Figure 2

Effects of GRK5-RH in vivo on tumor growth and biochemical analysis: treatment with adenovirus. A) To validate our in vitro results, we studied the effects of adenoviral mediated overexpression of GRK5-RH in a cancer model in vivo. Tumor growth was measured twice a week by a caliper during all the treatment long (17 days of treatment). Low doses (10⁸ pfu/ml in 200 ul) of AdGRK5-NT retard tumor growth compared to controls. High doses (10¹¹ pfu/ml in 200 μl) are more effective, inducing a complete inhibition of tumor growth. Figure also shows a representative image of tumors at the end of the treatment. B) 17 days from starting treatment, mice were sacrified and tumors were taken for histological analysis. To ascertain GRK5-RH expression, cryostat sections were analyzed by direct observation of the green light at the fluorescence microscope. LAC-Z expression in control tumors was evaluated by X-GAL staining at direct light (blue staining indicates LAC-Z expression; eosin was used for counterstaining in red). C) Tumors were homogenized to analyze IκBα levels, VEGF production and apoptosis. Adenoviral mediated overexpression of GRK5-RH induces an increase of IκBα and cleaved caspase 3 levels and a reduction of VEGF expression in treated tumors compared to controls treated with AdLac-Z. D) Nuclear extracts from tumors were analyzed by EMSA. AdGRK5-NT inhibits NFκB activity compared to controls. Lane 1 = probe alone; Lane 2 = empty; Lane 3-4 = AdLac-Z different treated tumors; Lane 5-6 = AdGRK5-NT treated tumors.