Effect of STAT-3-mediated non-dependent VEGFR-2 pathway on radiosensitivity of non-small cell lung cancer cells
Liu Bin,Hu Chenxi,Liu Liang,Liu Qingjing,Tang Jingjiao,Jiang Xiaodong
Affiliated Lianyungang Hospital of Xuzhou Medical University,Lianyungang 222002,China (Liu B,Liu QJ,Tang JJ)( Liu B' s Present' s unit: Department of Oncology,Binzhou Central Hospital of Shandong Province,Binzhou 251700,China); Department of Oncology,Affiliated Lianyungang Hospital of Xuzhou Medical University,Lianyungang 222002,China(Hu CX,Liu L,Jiang XD)
Abstract： Objective To investigate the effect of signal transducer and activator of transcription-3(STAT-3)-mediated non-dependent vascular endothelial growth factor receptor-2(VEGFR-2) pathway on the radiosensitivity of non-small cell lung cancer (NSCLC) cells and its possible mechanism. Methods NSCLC cells were divided into control group, apatinib (VEGFR-2 inhibitor) group, apatinib+S3I-201(STAT-3 inhibitor) group, radiation (RT) group, RT+apatinib group, and RT+apatinib+S3I-201 group. Q-PCR and Western blot were used to measure the mRNA and protein expression of VEGFR-2, STAT-3, other proteins involved in the signaling pathway, hypoxia-inducible factor 1α(HIF-1α), and cyclin D1. The cellular radiosensitivity was determined by colony-forming assay and cell apoptosis and cell cycle distribution were evaluated by flow cytometry. Results For Calu-1 cells, there was no significant difference in the expression of VEGFR-2 mRNA and STAT-3 mRNA between the apatinib group and the control group (P>0.05);the apatinib group had significantly lower expression of HIF-1α mRNA and cyclin D1 mRNA than the control group (P<0.05);the apatinib+S3I-201 group had significantly lower mRNA and phosphorylated protein expression of VEGFR-2, STAT-3, and related downstream target proteins than the control group (F=304.54, P<0.01;F=118.99, P<0.01, F=144.34, P<0.01;F=529.66, P<0.01);compared with the control group, the apatinib+S3I-201 group and the apatinib group showed increases in apoptosis rate and proportion of cells in G2+M phase, and the apatinib+S3I-201 group had significantly greater increases than the apatinib group (F=72.37, P<0.01). Compared with Calu-1 cells, the radiosensitizing effect of apatinib on A549 cells was limited (SER[sensitizer enhancement ratio]=1.39). The radiosensitizing effect of apatinib+S3I-201 on A549 cells was significantly higher than that of apatinib (SER:1.72 vs. 1.39, P=0.000). Conclusions The inhibition of STAT-3 can enhance the radiosensitivity of NSCLC cells. When VEGFR-2 is inhibited by apatinib, activated STAT-3 regulates the expression of cyclin D1 directly or indirectly to affect the radiosensitivity of NSCLC cells. The inhibition of VEGFR-2 and STAT-3 has a good radiosensitizing effect on NSCLC cells.
Liu Bin,Hu Chenxi,Liu Liang et al. Effect of STAT-3-mediated non-dependent VEGFR-2 pathway on radiosensitivity of non-small cell lung cancer cells[J]. Chinese Journal of Radiation Oncology, 2017, 26(8): 938-944.
