Bridging the gap between cytotoxic and biologic therapy with metronomic topotecan and pazopanib in ovarian cancer

Abstract

This study aimed to investigate the antitumor and antiangiogenic effects utilizing a novel therapy regimen of metronomic topotecan and pazopanib, a multireceptor tyrosine kinase inhibitor. In vitro (Western blot) and in vivo dose-finding experiments were done following pazopanib therapy in ovarian cancer models. Pazopanib and metronomic (daily) oral topotecan therapy was examined in an orthotopic model of ovarian cancer. Tumor weights, survival, and markers of the tumor microenvironment [angiogenesis (CD31 and pericyte coverage), proliferation (Ki-67), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)] were analyzed by immunostaining following therapy. Pazopanib therapy reduced vascular endothelial growth factor receptor 2 (VEGFR-2) activity in vitro and vivo in a dose-dependent manner. Compared with control mice, pazopanib reduced tumor weight by 28% to 82% (P < 0.01 in the SKOV3ip1 model) and metronomic topotecan reduced tumor weight by 40% to 59% in the HeyA8 (P = 0.13) and SKOV3ip1 (P = 0.07) models. Combination therapy had the greatest effect with 79% to 84% reduction (P < 0.01 for both models). In the SKOV3ip1 and A2780 models, mouse survival was significantly longer (P < 0.001 versus controls) with pazopanib and metronomic topotecan therapy. Pazopanib therapy reduced murine endothelial cell migration in vitro in a dose-dependent manner following VEGF stimulation and decreased tumor microvessel density and pericyte coverage when given in combination with metronomic topotecan. Tumor cell proliferation decreased in all treatment arms compared with controls (P < 0.01 for combination groups) and increased tumor cell apoptosis by 4-fold with combination therapy. Pazopanib therapy in combination with metronomic topotecan therapy showed significant antitumor and antiangiogenic properties in preclinical ovarian cancer models and warrants further investigation as a novel therapeutic regimen in clinical trials. Mol Cancer Ther; 9(4); 985-95. (c)2010 AACR.

Figure 1

Inhibition of VEGFR-2 phosphorylation in ovarian cancer cells. A) Western blot analysis of SKOV3ip1 cells treated with pazopanib for 3-4 hours followed by VEGF-A stimulation (10 ng/ml). Lysates were probed for total (tVEGFR-2) and phosphorylated (pVEGFR-2) VEGFR-2. Actin used as loading control. Densitometry analysis (below) demonstrating percent decrease in pVEGFR-2 relative to VEGF only stimulated cells. B) SKOV3ip1 tumor bearing mice were treated with pazopanib (100 mg/kg; daily × 3 days), sacrificed at respective time points. Photomicrographs (original magnification × 100) illustrate tumor cells (blue) and phosphorylated VEGFR-2 (red).

Figure 2

Effect of pazopanib and metronomic topotecan therapy in A) HeyA8 and B) SKOV3ip1 ovarian cancer models. Mice (n=10/group) were randomized to one of the following treatment regimens: vehicle (oral; daily), metronomic topotecan (0.5 mg/kg; oral; daily), pazopanib (100 mg/kg; oral; every other day), or metronomic topotecan plus pazopanib (same doses as single agent regimen). (Mean tumor weights (left panels) and tumor weight distribution (right panels) are shown. Error bars represent SEM. *p<0.05 compared to the control group.

Figure 3

Kaplan-Meier survival curve illustrating effects of pazopanib and metronomic topotecan therapy. A) SKOV3ip1 model comparing: vehicle, metronomic topotecan (0.5 mg/kg, daily), pazopanib 100 mg/kg every other day), combination (metronomic topotecan and pazopanib). p<0.01 combination arm vs. all other groups. B) A2780 model comparing: vehicle 1, pazopanib (100 mg/kg daily), maximum tolerated dose (MTD) topotecan 5 mg/kg (daily × 5d every 21 days), MTD topotecan 10 mg/kg (every 7d × 3 doses), metronomic topotecan (1 mg/kg daily), MTD topo 10 mg/kg + paz, metro topo 1 mg/kg + paz, vehicle 2. p=0.002 and p<0.001 for MTD topo 10 + paz and metro topo 1 + paz vs. paz alone, respectively. p=0.09 MTD topo 10 + paz vs. metro topo 1 + paz.

Figure 4

Anti-angiogenic effects of pazopanib therapy. A) Murine endothelial cell migration in vitro following pazopanib therapy with VEGF stimulation. Bars represent SD. * p<0.05 vs. VEGF treated cells B) Microvessel density analyses of HeyA8 (left) and SKOV3ip1 (right) tumors following pazopanib and metronomic topotecan therapy C) Representative photographs of CD31 staining in SKOV3ip1 tumors (original magnification 100 x) D) Pericyte coverage analysis of SKOV3ip1 tumors. Bars represent SEM. * p< 0.05 vs. control. δ p<0.05 vs. all other groups

Figure 5. Tumor cell proliferation and apoptosis analyses following in vivo therapy. A) Ki-67 staining in HeyA8 (left) and SKOV3ip1 (right) tumors following pazopanib and metronomic topotecan B) Representative photographs of Ki-67 staining in SKOV3ip1 tumors (original magnification 100 x). Bars represent SEM C) TUNEL analysis of tumor cell apoptosis in HeyA8 tumors. Bars represent standard deviation. * p< 0.05 vs. control. δp<0.05 vs. all other groups