Fig. 5.

Knockdown of MDM2 inhibits cancer cell growth and angiogenesis by reducing VEGF production. (A) Stable suppression of MDM2 by transfection of a pSUPER MDM2 siRNA plasmid. MDM2 expression in LA1-55N cells transfected with the pSUPER vector containing a scrambled 19-nt control (siRNA control) and in four clones transfected with pSUPER MDM2 (siMDM2), as seen by Western blotting. (B) VEGF protein levels as detected by ELISA in culture supernatant of LA1-55N cells transfected with either the siRNA control or siMDM2 during hypoxia. (C) Hypoxia-induced drug resistance in MDM2-overexpressing LA1-55N cells (siRNA control), but not in the same cells following MDM2 inhibition with siMDM2. The cells were cultured for 24 h in hypoxia or normoxia and then were treated with 10 μM cisplatin or without cisplatin, under normoxia, for 24 more hours. Cell viability was detected by the WST assay. Data represent the mean percentages (bars) plus SD (error bars) of cell survival relative to the control (normoxia without cisplatin was defined as 100). Values that are significantly different (P < 0.05) are indicated by an asterisk. (D) Effect of anti-VEGF antibody on hypoxia-mediated cisplatin resistance in LA1-55N cells. Cells were exposed to hypoxia and treated with cisplatin as described above for panel C in the absence (−) or presence (+) of increasing concentrations (25, 50, and 100 ng/ml) (indicated by the height of the black triangle) of anti-VEGF antibody or increasing concentrations (50 and 100 ng/ml) of control IgG. Cell survival was detected by the WST assay. (E) Representative histological comparison of tumor vessel content in xenografts of SCID mice that were inoculated with LA1-55N cancer cells containing siRNA control or LA1-55N siMDM2-transfected cells, after 4 weeks, as stained by CD34 immunohistochemistry.