Cell lines and culture conditions
Three poorly differentiated human NPC cell lines CNE-2, HONE-1, and HNE-1, were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco Invitrogen, CA), penicillin (100 units/ml), and streptomycin (100 units/ml) at 37 °C in a humidified 5% CO2 air atmosphere (normoxic condition) or in a humidified, 5% CO2, 0.1% O2 sealed chamber (hypoxic condition). Logarithmically growing cells were used in all experiments.
Drugs and reagents
Evofosfamide was provided by Merck KGaA (Darmstadt, Germany). For in vitro studies, evofosfamide was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 100 mmol/L and stored at − 20 °C. The stock was diluted in fresh culture medium immediately before use and the final concentration of DMSO never exceeded 0.1%. Evofosfamide was dissolved in sterile phosphate buffered saline (PBS) for in vivo studies. Cisplatin (DDP; Hospira Australia Pty Ltd, Victoria, Australia) was obtained as a commercial product from our hospital pharmacy. Cell counting kit-8 (CCK-8) was purchased from Dojindo (Tokyo, Japan). The antibody against HIF-1α was purchased from Becton–Dickinson and Company (Franklin, NJ, USA). Antibody against phospho-histone H2AX (Ser139), Alexa Fluor 488-conjugated antibody against phospho-histone H2AX (Ser139) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Pimonidazole and anti-pimonidazole antibodies were from HPI, Inc. (Burlington, MA, USA).
Cell viability assay
Cell viability was assessed by the CCK-8 assay according to the manufacturer’s instructions [19, 20]. Cells were seeded in 96-well plates and allowed to attach for 24 h. Evofosfamide was added at graded concentrations (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μmol/L) and the cells were incubated under the indicated hypoxic or normoxic conditions for 24 h. After removal of the drug the cells were cultured in complete medium under normoxic conditions for another 24 h. For sequential combination treatment, the cells were incubated with DDP under hypoxic or normoxic conditions for a further 48 h. Dose response curves and the 50% inhibition concentration (IC50) were calculated. The hypoxia cytotoxicity ratio (HCR) was calculated as IC50 under normoxia versus IC50 under hypoxia. Drug synergy was determined by the combination index (CI), which was calculated using Calcusyn software (Biosoft, Cambridge, UK) [21]. A CI of 1 indicates an additive effect between two agents, whereas a CI < 1 or > 1 indicates synergism or antagonism, respectively. All experiments were performed in triplicate in two or more independent experiments.
Clone formation assay
Cells were seeded in 6-well plates 24 h before drug treatment. Cells were incubated with the indicated concentration (0.01, 0.1, 1, 10, 100 μmol/L) of evofosfamide for 6 h under hypoxic or normoxic conditions. The drug was removed by replacing the medium with fresh complete medium and the cells were cultured under normoxic conditions for 7–10 day. Colonies were fixed and stained with crystal violet. Clonal colonies that contained more than 50 cells were counted and the surviving fraction was calculated by dividing the clonal efficiency of treated cells by that of untreated cells.
Flow cytometry
Cells were seeded in plates 24 h before drug treatment and then treated with the indicated concentrations of evofosfamide under hypoxic or normoxic conditions for 24 h. For cell cycle and apoptosis analysis, the cells were fixed in 70% ethanol and stored at − 20 °C overnight. The cells were stained with propidium iodide (PI) with protection from light at room temperature for 30 min and were detected using flow cytometry (Cytomics™ FC 500, Beckman Coulter, Inc., Brea, CA, USA). The DNA content was analyzed using CELL Quest software (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA). Apoptosis was assessed by sub-G1 phase analysis.
H2AX is required for checkpoint-mediated cell cycle arrest and DNA repair following induction of DNA double-strand breaks. DNA damage results in the rapid phosphorylation of H2AX at Ser139 [22]. To detect phospho-histone H2AX (γH2AX), cells were permeabilized with methanol, incubated with Alexa Fluor 488-conjugated γH2AX monoclonal antibody for 2 h, and analyzed by flow cytometry (Cytomics™ Gallios, Beckman Coulter, Inc.).
Western blotting
Cells were harvested and lysed in cell lysis buffer (Cell Signaling Technology, MA). The proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Basel, Switzerland). The membranes were incubated with primary antibodies against HIF-1α, phospho-histone H2AX (Ser139), and GAPDH overnight at 4 °C. After incubation with HRP-conjugated secondary antibody for 1 h at room temperature, bands were detected using an enhanced chemiluminescence (ECL) system (Cell Signaling Technology). GAPDH served as an internal reference.
Xenograft models and antitumor activity in vivo
All animal experiments were conducted in accordance with the Guidelines for the Welfare of Animals in Experimental Neoplasia [23]. Male BALB/c nude mice aged 6–8 weeks were supplied by Guangdong Medical Laboratory Animal Center (Guangzhou, Guangdong, China). HNE-1 cells (2 × 106 cells in PBS) were injected subcutaneously into the right flanks of nude mice. The body weight of the mice and tumor size were measured and recorded twice a week. The tumor volume was calculated by the following formula: Volume (mm3) = length × width2 × 0.5. When the mean tumor volume reached approximately 100–200 mm3, the mice were randomly assigned by the random number table method into six groups (n = 10–11/group) with approximately equivalent ranges of tumor volume between groups. Evofosfamide (50 or 75 mg/kg) was administered by intraperitoneal injection twice a week as a single agent or in combination. DDP (3 mg/kg) was administered by intraperitoneal injection once a week as a single agent or in combination. Intraperitoneal injection of 0.9% NaCl was administered to the controls. All groups were treated for 2 weeks. Antitumor activity was assessed by tumor growth inhibition (TGI; the ratio of the change in mean tumor volume of the treated group to that of the control group) and tumor growth delay (TGD; TGD500 and TGD1000 were determined as the average increase in time for the treated tumor to reach a size of 500 or 1000 mm3 compared with that of the control group). The mice were sacrificed 21 day after treatment and tumor tissues were harvested. The harvested tumor specimens were weighed, fixed in 10% buffered formalin, and embedded in paraffin.
Immunohistochemistry
Pimonidazole was intraperitoneally injected at 60 mg/kg body weight 1 h before animals were sacrificed. The tumor tissues were collected immediately after sacrifice and fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed to assess tumor morphology. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tumor tissue sections. The standard avidin–biotin complex–peroxidase method was used for pimonidazole staining. Slides were observed using a Nikon eclipse 80i microscope at 40× or 100× magnification. Pimonidazole-positive regions were extracted using Image-Pro Plus 6.0 (Media Cybernetics).
Statistical analysis
Statistical analysis was performed using SPSS version 16.0 software (SSPS, Chicago, IL, USA). The data were expressed as mean values ± standard deviation. Differences in the mean values were assessed using one-way analysis of variance. Two-sided P < 0.05 was considered statistically significant.