NCI-H460 ×enograft tumor growth in nude mice

A total of 18 male athymic BALB/c mice (8 weeks old; 18–19 g) were housed at a constant room temperature with a 12/12-h light/dark cycle and fed a standard rodent diet under standard pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Kunming Medical University. NCI-H460 cells (1×10⁶) were subcutaneously injected into the left dorsal flanks of the BALB/c mice. A total of 15 days after cell injection, mice were divided randomly into three groups (6 mice/group): Vehicle group (0.9% sodium chloride with 1% DMSO), low dose group (20 mg/kg hispidulin) and high dose group (40 mg/kg hispidulin) once every 2 days for 20 days. Mice body weight and tumor volumes were measured every 3 days for 21 days. All mice were euthanized with CO₂ and serum samples were aliquoted into sterile tubes, centrifuged at 1,100 × g for 10 min at room temperature for subsequent analysis. The activities of aspartate transaminase (AST) and alanine transaminase (ALT) were analyzed by using a VetScan analyzer (Abaxis, Inc.). Assay kits of AST and ALT were purchased from Abcam (cat. nos. ab105134 and ab105135). The harvested heart, liver and kidney tissues of mice were used for hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) staining were performed on NCI-H460 cell-derived xenograft tumors as previously described (20). The following primary antibodies were used for IHC: Proliferation marker protein Ki-67 (1:50; cat. no sc-7846; Santa Cruz Biotechnology, Inc.), cleaved-caspase 3 (1:100; cat. no. 9664; Cell Signaling Technology, Inc.) and p-EIF2α (1:1,000; cat. no. 3398; Cell Signaling Technology, Inc). HRP-conjugated secondary antibodies (cat. nos. sc-2317 and sc-2031) were purchased from Santa Cruz Biotechnology, Inc. Caspase-3 activity in tumor lysates was determined using a caspase-3 activity kit (Beyotime Institute of Biotechnology) according to the kit instructions.