Figure 3.

Effect of CPT on (A) glutaminolysis, (B-F) lipogenesis and (G and H) redox regulation in MIAPaCa-2 (mutant KRAS) and BxPC3 (wild-type KRAS) cancer cell lines. (A) Glutamine content in the nonadherent culture medium of MIAPaCa-2 (closed bars) and BxPC3 (open bars) cells treated with CPT. A total of 5×10⁵ cells were seeded into poly-HEMA-coated 60 mm dishes and incubated with 10 µM CPT for 48 h in triplicate. Glutamine content in the culture medium was determined using the Glutamine/Glutamate-Glo Assay kit. The bars indicate mean ± standard deviation. ^*P<0.05 using Welch's t-test. n.s., not significant. (B) Box plot of the areas of lipid droplets in MIAPaCa-2 (gray boxes) and BxPC3 (white boxes) cells treated with CPT. The cells were seeded in culture dishes at a density of 1.0×10⁵ cells/dish. When the cell density reached ~60%, CPT (0 or 5 µM) was added and incubated for 24 h. The cells were fixed in 4% paraformaldehyde and stained with LipiDyeII and DAPI. The integrated density of LipiDyeII staining (n=20 for each group) was measured using ImageJ software and normalized to the average of each control (0 µM) group. In the plots, the box indicates the lower and upper quartile, the horizontal bar represents the median and the whiskers are the highest and lowest data points that fall within 1.5 times the interquartile range from the lower and upper quartiles. ^***P<0.001 using Welch's t-test. n.s., not significant. (C-F) Representative images of lipid droplet formation in (C and D) MIAPaCa-2 and (E and F) BxPC3 cells. The cells were treated or untreated with 5 µM CPT for 24 h and then stained with LipiDyeII and DAPI. The nuclei of the cells were stained with DAPI (blue) and the lipid droplets were stained with LipiDyeII (red). Scale bars=10 µm. (G) Hydrogen peroxide content in the nonadherent culture medium of MIAPaCa-2 (closed bars) and BxPC3 (open bars) cells treated with CPT. A total of 5×10⁵ cells were seeded into poly-HEMA-coated 60 mm dishes and treated with 10 µM CPT for 48 h in triplicate. H₂O₂ contents in the culture medium were determined using the Amplite fluorimetric H₂O₂ kit. The bars indicate the mean ± standard deviation. ^* and ^***P<0.05 and P<0.001 using Welch's t-test, respectively. (H) GSH/GSSG in the nonadherent MIAPaCa-2 (closed bars) and BxPC3 (open bars) cells treated with CPT. A total of 5×10⁵ cells were seeded into poly-HEMA-coated 60 mm dishes and treated with 10 µM CPT for 48 h in triplicate. GSH/GSSG was determined by measuring GSH and GSSG using the GSH/GSSG-Glo Assay kit. The bars indicate mean ± standard deviation. ^***P<0.001 using Welch's t-test. n.s., not significant; CPT, cryptotanshinone; KRAS, Kirsten rat sarcoma viral oncogene homolog; GSH, glutathione-SH; GSSG, glutathione disulfide.