis ER, Kalber TL, Ashton SE, et al.Tumour overexpression of inducible nitric oxide synthase (iNOS) increases angiogenesis and may modulate the antitumour effects of the vascular disrupting agent ZD6126. Microvasc Res 2006; 71(2):76-84. doi:10.1016/j.mvr.2006.01.004. pmid: 16530791
25.	Ellie E, Loiseau H, Lafond F, et al.Differential expression of inducible nitric oxide synthase mRNA in humanbrain tumours. Neuroreport 1995; 7(1): 294-6. doi: 10.1097/00001756-199512290-00070. pmid: 8742473
26.	Loibl S, Buck A, Strank C, et al.The role of early expression of inducible nitric oxide synthase in human breast cancer. Eur J Cancer 2005; 41(2):265-71. doi:10.1016/j.ejca.2004.07.010. pmid: 15661552
27.	Ekmekcioglu S, Ellerhorst JA, Prieto VG, et al.Tumor iNOS predicts poor survival for stage III melanoma patients. Int J Cancer 2006; 119(4):861-6. doi:10.1002/ijc.21767. doi: 10.1002/ijc.21767 pmid: 16557582
28.	Dias S, Choy M, Alitalo K, et al.Vascular endothelial growth factor (VEGF)-C signaling through FLT-4 (VEGFR-3) mediates leukemic cell proliferation, survival, and resistance to chemotherapy. Blood 2002; 99(6): 2179-84. doi:10.1182/blood.V99.6.2179. doi: 10.1182/blood.v99.6.2179 pmid: 11877295
29.	Watanabe T, Sugaya M, Atkins AM, et al.Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen prolongs the life span of primary human umbilical vein endothelial cells. J Virol 2003; 77(11): 6188-96. doi:10.1128/JVI.77.11.6188-6196.2003.

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