Jiang Yu-Ping, Wu Xiao-Hua, Xing Han-Ying, DU Xing-Yan
Department of Obstetrics and Gynecology, Second Hospital of Hebei Medical University, Shijiazhuang 050000, China.
Zhonghua Fu Chan Ke Za Zhi. 2007 Jun;42(6):403-7.
To explore the effect of chemokine CXCL12 and its receptor CXCR4 on proliferation, migration and invasion of epithelial ovarian cancer cells.
CXCR4 and CXCL12 mRNA and protein expression of human ovarian cancer cell line CAOV3 was detected by RT-PCR and immunocytochemistry. Integrin beta1 and vascular endothelial growth factor-C (VEGF-C) mRNA expression were detected in CAOV3 cells stimulated by CXCL12. The CAOV3 cells were divided into 6 groups: control group (un-stimulated), experimental group 1 (stimulated by 100 ng/ml CXCL12), experimental group 2 (stimulated by 10 ng/ml CXCL12), experimental group 3 (100 ng/ml CXCL12 and 10 microg/ml neutralizing CXCR4 antibody), experimental group 4 (100 ng/ml CXCL12 and 1 microg/ml CXCR4 antagonist AMD3100), experimental group 5 (10 microg/ml neutralizing CXCR4 antibody or ascites). Methyl thiazolyl tetrazolium (MTT) was used to analyze the effects of different concentrations of CXCL12 on CAOV3 cell proliferation. Transwell invasion chamber and reconstructed basement membrane (Matrigel) were used to evaluate effect of various concentrations of CXCL12 and ascites on CAOV3 cell migration and invasion.
CAOV3 cells expressed CXCR4 mRNA (0.70 +/- 0.10) and protein, but did not express CXCL12 mRNA or protein. Immunostaining of CXCR4 was mainly located in cytoplasm. CXCR4 mRNA was up-regulated after 100 ng/ml CXCL12 stimulation (1.24 +/- 0.14; t = -7.1088, P = 0.0021). Integrin beta1 mRNA was greatly increased at 3 hours by stimulation of 100 ng/ml CXCL12 (before and after stimulation 0.53 +/- 0.10, 1.53 +/- 0.16; P < 0.01), and VEGF-C mRNA showed significant increase at 24 hours by treatment with CXCL12 (before and after stimulation 0.52 +/- 0.09, 1.11 +/- 0.15; P < 0.05). Under serum-free sub-optimal culture conditions, experimental group 1 greatly enhanced cell proliferation in CAOV3 cells compared with control group and experimental group 2 (respectively 0.428 +/- 0.051, 0.325 +/- 0.045, 0.328 +/- 0.039; P < 0.05). Experimental group 1 was strongly inhibited compared with experimental groups 3 and 4 (the latter two groups respectively 0.356 +/- 0.031, 0.373 +/- 0.029; P < 0.05). There was no significant difference between experimental group 5 (0.349 +/- 0.038) and control group (P > 0.05). Experimental group 1 stimulated the migration and invasion of CAOV3 cells in chemotaxis assay compared with control group and experimental group 2 (number of cell migration respectively 523.3 +/- 25.2, 108.0 +/- 7.2, 211.7 +/- 24.7, number of cell invasion respectively 39.3 +/- 4.0, 4.0 +/- 1.0, 15.7 +/- 3.1; P < 0.01). This enhancing effect of experimental group 1 was strongly inhibited compared with experimental groups 4 and 5 (P < 0.05). The number of migrating and invading cells in experimental group 5 (migration: 706.6 +/- 30.6, invasion: 61.7 +/- 7.6) was significantly higher than that of experimental group 1 (P < 0.05).
This in vitro study shows CXCL12 promote proliferation, migration, invasion of ovarian cancer cell line CAOV3, and up-regulate integrin beta1 and VEGF-C expression, and these effects are strongly inhibited by neutralizing CXCR4 antibody. It suggests CXCL12 and its receptor CXCR4 may play important roles in ovarian cancer growth and metastasis.
探讨趋化因子CXCL12及其受体CXCR4对上皮性卵巢癌细胞增殖、迁移及侵袭的影响。
采用逆转录-聚合酶链反应（RT-PCR）及免疫细胞化学法检测人卵巢癌细胞系CAOV3中CXCR4和CXCL12的mRNA及蛋白表达。检测CXCL12刺激后CAOV3细胞中整合素β1和血管内皮生长因子-C（VEGF-C）的mRNA表达。将CAOV3细胞分为6组：对照组（未刺激）、实验组1（100 ng/ml CXCL12刺激）、实验组2（10 ng/ml CXCL12刺激）、实验组3（100 ng/ml CXCL12与10 μg/ml中和性CXCR4抗体）、实验组4（100 ng/ml CXCL12与1 μg/ml CXCR4拮抗剂AMD3100）、实验组5（10 μg/ml中和性CXCR4抗体或腹水）。采用甲基噻唑基四氮唑（MTT）法分析不同浓度CXCL12对CAOV3细胞增殖的影响。利用Transwell侵袭小室和重组基底膜（Matrigel）评估不同浓度CXCL12及腹水对CAOV3细胞迁移和侵袭的影响。
CAOV3细胞表达CXCR4 mRNA（0.70±0.10）及蛋白，但不表达CXCL12 mRNA或蛋白。CXCR4免疫染色主要位于细胞质。100 ng/ml CXCL12刺激后CXCR4 mRNA上调（1.24±0.14；t=-7.1088，P=0.0021）。100 ng/ml CXCL12刺激3小时后整合素β1 mRNA显著增加（刺激前后分别为0.53±0.10、1.53±0.16；P<0.01），CXCL12处理24小时后VEGF-C mRNA显著增加（刺激前后分别为0.52±0.09、1.11±0.15；P<0.05）。在无血清次优培养条件下，与对照组和实验组2相比，实验组1显著增强了CAOV3细胞的增殖（分别为0.428±0.051、0.325±0.045、0.328±0.039；P<0.05）。与实验组3和4相比，实验组1受到强烈抑制（后两组分别为0.356±0.031、0.373±0.029；P<0.05）。实验组5（0.349±0.038）与对照组之间无显著差异（P>0.05）。在趋化实验中，与对照组和实验组2相比，实验组1刺激了CAOV3细胞的迁移和侵袭（细胞迁移数分别为523.3±25.2、108.0±7.2、211.7±24.7，细胞侵袭数分别为39.3±4.0、4.0±1.0、15.7±3.1；P<0.01）。与实验组4和5相比，实验组1的这种增强作用受到强烈抑制（P<0.05）。实验组5的迁移和侵袭细胞数（迁移：706.6±30.6，侵袭：61.7±7.6）显著高于实验组1（P<0.05）。
本体外研究表明CXCL12促进卵巢癌细胞系CAOV3的增殖、迁移和侵袭，并上调整合素β1和VEGF-C表达，且这些作用被中和性CXCR4抗体强烈抑制。提示CXCL12及其受体CXCR4可能在卵巢癌生长和转移中起重要作用。
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