Abstract
BackgroundBased on its role in angiogenesis and apoptosis, the inhibition of NFκB activity is considered an effective treatment for cancer, hampered by the lack of selective and safe inhibitors. We recently demonstrated that the RH domain of GRK5 (GRK5-RH) inhibits NFκB, thus we evaluated its effects on cancer growth.MethodsThe role of GRK5-RH on tumor growth was assessed in a human cancer cell line (KAT-4). RH overexpression was induced by adenovirus mediated gene transfer; alternatively we administered a synthetic protein reproducing the RH domain of GRK5 (TAT-RH), actively transported into the cells.ResultsIn vitro, adenovirus mediated GRK5-RH overexpression (AdGRK5-NT) in human tumor cells (KAT-4) induces IκB accumulation and inhibits NFκB transcriptional activity leading to apoptotic events. In BALB/c nude mice harboring KAT-4 induced neoplasias, intra-tumor delivery of AdGRK5-NT reduces in a dose-dependent fashion tumor growth, with the highest doses completely inhibiting it. This phenomenon is paralleled by a decrease of NFκB activity, an increase of IκB levels and apoptotic events. To move towards a pharmacological setup, we synthesized the TAT-RH protein. In cultured KAT-4 cells, different dosages of TAT-RH reduced cell survival and increased apoptosis. In BALB/c mice, the anti-proliferative effects of TAT-RH appear to be dose-dependent and highest dose completely inhibits tumor growth.ConclusionOur data suggest that GRK5-RH inhibition of NFκB is a novel and effective anti-tumoral strategy and TAT-RH could be an useful tool in the fighting of cancer.
Highlights
1) Adenoviral mediated overexpression of GRK5-RH a) Cell studies As previously demonstrated in endothelial cells [28], in KAT-4 cells AdGRK5NT causes transgene expression that can be visualized through GFP fluorescence at green light (Figure 1A)
Similar results were achieved by Annexin V cell staining (Figure 1C)
GRK5-RH overexpression causes apoptosis as shown by Annexin-V staining compared to live cells D) We evaluated GRK5 effects on NFκB activity by luciferase assay, in KAT-4 overexpressing GRK5-RH
Summary
Integrating cell-signalling pathways with NF-kappaB and IKK function. Based on its role in angiogenesis and apoptosis, the inhibition of NFκB activity is considered an effective treatment for cancer, hampered by the lack of selective and safe inhibitors. Activation of NFκB is initiated by the signal-induced phosphorylation of IκB proteins by IκB kinase (IKK), inducing IκB ubiquitination and degradation by the proteasome. At this time, NFκB is active and stably localized within the nucleus where it induces the expression of specific genes. NFκB is active and stably localized within the nucleus where it induces the expression of specific genes The activation of these genes by NFκB leads to inflammation, immune response, cell survival, or cellular proliferation depending on cell type. The pathogenetic role of NFκB has been clarified in many diseases [7,8,9], such as type II diabetes and insulin-resistance [10,11]; cardiac hypertrophy [12]; atherosclerosis [13]; chronic heart failure [14]; cancer and angiogenesis [15]
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