Ivacaftor Inhibits Glioblastoma Stem Cell Maintenance and Tumor Progression

Abstract

Glioblastoma (GBM) is the most common and malignant primary brain tumor. Glioblastoma stem cells (GSCs) not only initiate and sustain uncontrolled cell proliferation but also resistant to conventional clinical therapies including temozolomide (TMZ) dependent chemotherapy and radiotherapy, implying that there is an urgent need to identify new therapeutic strategies especially specific targeting GSCs. Here, we provide evidence showing that ivacaftor commonly applied in cystic fibrosis therapy acts as a potent inhibitor for GSCs maintenance. We found that ivacaftor promotes cellular apoptosis in vitro and represses patient-derived xenograft (PDX) tumor growth in vivo. In addition, we demonstrate that ivacaftor decreases stemness marker gene expressions of GSCs, including CD133, CD44, and Sox2. In summary, our findings reveal that ivacaftor inhibits glioblastoma progression via specifically eliminating GSCs, which opens a new avenue for GBM clinical therapy in the future.

Keywords: apoptosis; glioblastoma; glioblastoma stem cell; ivacaftor; stemness.

FIGURE 1

Ivacaftor inhibits GSCs proliferation and induces cellular apoptosis. (A) The chemical structure of FDA-approved drug ivacaftor. (B) Cell viability of indicated cells treated with various concentrations of ivacaftor. (C) The cells proliferation of GSC11 (left panel) and GBM1 (right panel) treated by 5 mM ivacaftor. (D,E) Representative images of BrdU incorporation assay in GSC11 (left panel) and GBM1 (right panel) treated with or without ivacaftor. Scale bar: 200 mm. (E) Quantification data for (D). (F,G) Ivacaftor treatment significantly increases cellular apoptosis in GSC11 and GBM1 cells determined by flow cytometry assay. (G) Quantification data for (F). (H) Apoptotic cell markers were dramatically increased following treatment with indicated concentrations of ivacaftor. tPARP, total PARP; cPARP, cleaved PARP. Data are shown as means ± SEM, *P < 0.05; ***P < 0.001; t-test.

FIGURE 2

Ivacaftor inhibits GSCs self-renewal ability. (A) Representative images of tumor sphere derived from GSC11 (top panel) and GBM1 (down panel) treated with indicated concentrations of ivacaftor. Scale bar: 500 μm. (B) Tumor sphere formation ability was significantly attenuated by treating GSC11 (left panel) and GBM1 (right panel) cells with different concentrations of ivacaftor. (C) Ivacaftor treatment significantly decreased GSC11 (left panel) and GBM1 (right panel) larger tumor sphere (diameter larger than 50 μm) formation ability. (D,E) CD133 membrane accumulated positive cells in GSC11 and GBM1 were decreased after treatment with 5 μM ivacaftor for 48 h determined by flow cytometry assay. (E) Quantification data for (D). (F) Effects of ivacaftor in indicated cells evaluated by in vitro limiting dilution assay. (G) GSC11 and GBM1 cells self-renewal ability and stemness marker gene expressions were inhibited by ivacaftor via time- or dosage-dependent manner. Data are shown as means ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; t-test.

FIGURE 3

Ivacaftor sensitizes GSCs response to TMZ. (A,B) Ivacaftor plus TMZ decreased GSC11 (top panel) and GBM1 (bottom panel) cells tumor sphere formation ability. Scale bar: 500 μm. (B) Quantification result of (A). (C) Tumor sphere derived from GSC11 (left panel) and GBM1 (right panel) cells were counted after treating indicated cells with 200 μM TMZ, 5 μM ivacaftor, or 5 μM ivacaftor combined with 200 μM TMZ. (D) Flow chart of xenograft tumor formation assay in nude mouse. (E) Representative images of tumors treated with 3 mg/kg ivacaftor alone or combined with 60 mg/kg TMZ. (F) Tumor masses of xenograft tumors treated with indicated drugs were shown. (G) Tumor volumes of xenograft tumors treated with indicated drugs were shown. (H,I) Representative images of immunohistochemical staining of Ki67 and CC3 in xenograft tumor sections. CC3 means cleaved caspase 3. (I) Quantification data for panel (H). Data are shown as means ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; t-test.

FIGURE 4

Ivacaftor inhibits PDX tumor growth in vivo. (A) Representative images of PDX tumors derived from three independent GBM clinical specimens treated with ivacaftor or vehicle. (B) Tumor mass and volume of three xenograft tumors treated with 3 mg/kg ivacaftor or vehicle. (C,D) Representative images of immunohistochemical staining of Ki67 and CC3 in three PDX tumors. (D) Quantification data for (C). Data are shown as means ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; t-test.