Ghrelin promotes oral tumor cell proliferation by modifying GLUT1 expression

Abstract

In our study, ghrelin was investigated with respect to its capacity on proliferative effects and molecular correlations on oral tumor cells. The presence of all molecular components of the ghrelin system, i.e., ghrelin and its receptors, was analyzed and could be detected using real-time PCR and immunohistochemistry. To examine cellular effects caused by ghrelin and to clarify downstream-regulatory mechanisms, two different oral tumor cell lines (BHY and HN) were used in cell culture experiments. Stimulation of either cell line with ghrelin led to a significantly increased proliferation. Signal transduction occurred through phosphorylation of GSK-3β and nuclear translocation of β-catenin. This effect could be inhibited by blocking protein kinase A. Glucose transporter1 (GLUT1), as an important factor for delivering sufficient amounts of glucose to tumor cells having high requirements for this carbohydrate (Warburg effect) was up-regulated by exogenous and endogenous ghrelin. Silencing intracellular ghrelin concentrations using siRNA led to a significant decreased expression of GLUT1 and proliferation. In conclusion, our study describes the role for the appetite-stimulating peptide hormone ghrelin in oral cancer proliferation under the particular aspect of glucose uptake: (1) tumor cells are a source of ghrelin. (2) Ghrelin affects tumor cell proliferation through autocrine and/or paracrine activity. (3) Ghrelin modulates GLUT1 expression and thus indirectly enhances tumor cell proliferation. These findings are of major relevance, because glucose uptake is assumed to be a promising target for cancer treatment.

Keywords: Glucose uptake; Oral squamous cell carcinoma cells; Orexigenic peptide.

Fig. 1

Occurrence and gene expression of ghrelin and its receptors in oral tissues. a Immunohistochemical staining of different oral biopsies with anti-ghrelin and anti-growth hormone secretagogue receptor (GHS-R)1a, and 1b antibodies. Primary magnification was 20× (40× for insets). b Relative gene expression of ghrelin, GHS-R1a, GHS-R1b, and Ki-67 in different oral biopsies (n = 15). Statistically significant differences (p < 0.05) between groups are marked with (#). HG healthy gingiva, IF irritation fibroma, LP leukoplakia, OSCC oral squamous cell carcinoma

Fig. 2

a Proliferation rates of BHY and HN cells after stimulation with ghrelin. b Western Blot of GSK-3β and phospho-GSK-3β (p-GSK-3β) in BHY and HN cells after ghrelin treatment. β-actin served as cytosolic loading control for comparison. For densitometric analysis, band intensities of p-GSK-3β and GSK-3β were normalized to β-actin. The numbers indicate relative ratios of p-GSK-3β vs. GSK-3β compared to the control (set to 1). Nuclear detection of β-catenin in BHY and HN cells after ghrelin stimulation. Lamin B1 was used as nuclear loading control. Protein band intensities were normalized to lamin B1. The numbers indicate relative ratios of β-catenin compared to the control (set to 1). c Relative cyclin D1 and d c-myc expression of BHY and HN cells after ghrelin stimulation. Statistically significant differences vs. control are marked with asterisks [p < 0.05 (*); p < 0.001 (***)]

Fig. 3

Inhibition of PKA signaling by H-89 in BHY and HN cells in the presence of externally applied ghrelin (100 nM). a Western Blot of GSK-3β and phospho-GSK-3β in BHY and HN cells after PKA inhibition. β-actin served as cytosolic loading control. For densitometric analysis, band intensities of p-GSK-3β and GSK-3β were normalized to β-actin. The numbers indicate relatives ratios of p-GSK-3β vs. GSK-3β compared to the unstimulated control (set to 1). b Relative gene expression of cyclin D1. c Relative gene expression of c-myc. d Corresponding proliferation rates of BHY and HN cells. Significant differences (p < 0.05) between groups are marked with (hash) and significant deviations from control with (asterisks)

Fig. 4

Effect of ghrelin mRNA silencing on the proliferation of BHY and HN cells. a Relative gene expression of ghrelin in BHY and HN cells after siRNA treatment. b Relative expression of cyclin D1 and c-myc. c Proliferation rates and cell numbers after treatment with 140 nM GHRL siRNA. Significant differences (p < 0.05) between groups are marked with (asterisks)

Fig. 5

Influence of exogenous and endogenous ghrelin on GLUT1. a Relative gene expression of GLUT1 after PKA inhibition by H-89 in the presence of ghrelin (100 nM). Statistically significant differences (p < 0.05) between groups are marked with (hash), to controls with (asterisks). b Western Blot of GLUT1 after stimulation of BHY and HN cells with various concentrations of ghrelin. Protein band intensities were normalized to β-actin. The numbers indicate relative ratios of GLUT1 compared to the control (set to 1). c Relative gene expression of GLUT1 after ghrelin siRNA treatment. Significant differences (p < 0.05) between groups are marked with (asterisks). d Western Blot of GLUT1 after incubation with 140 nM ghrelin siRNA in BHY cells. The numbers indicate the ratio of GLUT1 vs. β-actin. e Schematic summary of ghrelin signaling in oral tumor cells. Activation of GHS-R1a by ghrelin leads to nuclear translocation of β-catenin regulated through PKA and GSK-3β. As a consequence, nuclear β-catenin up-regulates its target genes cyclin D1 and c-myc, thus promoting proliferation. In addition, GLUT1 expression is also positively influenced leading to enhanced proliferative effects