Induction of acquired drug resistance in endothelial cells and its involvement in anticancer therapy.
Induction of acquired drug resistance in endothelial cells and its involvement in anticancer therapy.
复制标题
DOI：
10.1186/1756-8722-6-49
复制
发表时间：
2013-07-09
影响因子：
28.5
通讯作者：
Lu H
中科院分区：
文献类型：
--
作者：
Huang L;Perrault C;Coelho-Martins J;Hu C;Dulong C;Varna M;Liu J;Jin J;Soria C;Cazin L;Janin A;Li H;Varin R;Lu H
多药耐药（MDR）是癌症治疗中的主要问题之一。因此，克服它有望改善癌症患者的临床结果。MDR通常以ABC （atp结合盒）蛋白转运体如P-gp、MRP1和ABCG2过表达为特征。虽然ABC转运蛋白对癌细胞的重要性已被认识到，但很少有研究关注其对肿瘤血管生成至关重要的内皮细胞的影响。本研究探讨了这些ABC转运蛋白在体外内皮细胞中的表达和功能，以及它们在小鼠肿瘤生长中的潜在作用。将人微血管内皮细胞（HMEC-1）和人脐静脉内皮细胞（HUVEC）暴露于增加剂量的阿霉素（Dox）诱导ABC基因表达。用3h -胸腺嘧啶和MTS法测定细胞活力。采用流式细胞术、qPCR、western blot检测P-gp、MRP1、ABCG2 mRNA及蛋白表达。罗丹明123 （Rhodamine 123, Rho）在细胞内的积累被用来评估药物外排功能，P-gp、ABCG2和MRP1的抑制剂被用来验证它们各自在体外的作用。为了评估体内内皮细胞的耐药性，在MDA-MB-435肿瘤移植前，胸腺小鼠接受Dox治疗15天，观察Dox治疗后肿瘤生长抑制曲线的变化。此外，从这些小鼠的多个部位分离内皮细胞，通过流式细胞术估计其P-gp表达。在培养基中添加Dox成功诱导HMEC-1和HUVEC耐药。两个稳定的HMEC1亚细胞系（HMECd1和HMECd2）的耐药性分别增加了15倍和24倍。试验还表明，这些诱导的内皮细胞对结构无关的药物柔红霉素、长春花碱和依托泊苷具有交叉耐药。western blot结果显示，HMECd1和HMECd2中P-gp蛋白水平分别升高4倍和6倍。qPCR结果显示，P-gp基因表达增加了3.4倍和7.2倍，ABCG2基因表达略有增加。这些细胞内Rho的积累与P-gp的表达水平呈负相关。P-gp抑制剂，而ABCG2或MRP1抑制剂，能够阻断诱导的内皮细胞对Dox的耐药性。此外，我们还发现，向健康小鼠注射Dox可诱导内皮细胞中P-gp表达增加。在肿瘤生长实验中使用这些预处理小鼠，我们观察到Dox治疗的治疗效率显着降低，这表明支持肿瘤生长的小鼠内皮细胞具有耐药性。ABC转运蛋白可在体外诱导内皮细胞表达。本研究还表明，P-gp在内皮细胞获得对Dox的耐药性中起重要作用，这降低了化疗的效率。
Multidrug resistance (MDR) is one of the major problems in the treatment of cancer. Overcoming it is therefore expected to improve clinical outcomes for cancer patients. MDR is usually characterized by overexpression of ABC (ATP-binding cassette) protein transporters such as P-gp, MRP1, and ABCG2. Though the importance of ABC transporters for cancer cells is recognized, few studies have looked at its implications for the endothelial cells that are essential to tumor angiogenesis. This study investigated the expression and functions of these ABC transporters in endothelial cells in vitro and their potential contribution to cancer growth in mice. Human micro vessel endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC) were exposed to increasing doses of Doxorubicin (Dox) to induce ABC gene expression. Cell viability was then quantified by 3H-thymidine and MTS assay. Flow cytometry, qPCR, and western blot were used to detect mRNA and the protein expression of P-gp, MRP1, and ABCG2. The intracellular accumulation of Rhodamine 123 (Rho) was used to evaluate drug efflux function and the inhibitors for P-gp, ABCG2, and MRP1 were used to verify their respective roles in vitro. In an attempt to evaluate drug resistance in endothelial cells in vivo, athymic mice were treated with Dox for 15 days before a MDA-MB-435 tumor graft to observe subsequent changes in the inhibition curves of tumor growth in response to Dox treatment. Furthermore, endothelial cells from multiple sites in these mice were also isolated to estimate their P-gp expression by flow cytometry. Drug resistance in HMEC-1 and HUVEC was successfully induced by the addition of Dox to the culture media. Two stabilized subcell lines of HMEC1 (HMECd1 and HMECd2) showed 15- and 24-fold increases in resistance. Tests also showed that these induced endothelial cells were cross-resistant to the structurally unrelated drugs Daunorubicin, Vinblastine, and Etoposide. P-gp protein levels increased four and six fold in HMECd1 and HMECd2 as revealed by western blot. The qPCR demonstrated 3.4- and 7.2-fold increases in P-gp, and a slight increase in ABCG2, gene expression. The Rho accumulation within these cells was inversely correlated with the expression levels of P-gp. The inhibitors of P-gp, but not of ABCG2 or MRP1, were able to block the induced endothelial cell resistance to Dox. Furthermore, we also showed that injecting Dox into healthy mice induced an increase in P-gp expression in endothelial cells. Using these pretreated mice in a tumor growth experiment, we observed a dramatic diminution in the therapeutic efficiency of Dox treatment, suggesting implications for drug resistance in mice endothelial cells supporting tumor growth. ABC transporter expression can be induced in endothelial cells in vitro. This study also indicates that P-gp plays an important role in the acquisition of resistance to Dox in endothelial cells and that this reduces the efficiency of chemotherapy.
登录
查看更多内容
影响因子：
3.7
作者：
Goler-Baron V;Assaraf YG
通讯作者：
Assaraf YG
影响因子：
15.9
作者：
JAFFE, EA;NACHMAN, RL;MINICK, CR
通讯作者：
MINICK, CR
通过预防新血管形成，体内肿瘤休眠。
影响因子：
15.3
作者：
Gimbrone, M A Jr;Leapman, S B;Cotran, R S;Folkman, J
通讯作者：
Folkman, J
DOI：
10.1073/pnas.86.2.695
发表时间：
1989-01-01
影响因子：
11.1
作者：
CORDONCARDO, C;OBRIEN, JP;BERTINO, JR
通讯作者：
BERTINO, JR
影响因子：
6.1
作者：
Ji, BS;He, L;Liu, GQ
通讯作者：
Liu, GQ