Chen Bei, Cao Shanshan, Zhang Yingqi, Wang Xin, Liu Jie, Hui Xiaoli, Wan Yi, Du Wenqi, Wang Li, Wu Kaichun, Fan Daiming
State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, the Fourth Military Medical University, Xi'an, Shaanxi, PR China.
BMC Cell Biol. 2009 Sep 9;10:63. doi: 10.1186/1471-2121-10-63.
The discovery of the importance of angiogenesis in tumor growth has emphasized the need to find specific vascular targets for tumor-targeted therapies. Previously, using phage display technology, we identified the peptide GX1 as having the ability to target the gastric cancer vasculature. The present study investigated the bioactivities of GX1, as well as its potential ability to cooperate with recombinant mutant human tumor necrosis factor alpha (rmhTNFalpha), in gastric cancer therapy.
Tetrazolium salt (MTT) assay showed that GX1 could inhibit cell proliferation of both human umbilical vein endothelial cells (HUVEC) (44%) and HUVEC with tumor endothelium characteristics, generated by culturing in tumor-conditioned medium (co-HUVEC) (62%). Flow-cytometry (FCM) and western blot assays showed that GX1 increased the rate of apoptosis from 11% to 31% (p < 0.01) by up-regulating caspase 3 expression level. A chorioallantoic membrane assay indicated that GX1 could suppress neovascularization in vivo, with the microvessel count decreasing from 21 to 11 (p < 0.05). When GX1 was fused to rmhTNFalpha, GX1-rmhTNFalpha selectively concentrated in the gastric cancer vasculature, as shown by enzyme-linked immunosorbent assay, immunofluorescence and emission-computed tomography. In vitro MTT and FCM assays showed that, compared to rmhTNFalpha alone, GX1-rmhTNFalpha was more effective at suppressing co-HUVEC proliferation (45% vs. 61%, p < 0.05) and inducing apoptosis (11% vs. 23%, p < 0.05). In a tumor formation test, GX1-rmhTNFalpha more effectively inhibited tumor growth than rmhTNFalpha (tumor volume: 271 mm3 vs. 134 mm3, p < 0.05), with less systemic toxicity as measured by body weight (20.57 g vs. 19.30 g, p < 0.05). These therapeutic effects may be mediated by selectively enhanced tumor vascular permeability, as indicated by Evan's blue assay.
GX1 had both homing activity and the ability to inhibit vascular endothelial cell proliferation in vitro and neovascularization in vivo. Furthermore, when GX1 was conjugated to rmhTNFalpha, the fusion protein was selectively delivered to targeted tumor sites, significantly improving the anti-tumor activity of rmhTNFalpha and decreasing systemic toxicity. These results demonstrate the potential of GX1 as a homing peptide in vascular targeted therapy for gastric cancer.
血管生成在肿瘤生长中的重要性被发现后，人们愈发强调需要找到针对肿瘤靶向治疗的特定血管靶点。此前，我们利用噬菌体展示技术鉴定出肽GX1具有靶向胃癌血管的能力。本研究调查了GX1的生物活性及其在胃癌治疗中与重组突变型人肿瘤坏死因子α（rmhTNFα）协同作用的潜在能力。
四氮唑盐（MTT）试验表明，GX1可抑制人脐静脉内皮细胞（HUVEC）的细胞增殖（44%）以及在肿瘤条件培养基中培养产生的具有肿瘤内皮细胞特征的HUVEC（co-HUVEC）的细胞增殖（62%）。流式细胞术（FCM）和蛋白质印迹分析表明，GX1通过上调半胱天冬酶3表达水平使细胞凋亡率从11%提高到31%（p<0.01）。鸡胚绒毛尿囊膜试验表明，GX1可在体内抑制新血管形成，微血管计数从21降至11（p<0.05）。当GX1与rmhTNFα融合时，酶联免疫吸附测定、免疫荧光和发射计算机断层扫描显示，GX1-rmhTNFα选择性地聚集在胃癌血管中。体外MTT和FCM试验表明，与单独的rmhTNFα相比，GX1-rmhTNFα在抑制co-HUVEC增殖（45%对vs . 61%，p<0.05）和诱导细胞凋亡（11%对vs .）方面更有效23%，p<0.05）。在肿瘤形成试验中，GX1-rmhTNFα比rmhTNFα更有效地抑制肿瘤生长（肿瘤体积：271mm³对vs . 134mm³，p<0.05），以体重衡量的全身毒性较小（20.57g对vs . 19.30g，p< Evans蓝试验表明，这些治疗效果可能是由选择性增强的肿瘤血管通透性介导的。
GX1具有归巢活性，在体外具有抑制血管内皮细胞增殖和在体内抑制新血管形成的能力。此外，当GX1与rmhTNFα偶联时，融合蛋白被选择性地递送至靶向肿瘤部位，显著提高了rmhTNFα的抗肿瘤活性并降低了全身毒性。这些结果证明了GX1作为胃癌血管靶向治疗中归巢肽的潜力。 05）。
