Figure 6. sMEK1 suppressed tumor growth by inhibiting angiogenesis in vivo.

A. Growth curves of human ovarian xenografts treated with sMEK1. Human SKOV-3 ovarian cancer cells were injected subcutaneously into nude mice and allowed to form tumors 70∼100 mm³ in size. Mice were injected i.p. with the control or sMEK1, and tumor size was calculated every 3 days. *, P < 0.05; **, P < 0.01 vs. control. B. Endothelial cells within paraffin-embedded tumor sections were stained using anti-CD31 antibodies. sMEK1-treated tumors exhibited an ∼3-fold reduction in the number of blood vessels stained with CD31. Bar = 50 μm. *, P < 0.05 vs. control. C. Soluble protein extracts were isolated from the tumors of xenografted mice and assayed by immunoblotting using antibodies against VEGF, HIF-1α, p-VEGFR-2, PCNA, Ki-67, cyclin D1, p53, XIAP, and caspase-3. VEGFR-2 and β-actin were used to verify equal loading amounts among the samples. D. sMEK1 treatment decreased the phosphorylation of major regulators of the PI3K/Akt signaling pathway. The levels of phospho-PI3K, phospho-PDK1, phospho-Akt, and phospho-eNOS in total tumor lysates were detected using immunoblot analysis. The unphosphorylated proteins were used as controls to verify equal loading amounts (PI3K, PDK1, Akt, and eNOS). The intensities of the protein bands were analyzed using densitometry.