[1] Bayman N,Blackhall F,McCloskey P,et al. How can we optimise concurrent chemoradiotherapy for inoperable stage Ⅲ non-small cell lung cancer?[J].Lung Cancer,2014,83(2):117-125.DOI:10.1016/j.lungcan.2013.11.017. [2] Jiang XD,Qiao Y,Dai P,et al. Enhancement of recombinant human endostatin on the radiosensitivity of human pulmonary adenocarcinoma A549 cells and its mechanism[J].J Biomed Biotechnol,2012,2012:301931.DOI:10.1155/2012/301931. [3] Jiang XD,Dai P,Wu J,et al. Inhibitory effect of radiotherapy combined with weekly recombinant human endostatin on the human pulmonary adenocarcinoma A549 xenografts in nude mice[J].Lung Cancer,2011,72(2):165-171.DOI:10.1016/j.lungcan.2010.09.003. [4] Jiang XD,Qiao Y,Dai P,et al. Preliminary clinical study of weekly recombinant human endostatin as a hypoxic tumour cell radiosensitiser combined with radiotherapy in the treatment of NSCLC[J].Clin Transl Oncol,2012,14(6):465-470.DOI:10.1007/s12094-012-0825-z. [5] Jiang XD,Dai P,Wu J,et al. Effect of recombinant human endostatin on radiosensitivity in patients with non-small-cell lung cancer[J].Int J Radiat Oncol Biol Phys,2012,83(4):1272-1277.DOI:10.1016/j.ijrobp.2011.09.050. [6] Jiang XD,Dai P,Qiao Y,et al. Clinical study on the recombinant human endostatin regarding improving the blood perfusion and hypoxia of non-small-cell lung cancer[J].Clin Transl Oncol,2012,14(6):437-443.DOI:10.1007/s12094-012-0821-3. [7] Liu L,Qiao Y,Hu C,et al. Endostatin exerts radiosensitizing effect in non-small cell lung cancer cells by inhibiting VEGFR-2 expression[J].Clin Transl Oncol,2016,18(1):18-26.DOI:10.1007/s12094-015-1319-6. [8] Liu Y,Qiao Y,Hu C,et al. VEGFR-2 inhibition by RNA interference affects cell proliferation,migration,invasion,and response to radiation in Calu-1 cells[J].Clin Transl Oncol,2016,18(2):212-219.DOI:10.1007/s12094-015-1358-z. [9] Jiang XD,Ding MH,Qiao Y,et al. Recombinant human endostatin combined with radiotherapy in the treatment of brain metastases of non-small cell lung cancer[J].Clin Transl Oncol,2014,16(7):630-636.DOI:10.1007/s12094-013-1129-7. [10] Ellis PM,Al-Saleh K.Multitargeted anti-angiogenic agents and NSCLC:clinical update and future directions[J].Crit Rev Oncol Hematol,2012,84(1):47-58.DOI:10.1016/j.critrevonc.2012.02.004. [11] Lu S,Gao Y,Huang XL,et al. GYY4137,a hydrogen sulfide (H2S) donor,shows potent anti-hepatocellular carcinoma activity through blocking the STAT-3 pathway[J].Int J Oncol,2014,44(4):1259-1267.DOI:10.3892/ijo.2014.2305. [12] Islam M,Sharma S,Teknos TN.RhoC regulates cancer stem cells in head and neck squamous cell carcinoma by overexpressing IL-6 and phosphorylation of STAT-3[J].PLoS One,2014,9(2):e88527.DOI:10.1371/journal.pone.0088527. [13] Siveen KS,Sikka S,Surana R,et al. Targeting the STAT-3 signaling pathway in cancer:role of synthetic and natural inhibitors[J].Biochim Biophys Acta,2014,1845(2):136-154.DOI:10.1016/j.bbcan.2013.12.005. [14] Nechemia-Arbely Y,Khamaisi M,Rosenberger C,et al. In vivo evidence suggesting reciprocal renal hypoxia-inducible factor-1 upregulation and signal transducer and activator of transcription 3 activation in response to hypoxic and non-hypoxic stimuli[J].Clin Exp Pharmacol Physiol,2013,40(4):262-272.DOI:10.1111/1440-1681.12064. [15] Zhang XM,Zhou C,Gu H,et al. Correlation of RKIP,STAT-3 and cyclin D1 expression in pathogenesis of gastric cancer[J].Int J Clin Exp Pathol,2014,7(9):5902-5908. [16] 张莹,蒋晓东,乔云,等.