Li Minglun, Ping Gong, Plathow Christian, Trinh Thuy, Lipson Kenneth E, Hauser Kai, Krempien Robert, Debus Juergen, Abdollahi Amir, Huber Peter E
Department of Radiation Oncology, German Cancer Research Centre (DKFZ), Heidelberg, Germany.
BMC Cancer. 2006 Mar 24;6:79. doi: 10.1186/1471-2407-6-79.
Several small receptor tyrosine kinase inhibitors (RTKI) have entered clinical cancer trials alone and in combination with radiotherapy or chemotherapy. The inhibitory spectrum of these compounds is often not restricted to a single target. For example Imatinib/Gleevec (primarily a bcr/abl kinase inhibitor) or SU11248 (mainly a VEGFR inhibitor) are also potent inhibitors of PDGFR and other kinases. We showed previously that PDGF signaling inhibition attenuates radiation-induced lung fibrosis in a mouse model. Here we investigate effects of SU9518, a PDGFR inhibitor combined with ionizing radiation in human primary fibroblasts and endothelial cells in vitro, with a view on utilizing RTKI for antifibrotic therapy.
Protein levels of PDGFR-alpha/-beta and phosphorylated PDGFR in fibroblasts were analyzed using western and immunocytochemistry assays. Functional proliferation and clonogenic assays were performed (i) to assess PDGFR-mediated survival and proliferation in fibroblasts and endothelial cells after SU9518 (small molecule inhibitor of PDGF receptor tyrosine kinase); (ii) to test the potency und selectivity of the PDGF RTK inhibitor after stimulation with PDGF isoforms (-AB, -AA, -BB) and VEGF+bFGF. In order to simulate in vivo conditions and to understand the role of radiation-induced paracrine PDGF secretion, co-culture models consisting of fibroblasts and endothelial cells were employed.
In fibroblasts, radiation markedly activated PDGF signaling as detected by enhanced PDGFR phosphorylation which was potently inhibited by SU9518. In fibroblast clonogenic assay, SU9518 reduced PDGF stimulated fibroblast survival by 57%. Likewise, SU9518 potently inhibited fibroblast and endothelial cell proliferation. In the co-culture model, radiation of endothelial cells and fibroblast cells substantially stimulated proliferation of non irradiated fibroblasts and vice versa. Importantly, the RTK inhibitor significantly inhibited this paracrine radiation-induced fibroblast and endothelial cell activation.
Radiation-induced autocrine and paracrine PDGF signaling plays an important role in fibroblast and endothelial cell proliferation. SU9518, a PDGFR tyrosine kinase inhibitor, reduces radiation-induced fibroblast and endothelial cell activation. This may explain therapeutic anticancer effects of Imatinib/Gleevec, and at the same time it could open a way of attenuating radiation-induced fibrosis.
几种小分子受体酪氨酸激酶抑制剂（RTKI）已单独或与放疗或化疗联合进入癌症临床试验。这些化合物的抑制谱通常不限于单一靶点。例如，伊马替尼/格列卫（主要是一种bcr/abl激酶抑制剂）或SU11248（主要是一种VEGFR抑制剂）也是PDGFR和其他激酶的有效抑制剂。我们之前表明，在小鼠模型中，抑制PDGF信号可减轻辐射诱导的肺纤维化。在此，我们研究PDGFR抑制剂SU9518与电离辐射联合对人原代成纤维细胞和内皮细胞的影响，以期将RTKI用于抗纤维化治疗。
使用western和免疫细胞化学分析方法分析成纤维细胞中PDGFR-α/-β和磷酸化PDGFR的蛋白水平。进行功能增殖和克隆形成分析：（i）评估SU9518（PDGF受体酪氨酸激酶小分子抑制剂）作用后，成纤维细胞和内皮细胞中PDGFR介导的存活和增殖情况；（ii）在用PDGF异构体（-AB、-AA、-BB）和VEGF+bFGF刺激后，测试PDGF RTK抑制剂的效力和选择性。为模拟体内情况并了解辐射诱导的旁分泌PDGF分泌的作用，采用了由成纤维细胞和内皮细胞组成的共培养模型。
在成纤维细胞中，辐射通过增强PDGFR磷酸化显著激活了PDGF信号，而SU9518可有效抑制该磷酸化。在成纤维细胞克隆形成分析中，SU9518使PDGF刺激的成纤维细胞存活率降低了57%。同样，SU9518可有效抑制成纤维细胞和内皮细胞的增殖。在共培养模型中，内皮细胞和成纤维细胞的辐射显著刺激了未辐射的成纤维细胞的增殖，反之亦然。重要的是，RTK抑制剂显著抑制了这种旁分泌辐射诱导的成纤维细胞和内皮细胞激活。
辐射诱导的自分泌和旁分泌PDGF信号在成纤维细胞和内皮细胞增殖中起重要作用。PDGFR酪氨酸激酶抑制剂SU9518可减少辐射诱导的成纤维细胞和内皮细胞激活。这可能解释了伊马替尼/格列卫的抗癌治疗效果，同时也可能为减轻辐射诱导的纤维化开辟一条途径。
