Reversal of Chemoresistance in Ovarian Cancer by Co-Delivery of a P-Glycoprotein Inhibitor and Paclitaxel in a Liposomal Platform

Abstract

The overexpression of permeability-glycoprotein (P-gp), an ABC transporter involved in the cellular exclusion of chemotherapeutic drugs, is a major factor in paclitaxel-resistant ovarian cancer. However, in clinical trials, co-administration of P-gp inhibitors and anticancer drugs has not resulted in the efficient reversal of drug resistance. To improve administration, we encapsulated the third-generation P-gp inhibitor tariquidar (XR-9576, XR), alone or in combination with paclitaxel (PCT) in liposomes (LP). After optimization, the liposomes demonstrated favorable physicochemical properties and the ability to reverse chemoresistance in experiments using chemosensitive/chemoresistant ovarian cancer cell line pairs. Analyzing publicly available datasets, we found that overexpression of P-gp in ovarian cancer is associated with a shorter progression-free and overall survival. In vitro, LP(XR) significantly increased the cellular retention of rhodamine 123, a P-gp substrate. LP(XR,PCT) synergistically inhibited cell viability, blocked proliferation, and caused G₂-M arrest in paclitaxel-resistant SKOV3-TR and HeyA8-MDR cell lines overexpressing P-gp. Holographic imaging cytometry revealed that LP(XR,PCT) treatment of SKOV3-TR cells induced almost complete mitotic arrest, whereas laser scanning cytometry showed that the treatment induced apoptosis. In proof-of-concept preclinical studies, LP(XR,PCT), when compared with LP(PCT), significantly reduced tumor weight (43.2% vs. 16.9%, P = 0.0007) and number of metastases (44.4% vs. 2.8%, P = 0.012) in mice bearing orthotopic HeyA8-MDR ovarian tumors. In the xenografts, LP(XR,PCT) efficiently induced apoptosis and impaired proliferation. Our findings suggest that co-delivery of a P-gp inhibitor and paclitaxel using a liposomal platform can sensitize paclitaxel-resistant ovarian cancer cells to paclitaxel. LP(XR,PCT) should be considered for clinical testing in patients with P-gp-overexpressing tumors. Mol Cancer Ther; 15(10); 2282-93. ©2016 AACR.

Figure 1. Correlation between P-gp expression and ovarian cancer patient survival using two datasets

The cutoff level of expression dividing the high or low groups was calculated by an algorithm of the Kaplan Meier Plotter. (A) Progression-free survival (PFS) analysis of P-gp/ABCB1 gene (n = 1106; log rank P = 1.3 × 10⁻⁵). The cutoff level was determined at 43 to divide high (n = 420) and low (n = 686) groups. (B) Overall survival analysis of ABCB1 gene which encodes for the P-gp protein (n = 1339; log rank P=0.016). The cutoff level was determined at 64 to divide high (n = 520) and low (n=819) expression groups. (C) PFS analysis of MRP1/ABCC1 gene which encodes for the MRP1 protein (n = 1056; log rank P = 0.012). The cutoff level was determined at 393 to divide high (n = 595) and low (n = 461) expression groups. (D) PFS analysis of MRP3/ABCC3 gene (n = 1056; log rank = 0.19). The cutoff level was determined at 101 to divide high (n = 653) and low (n = 403) expression groups.

Figure 2. Synthesis and characterization of drug-loaded liposomes

(A) Schematic of liposome preparation. (B) Transmission electron micrographs of liposomes (LP) encapsulated with no drugs (blank), tariquidar (XR) or paclitaxel (PCT), using a negative staining technique with uranyl acetate. Scale bar, 500 nm.

Figure 3. Tariquidar inhibits p-glycoprotein (P-gp) activity in chemoresistant ovarian cancer cells

(A) Flow cytometric analysis of P-gp and MRP-1 expression. Three pairs of chemotherapy-sensitive (HeyA8, SKOV3ip1, Tyk-Nu) and -resistant (HeyA8-MDR, SKOV3ip1-TR, Tyk-Nu-R) OvCa cell lines were stained with either FITC-labeled anti-P-gp or PE-labeled anti-MRP1 antibody. (B) Immunoblot analysis of P-gp expression in HeyA8, HeyA8-MDR, SKOV3ip1 and SKOV3-TR cells. (C) Rhodamine123 exclusion assay to measure efflux. The sensitive and resistant cell line pairs, HeyA8/HeyA8-MDR and SKOV3ip1/SKOV3-TR cells, were treated with free tariquidar (140 nM, XR) or liposomal XR (14 nM, XR) for 2 days, stained with rhodamine123 and analyzed by flow cytometry. MFI, mean fluorescence intensity. Columns represent means ± SEM from three independent experiments. * p<0.05; ns, not significant, two-tailed t-test.

