Adiponectin inhibits the growth and peritoneal metastasis of gastric cancer through its specific membrane receptors AdipoR1 and AdipoR2

Abstract

Adiponectin, a circulating peptide hormone produced in adipose tissue, has been shown to be reduced in the plasma of patients with cancer, suggesting that this adipokine may be mechanically involved in the pathogenesis of adiposity-related carcinogenesis. In this study, we examined the expression of adiponectin receptors (AdipoR1 and AdipoR2) and assessed the function of adiponectin in gastric cancer. All of the six gastric cancer cell lines significantly expressed mRNA and protein of both receptors with variable levels. Addition of 30 microg/mL adiponectin potently induced apoptosis and inhibited the proliferation of AZ521 and HCG27. Down-regulation of either AdipoR1 or AdipoR2 by specific siRNA significantly suppressed the growth inhibitory effects of adiponectin in both cell lines. Moreover, a local injection of adiponectin markedly inhibited the growth of AZ521 inoculated subcutaneously in nude mice. Similarly, the continuous intraperitoneal infusion of adiponectin effectively suppressed the development of peritoneal metastasis of AZ521. Adiponectin negatively regulates the progression of gastric cancer cells possibly through both AdipoR1 and AdipoR2. Although adiponectin was already reported to have antiangiogenic effects, our results suggest that the antitumor effect of adiponectin was, at least partially, dependent on the direct effects on tumor cells.

Figure 1

The expression of AdipoR1/R2 in human gastric cancer. (a) Northern blot analysis of AdipoR1 (left panel) and AdipoR2 (right panel) mRNA by different gastric cancer cells. Both receptors were detectable at 2.0 and 4.0 kb, respectively. (b) Reverse transcription‐polymerase chain reaction (RT‐PCR) was carried out on the total RNA as described in the material and methods section. The relative amounts of each AdipoR1/R2 transcript were normalized against the amount of cyclophilin transcript in the same cDNA.

Figure 2

Immunostaining of AdipoR1/R2 in human gastric tisuue. Immunohistochemical staining of AdipoR1 (a; normal mucosa, b; carcinoma) and AdipoR2 (c; normal mucosa, d; carcinoma) using surgically resected gastric cancer tissues. AdipoR1 and R2 were positively detected in the cytoplasm as well as the cell membrane of carcinoma cells.

Figure 3

The effect of adiponectin on cell proliferation of AZ521 and HGC27. These cells were cultured with indicated concentrations of adiponectin in the absence (a) or presence (b) of 100 µM epidermal growth factor (EGF). After incubation for 120 h, cell proliferation was measured by a MTS assay. Data are expressed as a percentage of the control, where control indicates cell proliferation without adiponectin (100%). Columns indicate the mean of five different experiments carried out in triplicate; bars indicate SD. *P < 0.05, compared with control by one‐way anova combined with Bonferroni's test.

Figure 4

Apoptosis of AZ521 cancer cell with adiponectin. (a) AZ521 cell was cultured with (lower panel) or without (upper panel) 30 µg/mL adiponectin for 6 h (left panel) and 24 h (right panel), and then was double‐stained by Annexin‐V (x‐axis) and PI (y‐axis). The representative FACS profile was shown. (b) AZ521 and HGC 27 were cultured for 24 h with (white bar) or without (black bar) 30 µg/mL adiponectin for 24 h and Annexin‐V positive percentages were calculated as apoptotic cells. Columns indicate the mean of three different experiments performed in triplicate; bars indicate SD.

Figure 5

The effect of adiponectin in AdipoR1/R2 down‐regulated cells. AZ521 was cultured with 3 nM siRNA of AdipoR1/R2 or control siRNA for 48 h. (a) The relative amounts of each AdipoR1/R2 mRNA against cyclophilin were measured with real‐time‐polymerase chain reaction (RT‐PCR). (b) The protein expressions of AdipoR1/R2 were examined with Western blotting. (c) AZ521 and HGC27 treated with the same condition described above were cultured with or without 10 or 30 µg/mL adiponectin for additional 24 h and then the cell proliferation was evaluated as described in the legend of Figure 3. Columns indicate the mean of five studies performed in triplicate; bars indicate SD. *P < 0.05, compared with control by one‐way ANOVA combined with Bonferroni's test.

Figure 6

Growth inhibition of subcutaneous tumor by the treatment with adiponectin. AZ521 (1 × 10⁶ in 100 µL phosphate‐buffered saline [PBS]) were subcutaneously implanted in nude mice. Adiponectin (5 µg or 50 µg per mouse) dissolved in 100 µL of PBS or PBS alone were intratumorally injected every day from 3 days after tumor inoculation throughout the experiment. Dots and bars indicate mean and SD in five mice treated with high or low concentrations of adiponectin and PBS in a representative one in three different sets of experiments.

Figure 7

Inhibition of peritoneal metastasis by continuous intraperitoneal infusion of adiponectin. AZ521 (5 × 10⁶ in 1 mL phosphate‐buffered saline [PBS]) was injected into the peritoneal cavity of each mouse, and 3 days later, 50 µg adiponectin or PBS were continuously infused into peritoneal cavity using an Alzet micropump. (a) On day 14, all mice were killed, and the number (left) and volume (right) of macroscopic nodules on the peritoneal surface were calculated. Columns and bars indicate mean and SD of five mice in a representative one in three different sets of experiments. (b) The representative peritoneal cavity of adiponectin (right) or PBS (left) injected mice. Arrow indicates peritoneal metastatic tumor.

Figure 8

Inhibition of neovascularization in peritoneal tumors by adiponectin. (a) In peritoneal tumors, endothelial cells were immunohistochemically detected using a rat antimouse CD31 and vascular density were quantified by counting CD31 positive capillary in eight different fields randomly selected in four different tumors under ×400 magnification. Columns indicate the mean; bars indicate SD. Immunohistochemical expression of blood vessel endothelial cells in disseminated tumor with treated PBS (b) and adiponectin (c). Bar, 100 µm.

Figure 9

Induction of tumor apoptosis cells in peritoneal tumors by adiponectin. (a) In peritoneal tumors, apoptotic cells were immunohistochemically detected by the terminal deoxynucleotidyltransferase‐mediated dUTP nick end labeling (TUNEL) method and the number of apoptotic cells was quantified by counting TUNEL‐positive cells in eight different fields randomly selected in four different tumors under ×400 magnification. Columns indicate the mean; bars indicate SD. Apoptotic cells in tumors treated with phosphate‐buffered saline (PBS) (b) or adiponectin (c) were visualized as green staining. Bar, 100 µm.