Figure 1.
PI3K/AKT pathway analysis in cervical cancer cell lines.
(A–B) mTOR pathway components and phosphorylated forms of AKT were tested using commercially available antibodies on eight cervical cancer cell lysates prepared without any treatment. (C) PIK3CA, AKT, and PTEN gene mutational status of 8 human cervical cancer cell lines.
Figure 2.
Cervical cancer patients with E545K or E542K mutant tumors have inferior survival outcomes after standard chemoradiation (cisplatin plus pelvic RT).
Kaplan Meier curve for progression-free survival for cervical cancer patients with wild type PIK3CA versus E545K or E542K mutant tumors (p = .05).
Figure 3.
Effects of AKT inhibitors on cell viability.
(A–B) C33A cells were seeded on to 48 well plates and treated with increasing doses of SC-66 (0.0001–5 µg/ml) and MK-2206 (1.25–30 µM) in triplicates for 24 and 48 hrs. Viability was measured using Alamar Blue. Percent viability was calculated based on vehicle treated controls. (C) C33A cells were seeded on to 48 well plates and transfected with oligos against AKT1, AKT2 and RICTOR and treated with SC-66 (1 µg/ml) and MK-2206 (20 µM) in triplicates for 24. Viability was measured using Alamar Blue. Percent viability was calculated based on vehicle treated controls and control siRNA transfected controls, p<0.001 for the comparison of control siRNA versus siRNA for AKT1, AKT2 and RICTOR, SC-661 µg/ml, MK-2206 20 µM. (D) C33A cells were seeded on to 48 well plates and transfected with oligos against AKT1, AKT2 and RICTOR and lysates were prepared after 48 h and western blots were performed. (E) C33A cells were treated with SC-66 (2.5 µg/ml) and MK-2206 25 µM for 24 h then stained with Annexin/7-AAD and analyzed by flow cytometry. The graph represents % cell viability. (F) C33A cells were treated with SC-66 (0.0001 µg/ml–0.1 µg/ml) with or without 20 mM 2-DG for 48 h.
Figure 4.
Effect of SC-66 on mTOR signaling.
(A–C) C33A cells were treated with increasing concentrations of SC-66(6–10 µg/ml) for 3 h and lysates were prepared for western blot.
Figure 5.
Effects of SC-66 on glucose transport.
A) C33A cells were treated with SC-66 (35 µg/ml) or block (cytochalasin B) for 30 minutes prior to incubation with 18F-fluorodeoxyglucose as described in the methods section. The graph represents counts per minute values, p<0.01 for the comparison of FDG alone (cells only) versus FDG + SC-66. B) C33A cells were treated with SC-66 (0–5 µg/ml) for 3 and 24 h and Glut1 levels were analyzed by western blot. C) C33A cells were treated with SC-66 (0, 1 and 5 µg/ml) for 24 h. Membrane and cytosol fractions were prepared using a kit (MemPER) from Peirce Biotechnology. These subcellular fractions were then mixed with sample buffer and incubated at 65°C for 20 mins before loading onto the gels for western blot for Glut1 and Glut4. D) Immunofluorescence was performed on C33A cells after treating them with SC-66 (1 µg/ml) for 3 hours in a chamber slide.
Figure 6.
Effects of SC-66 on glucose metabolism.
(A–B) C33A cells were seeded in T 25 cm2 tissue culture flasks, treated with SC-66 and intracellular NADPH levels and ATP were determined. The graph represents ATP and NADPH µM levels based on standard curve, p<0.01 for the comparison of control versus SC-66 10 µg/ml 1 and 3 h and p<0.001 for control versus 30 µg/ml for ATP levels; p<0.01 for the comparison of control versus SC-66 5 µg/ml 3 h and p<0.01 control versus 10 µg/ml 3 h for NADPH levels. (C–D) C33A cells were seeded in T 25 cm2 tissue culture flasks, treated with SC-66 and excreted lactate levels were measured in nM concentrations using a standard curve, p<0.001 for the comparison of control versus SC-66 5 and10 µg/ml. (E) C33A cells were treated with increasing concentrations of SC-66 (0–5 µg/ml) for 5 h and 24 h and lysates were prepared for western blot.
Figure 7.
Effects of SC-66 and MK-2206 on cell migration.
C33A cells were treated with A) SC-66 (1 and 2.5 µg/ml) and B) MK-2206 (2.5 and 5 µM) for 24 h. Percent wound healing was calculated as described in methods section, p<0.0001 for the comparison of control versus 1 ug SC-66 and p<0.0001 for control versus 2.5 ug SC-66.