Growth inhibition of human hepatocellular carcinoma cells by overexpression of G-protein-coupled receptor kinase 2

Abstract

Hepatocellular carcinoma (HCC) is one of the deadliest forms of human liver cancer and does not respond well to conventional therapies. Novel effective treatments are urgently in need. G-protein-coupled kinase 2 (GRK2) is unique serine/threonine kinase that involves in many signaling pathways and regulates various essential cellular processes. Altered levels of GRK2 have been linked with several human diseases including cancer. In this study, we investigated a novel approach for HCC treatment by inducing overexpression of GRK2 in human HCC cells. We found that overexpression of GRK2 through recombinant adenovirus transduction inhibits the growth of human HCC cells. BrdU incorporation assay showed that the growth inhibition caused by elevated GRK2 level was due to reduced cell proliferation but not apoptosis. To examine the anti-proliferative function of increased GRK2 level, we performed cell cycle analysis using propidium iodide staining. We found that the proliferation suppression was associated with G2/M phase cell cycle arrest by the wild-type GRK2 but not its kinase-dead K220R mutant. Furthermore, increased levels of wild-type GRK2 induced upregulation of phosphor-Ser(15) p53 and cyclin B1 in a dose-dependent manner. Our data indicate that the anti-proliferative function of elevated GRK2 is associated with delayed cell cycle progression and is GRK2 kinase activity-dependent. Enforced expression of GRK2 in human HCC by molecular delivery may offer a potential therapeutic approach for the treatment of human liver cancer.

Fig. 1

Basal GRK2 protein levels in HCC cell lines. A: Cytosolic distribution of GRK2. B: Basal GRK2 protein levels in all five HCC cell lines detected by Western blotting. Cell lysates (25 μg total protein) were subjected to immunoblotting with antibodies as indicated. GRK2 levels were normalized to GAPDH. The immunoblot shown is a representative of two independent experiments.

Fig. 2

Overexpression of GRK2 reduces cell viability in HCC cells. A: Western blots showing overexpression of wild-type GRK2 and its kinase-dead K220R mutant in Mahlavu and Huh7 cells transduced with recombinant adenovirus containing WT GRK2 or GRK2 K220R mutant. β-Actin was used as total protein loading control. B: MTT method was used to detect cell viability with overexpression of GRK2 and K220R mutant during 4-day growth period in 10% FBS medium. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/jcp]

Fig. 3

Overexpression of GRK2 suppresses cell proliferation in HCC cells. A: Immunofluorescent staining of BrdU incorporation in Huh7 cells. DAPI was used for staining nucleus. Antibody against BrdU was immunoblotted with a secondary antibody labeled with Tritc. Overexpression of GRK2 and K220R mutant was achieved by adenovirus transduction as indicated in Figure 2. B: BrdU incorporation in Huh7 cells as shown in A. C: BrdU incorporation in Mahlavu cells. GRK2 H represents at least twofold higher of MOI of adenovirus used for achieving GRK2 overexpression. *P < 0.01.

Fig. 4

Overexpression of GRK2 leads to G2/M phase arrest in HCC cells. A: Representative cell cycle his to grams with over expression of GRK2 and K220R mutant 2 days post-adenovirus transduction. Cells were analyzed by FACS for DNA content by PI staining. GRK2 H represents at least twofold higher of MOI of adenovirus used for achieving GRK2 overexpression. B: Cells distributed in different phases as shown in A.

Fig. 5

Overexpression of GRK2 induces upregulation of phosphor-p53, cyclin B1, and phosphor-Akt. A. Overexpression of GRK2 in Huh7 was achieved by increasing MOI of adenovirus for examining dose-dependent effects. Cell lysates were subjected to immunoblotting with antibodies as indicated on the penal. In Mahlavu cells, Western blotting was compared between overexpression of GRK2 and K220 R mutant. B: Western blotting with overexpression of GRK2 K220R mutant in a dose-dependent manner in Huh7 and Mahlavu cells.