Glycolytic inhibitor 2-deoxyglucose simultaneously targets cancer and endothelial cells to suppress neuroblastoma growth in mice

Abstract

Neuroblastoma is characterized by a wide range of clinical manifestations and associated with poor prognosis when there is amplification of MYCN oncogene or high expression of Myc oncoproteins. In a previous in vitro study, we found that the glycolytic inhibitor 2-deoxyglucose (2DG) could suppress the growth of neuroblastoma cells, particularly in those with MYCN amplification. In this study, we established a mouse model of neuroblastoma xenografts with SK-N-DZ and SK-N-AS cells treated with 2DG by intraperitoneal injection twice a week for 3 weeks at 100 or 500 mg/kg body weight. We found that 2DG was effective in suppressing the growth of both MYCN-amplified SK-N-DZ and MYCN-non-amplified SK-N-AS neuroblastoma xenografts, which was associated with downregulation of HIF-1α, PDK1 and c-Myc, and a reduction in the number of tumor blood vessels. In vitro study showed that 2DG can suppress proliferation, cause apoptosis and reduce migration of murine endothelial cells, with inhibition of the formation of lamellipodia and filopodia and disorganization of F-actin filaments. The results suggest that 2DG might simultaneously target cancer cells and endothelial cells in the neuroblastoma xenografts in mice regardless of the status of MYCN amplification, providing a potential therapeutic opportunity to use 2DG or other glycolytic inhibitors for the treatment of patients with refractory neuroblastoma.

Keywords: 2-deoxyglucose; Endothelial cell; MYCN amplification; Neuroblastoma; Xenograft.

Fig. 1.

2DG downregulates the expression of HIF-1α, PDK1 and c-Myc in NB xenograft. The mice received subcutaneous injection of SK-N-DZ NB cells (A,B) and SK-N-AS NB cells (C-E) and received a total of six doses of normal saline or 2DG. The xenografts were harvested on the 27th day and then subjected to western blotting using the indicated antibodies. Graph data are the means±s.e.m.; *P<0.05. n=4 (A,C,E); n=7 for normal saline group and n=4 for 2DG groups (B); and n=7 (D).

Fig. 2.

Immunohistochemical staining for PDK1 expression in neuroblastoma xenografts. Representative xenografts of SK-N-DZ NB cells (A-C) and SK-N-AS NB cells (D-F) receiving a total of six doses each of normal saline (A,D), 100 mg/kg of 2DG (B,E) or 500 mg/kg of 2DG (C,F) were immunostained with anti-PDK1 antibody after formalin fixation and paraffin embedding. PDK1 expression is present in both the nuclei and the cytoplasm of the tumor cells. Decreased PDK1 immunostaining is present with 2DG treatment. Scale bars, 50 μm.

Fig. 3.

2DG significantly suppresses cell proliferation and induces apoptosis in murine endothelial cells. (A) SVEC4-10 cells were treated with six different dosages of 2DG for 24 or 48 h, and then a cell proliferation assay and Trypan Blue exclusion assay were conducted. (B) After 2DG treatment for 24 or 48 h, SVEC4-10 cells were stained with annexin V (fluorescein isothiocyanate)/PI for apoptosis analysis by flow cytometry. (C) The ratio of cells with apoptosis (annexin V⁺, PI⁻ and annexin V⁺, PI⁺) was measured. (D) Cleaved caspase-3 expression was measured by western blotting. Data are means±s.d.; *P<0.05, n=4 (A-C); n=3 (D).

Fig. 4.

2DG decreases wound closure and inhibits cell migration in murine endothelial cells. (A) SVEC4-10 cells treated with 2DG fill the wound area (area between the two dotted lines) more slowly than those untreated at 8 and 24 h. The wound-healing assay was expressed as relative wound width (8 or 24 h average wound width divided by 0 h wound width). (B) For the migration assay, SVEC4-10 cells were cultured in a Boyden chamber and treated with 2DG. After 24 and 48 h, cells were fixed and stained with crystal violet. Stained cells on the bottom membrane were counted on five microscopic fields per sample. Graph data are means±s.d.; *P<0.05.

Fig. 5.

2DG suppresses of lamellipodia and filopodia and causes disorganization of F-actin filaments in murine endothelial cells. After 24 h of 2DG treatment, SVEC4-10 cells were stained with CellMask, F-actin and DAPI. Formation of lamellipodia (arrows in left column) was impaired and formation of filopodia (middle column) inhibited by 2DG treatment. Scale bars, 10 μm.

Fig. 6.

2DG reduces the number of tumor vessels in both AS and DZ xenografts after six doses of treatment. The slides with 3-µm-thick tissue sections of AS and DZ xenografts were stained with IB₄ and counterstained with Hematoxylin (top panels). Five microscopic fields were taken per sample. The IB₄-stained area was selected and highlighted by ImageJ (bottom panels) to determine the percentage of endothelial coverage in the xenograft. Graph data are means±s.e.m.; *P<0.05, n=5 for normal saline group and n=7 for 2DG groups (AS xenografts); n=5 for normal saline group and n=4 for 2DG groups (DZ xenografts).