不同肺癌细胞系中VEGFR-2 mRNA表达的实验研究[J].临床肿瘤学杂志,2013,18(10):879-881.DOI:10.3969/j.issn.1009-0460.2013.10.004. Zhang Y,Jiang XY,Qiao Y,et al. The expressions of VEGFR-2 mRNA in different lung cancer cell lines[J].Chin Clin Oncol,2013,18(10):879-881.DOI:10.3969/j.issn.1009-0460.2013.10.004. [17] Jiang XD,Ding MH,Qiao Y,et al. Study on lung cancer cells expressing VEGFR-2 and the impact on the effect of RHES combined with radiotherapy in the treatment of brain metastases[J].Clin Lung Cancer,2014,15(2):e23-e29.DOI:10.1016/j.cllc.2013.11.012. [18] Wang L,Yi TS,Zhang W,et al. IL-17 enhances tumor development in carcinogen-induced skin cancer[J].Cancer Res,2010,70(24):10112-10120.DOI:10.1158/0008-5472.CAN-10-0775. [19] Yan B,Wei JJ,Yuan Y,et al. IL-6 cooperates with G-CSF to induce protumor function of neutrophils in bone marrow by enhancing STAT-3 activation[J].J Immunol,2013,190(11):5882-5893.DOI:10.4049/jimmunol.1201881. [20] Matsumura A,Kubota T,Taiyoh H,et al. HGF regulates VEGF expression via the c-Met receptor downstream pathways,PI3K/Akt,MAPK and STAT-3,in CT26 murine cells[J].Int J Oncol,2013,42(2):535-542.DOI:10.3892/ijo.2012.1728. [21] Won C,Lee CS,Lee JK,et al. CADPE suppresses cyclin D1 expression in hepatocellular carcinoma by blocking IL-6-induced STAT-3 activation[J].Anticancer Res,2010,30(2):481-488. [22] Chen SH,Murphy D,Lassoued W,et al. Activated STAT-3 is a mediator and biomarker of VEGF endothelial activation[J].Cancer Biol Ther,2008,7(12):1994-2003.DOI:10.4161/cbt.7.12.6967. [23] Su L,Rao K,Guo F,et al. In ovo leptin administration inhibits chorioallantoic membrane angiogenesis in female chicken embryos through the STAT-3-mediated vascular endothelial growth factor (VEGF) pathway[J].Domest Anim Endocrinol,2012,43(1):26-36.DOI:10.1016/j.domaniend.2012.01.007. [24] Wen W,Ding J,Sun W,et al. Suppression of cyclin D1 by hypoxia-inducible factor-1 via direct mechanism inhibits the proliferation and 5-fluorouracil-induced apoptosis of A549 cells[J].Cancer Res,2010,70(5):2010-2019.DOI:10.1158/0008-5472.CAN-08-4910. [25] Kim YM,Geiger TR,Egan DI,et al. The HTLV-1 tax protein cooperates with phosphorylated CREB,TORC2 and p300 to activate CRE-dependent cyclin D1 transcription[J].Oncogene,2010,29(14):2142-2152.DOI:10.1038/onc.2009.498. [26] Zhang HS,Yan B,Li XB,et al. PAX2 protein induces expression of cyclin D1 through activating AP-1 protein and promotes proliferation of colon cancer cells[J].J Biol Chem,2012,287(53):44164-44172.DOI:10.1074/jbc. M112.401521. [27] 崔海鹏,董建一,李慧玲,等.Ras/MAPK/ERK通过协同NF-κB促进肝癌细胞中CyclinD1的表达[J].实验动物科学,2014,31(6):1-6,10.DOI:10.3969/j.issn.1006-6179.2014.06.001. Cui HP,Dong JY,Li HL,et al. Ras/MAPK/ERK promote expression of CyclinD1 collaborating with NF-κB in hepatoma cells[J].Lab Anim Sci,2014,31(6):1-6,10.DOI:10.3969/j.issn.1006-6179.2014.06.001. [28] Sun Y,Luo DZ,Liao DJ,et al. CyclinD1 protein plays different roles in modulating chemoresponses in MCF7 and MDA-MB231 cells[J].J Carcinog,2012,11:12.DOI:10.4103/1477-3163.100401. [29] 何向明,黄润,俞洋,等.CDK2-AP1通过调控细胞周期抑制乳腺癌生长[J].浙江临床医学,2015,17(7):1059-1061. He XM,Huang R,Yu Yet al. CDK2-AP1 through the regulation of cell cycle inhibiting the growth of breast cancer[J].Zhejiang Clin Med J,2015,17(7):1059-1061.
2017, Vol. 26 
