Antivascular therapy for multidrug-resistant ovarian tumors by macitentan, a dual endothelin receptor antagonist

Abstract

Endothelin receptors (ETRs) are often overexpressed in ovarian tumors, which can be resistant to conventional therapies. Thus, we investigated whether blockage of the ETR pathways using the dual ETR antagonist macitentan combined with taxol or cisplatinum can produce therapy for orthotopically growing multidrug-resistant (MDR) human ovarian carcinoma. In several studies, nude mice were injected in the peritoneal cavity with HeyA8-MDR human ovarian cancer cells. Ten days later, mice were randomized to receive vehicle (saline), macitentan (oral, daily), taxol (intraperitoneal, weekly), cisplatinum (intraperitoneal, weekly), macitentan plus taxol, or macitentan plus cisplatinum. Moribund mice were killed, and tumors were collected, weighed, and prepared for immunohistochemical analysis. The HeyA8-MDR tumors did not respond to taxol, cisplatinum, or macitentan administered as single agents. In contrast, combination therapy with macitentan and taxol or macitentan and cisplatinum significantly decreased the tumor incidence and weight and significantly increased the survival of mice and their general condition. Multiple immunohistochemical analyses revealed that treatment with macitentan and macitentan plus taxol or cisplatinum inhibited the phosphorylation of ETRs, decreased the levels of pVEGFR2, pAkt, and pMAPK in tumor cells after 2 weeks of treatment and induced a first wave of apoptosis in tumor-associated endothelial cells followed by apoptosis in surrounding tumor cells. Our study shows that ovarian cancer cells, which express the endothelin axis and are multidrug resistant, are exquisitely sensitive to treatment with a dual ET antagonist and can be resensitized to both taxol and cisplatinum. This combined therapy led to a significant reduction in tumor weight.

Figure 1

Immunohistochemical analyses of orthotopic HeyA8-MDR ovarian tumors. pVEGFR2, pAkt, pMAPK, and TUNEL-positive cells are stained green. CD31-positive endothelial cells are stained red and endothelial cells positive for pAkt, pMAPK, and TUNEL are stained yellow. Treatment with macitentan at 10 or 50 mg/kg (but not 1 mg/kg) alone or in combination with taxol inhibited phosphorylation of VEGFR2, Akt, and MAPK. The combination of macitentan at 10 or 50 mg/kg and taxol induced apoptosis in tumor-associated endothelial cells (yellow) and tumor cells (green).

Figure 2

Double immunofluorescence staining for phosphorylated ETRs. HeyA8-MDR tumor sections were stained with an anti-phosphoserine antibody (red), (A) anti-ET_AR antibody (green), or (B) ET_BR antibody; the phosphorylated receptors are stained yellow. Treatment with macitentan at 10 or 50 mg/kg (but not 5 mg/kg), administered alone or with taxol, inhibited phosphorylation of ET_AR and ET_BR. Macitentan (10 mg/kg) plus cisplatinum also inhibited the phosphorylation of the receptors. Therapeutic effective inhibition of receptor phosphorylation was achieved after 2 weeks of treatment.

Figure 3

Inhibition of cell proliferation in orthotopic HeyA8-MDR tumors. Nuclei 4′,6-diamidino-2-phenylindole, dihydrochloride are stained dark blue and Ki-67-positive tumor cells are green, dark blue, and green, yielding a sky blue color for dividing cells. CD31-positive vessels are stained red, and dividing endothelial cells are white (red and green results in white color, arrows). Note the significant decrease in the number of Ki-67-positive cells in tumor and endothelial cells (arrows) in tumors of mice treated with taxol and macitentan (10 or 50 mg/kg), as well as those treated with macitentan (10 mg/kg) and cisplatinum.

Figure 4

Induction of apoptosis in orthotopic HeyA8-MDR tumors. To detect apoptotic cells, tissues were stained with CD31 antibodies (red) and TUNEL (green). Treatment with macitentan (10 or 50 mg/kg, but not 5 mg/kg) and taxol induced apoptosis in tumor (green) and endothelial cells (yellow). Similar findings were found for macitentan (10 mg/kg) and cisplatinum. Two weeks after the start of treatment with macitentan and taxol and 3 weeks after the start of macitentan and cisplatinum treatment, the first wave of apoptosis was induced in tumor-associated endothelial cells (yellow) and surrounding tumor cells.