Figure 5.

Inhibition of ASNS suppresses in vivo tumor growth of KRAS-mutant CRC cells.

(A) Cell proliferation measured by CCK-8 assay. HCT116 cells transfected with control or two independent shASNS vectors were treated with various concentrations of L-Asp for 72 hours. Viability in each condition was normalized to the control. Mean; bars, ± SD, n = 3 (Student's t test; *P < .05). (B) Clonogenic assay with HCT116 transfected with control or two independent shASNS vectors. Cells were treated by various concentrations of L-Asp for 10 days. Each colony number was normalized to that under no treatment of L-Asp. Mean; bars, ± SD, n = 3 (Student's t test; *P < .05). (C) Cell proliferation measured by CCK-8 assay. HCT116 and DLD-1 cells were treated by DMSO, rapamycin (20 nM), L-Asp (0.25 U/ml), or rapamycin (20 nM) + L-Asp (0.25 U/ml) for 48 hours. Viability in each condition was normalized to that of DMSO treatment group. Mean; bars, ± SD, n = 3 (Student's t test; *P < .05, compared with DMSO treatment group; **P < .05, compared with combination treatment group [rapamycin + L-Asp]). (D) Clonogenic assay. HCT116 and DLD-1 cells were treated by DMSO, rapamycin (20 nM), L-Asp (0.1 U/ml), or rapamycin (20 nM) + L-Asp (0.1 U/ml). Each colony number was normalized to that of DMSO treatment group. Mean; bars, ± SD, n = 3 (Student's t test; *P < .05, compared with DMSO treatment group; **P < .05, compared with combination treatment group [rapamycin + L-Asp)]. (E) Xenograft size of HCT116 cells treated with vehicle, L-Asp, rapamycin, or L-Asp + rapamycin. HCT116 subcutaneously injected into the flank of 4- to 6-week-old female nude mice. Tumors of 150 to 300 mm³ were randomly divides into 4 groups: vehicle, L-Asp alone (1500 U/kg), rapamycin alone (3 mg/kg), and combination of both drugs groups. We injected L-Asp daily and rapamycin every 5 days intraperitoneally for 15 days. Relative tumor volume was calculated based on the tumor volume at the start day of each treatment. Each group, n = 5. Mean; bars, ± SD, Student's t test; *P < .05, compared with combination treatment group (rapamycin + L-Asp).