CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver

Abstract

The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions.

Copyright © 2011 Wiley-Liss, Inc.

Figures

Fig. 1

Modulation of A3AR expression levels in liver tissue derived from Con. A-induced hepatitis in mice. Acute hepatitis was induced in mice by I.V. injection of Concanavalin A(Con.A). Liver tissue was collected 21 h later and protein extracts derived from naïve-and Con. A-induced hepatitis mice were subjected to WB analysis. A3AR expression levels were found to be up-regulated in the liver tissue derived from the mice suffering from liver inflammation in comparison to the naïve animals.

Fig. 2

Effect of CF102 on the development of Con. A-induced hepatitis in mice. Acute hepatitis was induced in mice by I.V. injection of Con.A. CF102 (100 μg/kg) was administered orally twice daily, starting 8 h after Con. A injection. Serum levels of liver enzymes were measured 21 h after Con. A injection. CF102 markedly decreased SGOT and SGPT levels in comparison to the vehicle-treated group (P < 0.05).

Fig. 3

CF102 protected the liver tissue from Con. A-induced damage. Acute hepatitis was induced in mice by I.V. injection of Con.A. Tissue sections of the livers were withdrawn from the mice 21 h after Con. A injection, fixed in formalin and subjected to H&E staining. In the vehicle-treated group, an extensive area of necrosis was observed while in the CF102-treated group no sign of necrosis was noted.

Fig. 4

CF102 acted as an anti-inflammatory agent in Con. A-induced hepatitis. Acute hepatitis was induced in mice by I.V. injection of Con.A. Liver tissues were collected 21 h later and protein extracts were derived from naïve- and Con. A-induced hepatitis mice were subjected to WB analysis. CF102 treatment induced (A) down-regulation in the expression levels of phosphorylated GSK-3β while the total GSK-3β expression levels remained almost unchanged in comparison to the vehicle-treated group. B: Down-regulation in the expression levels of the pro-inflammatory proteins NF-κB and TNF-α in comparison to the vehicle-treated group P < 0.05).

Fig. 5

CF102 prevented apoptosis in the liver upon Con. A-induced hepatitis. Acute hepatitis was induced in mice by I.V. injection of Con.A. Liver tissue was collected 21 h later and protein extracts from derived from naïve- and Con. A-induced hepatitis mice were subjected to WB analysis. CF102 treatment induced down-regulation in the pro-apoptotic proteins FasR, Bax, and Bad in comparison to the vehicle-treated group (P = 0.05).

Fig. 6

CF102 inhibits the proliferation of human HCC Hep-3B cells. Hep-3B cells were incubated for 48 h with CF102 at concentrations of 1 and 10 nM. 3[H]-thymidine incorporation assay revealed that CF102 inhibited linearly the proliferation of the Hep-3B cells.

Fig. 7

Modulation of A3AR expression levels in Hep-3B treated with CF102. Hep-3B cells were incubated for 48 h with CF102 at a concentration of 10 nM. Protein extracts derived from the Hep-3B cells were subjected to WB analysis. CF102 treatment resulted in down-regulation of A3AR expression levels.

Fig. 8

Modulation of down-stream signaling proteins expression levels in Hep-3B treated with CF102. Hep-3B cells were incubated for 48 h with CF102 at a concentration of 10 nM. Protein extracts derived from the Hep-3B cells were subjected to WB analysis. CF102 treatment induced down-regulation of PI3K, PKB/Akt, and NF-κB expression levels and increased the expression levels of the pro-apoptotic protein caspase-3.

Fig. 9

CF102 inhibited the development of Hep-3B tumors in a xenograft model. Hep-3B cells were injected subcutaneously into the flank of balb/c nude mice. Oral treatment with CF102 (100 μg/kg, three times per day) was initiated when the tumor reached ~50–100 mm3 in size and lasted until study termination. A: CF102 treatment inhibited tumor growth in comparison to the vehicle-treated group. B,C: At the end of the study an inhibition of 46% in tumor volume was observed in the CF102-treated group (P < 0.05).

Fig. 10

CF102 up-regulated the apoptotic pathway in Hep-3B tumor cells. Hep-3B cells were injected subcutaneously into the flank of Balb/c nude mice. Oral treatment with CF102 (100 μg/kg, three times per day) was initiated when the tumor reached ~50–100 mm3 in size and lasted until study termination. Upon study termination, the tumors were excised and subjected to WB analysis. CF102 treatment induced up-regulation in the expression levels the pro-apoptotic proteins FasR, caspase-8, Bax, Bad, cytochrome-c, and caspase-3 in comparison to the vehicle-treated group (P = 0.05).