A new synthetic protein, TAT-RH, inhibits tumor growth through the regulation of NFκB activity

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.

Intracardiac Injection of AdGRK5-NT Reduces Left Ventricular Hypertrophy by Inhibiting NF-κB–Dependent Hypertrophic Gene Expression

“…This effect is attributed to the sterical inhibition of NF-B activity in the cardiac myocyte through means of the stabilization of IB␣. Indeed, GRK5-NT contains the kinase RH domain, which is sufficient for GRK5-NT effects 12 but lacks both the catalytic domain and the nuclear localization sequence of GRK5. Thus, this mutant cannot participate in the regulation of hypertrophy that depends on GRK5-mediated phosphorylative events.…”

“…24 Rather, we show NF-B inhibition induced by IB␣ accumulation in cardiac myocytes, a mechanism demonstrated previously in other cell types. 11,12 In the in vivo study, we used a genetic model of LVH, the SHR, in which LVH is sustained by high BP levels. It has been described that the inhibition of NF-B reduces LVH in SHRs in a BP-independent manner.…”

“…In H9C2 cardiomyoblasts, AdGRK5-NT caused an increase of total IB␣ levels ( Figure 1A), analyzed by Western blot, as described previously in other cellular types. 11,12 The effect of GRK5-NT on NF-B activity was evaluated by luciferase assay. PE increased NF-B transcription activity, and the overexpression of GRK5-NT reduced such effect both in basal conditions and after PE stimulation ( Figure 1B), suggesting that NF-B is induced by hypertrophic stimuli in cardiac cells and that GRK5-NT is able to inhibit such phenomenon.…”

“…AdGRK5-NT was a kind gift of Prof Walter J. Koch (Thomas Jefferson University). 12 Transient transfection was performed using the Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. Cells were stimulated with the alpha 1 adrenergic receptor agonist phenylephrine (PE; 10 Ϫ7 M) for 24 hours.…”

“…To evaluate adenovirus expression, paraffined sections were analyzed directly at fluorescent microscopy or the green fluorescent protein (GFP) tag was evaluated by immunohistochemistry, as previously described. 12 For the analysis of cardiomyocytes size, Masson's trichrome staining sections were used. 21 The areas (squared micrometers) of 100 cardiac myocytes per heart were measured with the public domain Java image processing program ImageJ.…”