Induction of acquired drug resistance in endothelial cells and its involvement in anticancer therapy

Abstract

BackgroundMultidrug 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.MethodsHuman 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.ResultsDrug 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.ConclusionsABC 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.

In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines

“…25,26 In any case, DOX has an antiproliferative effect and presumably this is the cause of lower C-MYC and C-FOS expression (as genes involved in cell proliferation and survival). There are many publications on gene expression in the course of treatment with DOX [27][28][29][30][31] but, this is the first report of changes in expression pattern in cell cultures NIH3T3 and MCF7 with magnetic NP-conjugated DOX. Our observations on gene expression level are in accordance with our other tests and indicate that NPs can attenuate or even inhibit the effect of DOX, in particular in tumor MCF7 cell line.…”

La résistance aux traitements antiangiogéniques

“…Nous avons récemment montré qu'ABCB1 était présent sur les cellules HMEC traitées à la doxorubicine. [38]. Ces pompes sont en effet exprimées à la surface des cellules endothéliales isolées de xénogreffes de cellules tumorales humaines (lignée MDA-MB-435) dans des souris nudes (dépourvues de système immunitaire) pré-traitées avec des petites doses de doxorubicine avant l'expression de plusieurs chimiokines proangiogéniques comme CXCL7 [30], CXCL8 ou IL-8 [29], et de leur récepteurs CXCR1-2, exprimés par les cellules endothéliales mais aussi au niveau des cellules tumorales et des cellules du système immunitaire, est observée [18,30].…”

“…Cette multi-origine, de même que la plasticité de ces cellules, semblent contribuer fortement à l'acquisition de la résistance aux agents antiangiogéniques [8]. Nous développons de nouveaux modèles d'étude de ces cellules endothéliales tumola greffe [38]. Ces cellules endothéliales, traitées à la doxorubicine et exprimant ABCB1, présentent des résistances croisées aux agents de chimiothérapie comme l'étoposide (Vépéside ® ou Etopophos ® ou Celltop ® ), mais également au sunitinib [39,40].…”

Discovery of substituted 1,4-dihydroquinolines as novel class of ABCB1 modulators

“…11,17 Moreover, the efflux-pump inducing properties of both substrates and inhibitors gained more interest as they strengthen the mdr problem under therapy. [18][19][20] So there is a great challenge to find novel classes of efflux pump inhibitors. Beside their inhibiting properties they shoud not be substrates of the efflux pump to avoid an unfavourable higher dosage to achieve cellular effects.…”