(a) Average +SA tumor volume in the treatment groups at the start (Day 0) and end (Day 11) of the treatment period. +SA mammary cells () suspended in 100 μL of 0.05 M PBS were injected into the number 4 abdominal mammary fat pad of syngeneic female BALB/c mice. Once tumors reached 4-5 mm in diameter, mice were divided into different treatment groups (8 mice/group) and treated with intralesional injections of lipid nanoemulsion formulations of α-tocopherol (αT), -tocotrienol (T3), or -tocotrienol oxazine derivative, compound 44 (44), at a dose of 0–120 μg/20 μL every other day throughout the experimental period. The untreated control group (C) was added to ensure that intralesional injection of the α-tocopherol nanoemulsion did not influence tumor growth in a nonspecific manner. Data points indicate the average tumor volume (cm3 ± SEM) for 8 mice/group ± SEM in each treatment group. , as compared with the α-tocopherol-treated negative control group. (b) Western blot analysis of HIF-1α, phospho-Akt (p-Akt, Ser473), phospho-mTOR (p-mTOR, Ser2448), phospho-p70S6K (p-p70S6K, Ser424), phospho-eIF-4E1 (p-eIF-4E1, Ser209), phosphor-4E-BP1 (p-4E-BP1, Thr37), and phospho-ERK1/2 (p-ERK1/2, Thr202/Tyr204) in +SA mammary tumors grown in syngeneic BALB/c mice exposed to the various treatments. Lysates prepared from each tumor were separated by polyacrylamide gel electrophoresis (40 μg/lane) followed by Western blot analysis. α-Tubulin was visualized to ensure equal sample loading in each lane. Each Western blot is a representative image of data obtained for experiments that were repeated at least three times. Scanning densitometric analysis was performed on all blots in triplicate and the optical density of each band was normalized with that of the corresponding α-tubulin, as shown in bar graphs. Vertical bars indicate the normalized integrated optical density of bands visualized in each lane SEM. , as compared with the α-tocopherol-treated negative control group. C: Untreated control; α-T: α-tocopherol-treated negative control; T3: -tocotrienol; 44: -tocotrienol oxazine derivative, compound 44.