Figure 4. Liposomal tariquidar/paclitaxel blocks proliferation, migration, and colony formation of chemoresistant ovarian cancer cells

The chemosensitive (HeyA8, SKOV3iP1) and chemoresistant cell lines (HeyA8-MDR, SKOV3-TR) were seeded and treated with tariquidar (XR), paclitaxel (PCT), liposomal (LP) XR [LP(XR)], LP(PCT), LP(XR,PCT), or left untreated (UT). (A) Cell viability. Cells were treated with paclitaxel for 7 days and viability measured. (B) Cell cycle analysis. Top: cells were treated with paclitaxel (100 nM) or tariquidar (90 nM) for 18 hrs before fixation, propidium iodide staining and flow cytometric analysis. Bottom: immunoblot analysis of phosphorylated (Y15) CDC2, total CDC2, CDC25C, Cyclin B1 and p21 in HeyA8-MDR cells upon liposomes treatments (PCT, 100 nM; XR, 9 0nM; 18 hrs). (C) Cell migration. Cells were scratched, treated with drugs (50 nM, PCT; 45 nM, XR) and imaged every 2 hrs for 24 hrs using the IncuCyte^® live cell imaging system. Wound confluence was determined using Zoom software. Columns represent means ± SEM from two independent experiments. (D) Colony formation. Top: monolayer colony formation. Cells were treated (100 nM, PCT; 90 nM, XR) for 7 days before fixation, crystal violet staining and imaging. Bottom: soft agar colony formation. Cells were treated with the indicated drugs at the same concentrations for 40 days before fixation, crystal violet staining and imaging. *** p<0.001; ns, not significant, two-tailed t-test.

Figure 5. Liposomal tariquidar/paclitaxel mediated apoptosis following mitotic arrest due to paclitaxel-induced tubulin polymerization

(A) Confocal microscopy. Immunofluorescent detection of β-tubulin. Images show paclitaxel (PCT)-induced β-tubulin dysfunction in paclitaxel-resistant SKOV3-TR cells treated with liposomal tariquidar/paclitaxel [LP(XR,PCT)]. Blue: Hoechst; green: β-tubulin. Scale bar, 25 µm. (B) Holographic imaging cytometry. SKOV3-TR cells were treated with either LP(PCT) or LP(XR,PCT) and visualized by HoloMonitopr^®M4 every 5 min for 48 hrs. 3D representation of holographic images of cells at 24- or 48-hr treatment is shown. Pseudo-coloring of cells allows gauging of cell thickness. Green depicts cells with low optical thickness, such as flat interphase cells; whereas red indicates cells with high thickness such as rounded cells in mitosis. Middle: 4D projection. ‘Longer streaks’ as seen in the LP(XR,PCT) group show mitotic arrest, since these cells are unable to divide and progressively increase in volume and thickness over a long period of time until cell death. Scale bar, 25 µm. Right: quantification of mitotic arrest with data shown in the left panels (2500 µm³, a threshold for average volume). (C) Laser scanning cytometry to detect apoptosis. SKOV3-TR cells were treated (150 nM, PCT) for 24 hrs and recovered for 24 hrs. The cells were stained with Hoechst (cyan, nuclei), Yo-Pro (green, early apoptosis) and propidium iodide (magenta, late apoptosis). Insert, apoptotic bodies - yellow arrows. Scale bar, 50 µm. In the LP(XR,PCT) panel there are fewer cells because of the combination of reduced cell proliferation and induced apoptosis. Two-way ANOVA.

Figure 6. Paclitaxel and tariquidar-loaded liposomes reduced tumor burden in a chemoresistant ovarian cancer xenograft mouse model

(A) Timeline. One million paclitaxel-resistant HeyA8-MDR cells were intraperitoneally (IP) inoculated into female athymic nude mice (arrow). Eight days later 15 mice were randomized into 3 groups (n = 5 mice), and treated IP with either liposomal paclitaxel [LP(PCT)] (■), liposomal tariquidar/paclitaxel [LP(XR,PCT)] (▲) (1.5 mg/kg, PCT; ~ 1mg/kg, XR) or PBS (●). (B) Body weight. (C) Tumor weight. (D) Metastasis. Columns represent means ± SEM from two independent experiments (n > 8 mice). Two and one mouse were failed to form tumor in LP(PCT) and LP(XR,PCT) groups respectively, and therefore excluded from the assessment. (E) Hematoxylin and eosin staining and immunohistochemistry for cleaved caspase-3 (apoptosis) and Ki-67 (proliferation). Scale bar, 100 µm. The percentage of Ki-67 positive cells and cleaved caspase 3 positive cells over total cells was quantified by ImageJ (right). Bars represent means ± SEM (n = 5 mice). *** p<0.001; ** p<0.01; * p<0.05; ns, not significant, two-tailed t-test.