Leptin induces functional activation of cyclooxygenase-2 through JAK2/STAT3, MAPK/ERK, and PI3K/AKT pathways in human endometrial cancer cells

Abstract

Hyperleptinemia is a common feature of obese women who have a higher risk of endometrial cancer than women with normal weights, and epidemiologic studies have suggested a correlation between obesity and endometrial carcinoma. Therefore, understanding of the molecular mechanism involved in leptin signaling transduction is important in endometrial cancer prevention and treatment. In this study, both isoforms of the leptin receptor (Ob-R), the long form (Ob-Rb) and short form (Ob-Ra), were detected as being expressed in six endometrial cancer cell lines with various differentiation status by western blotting, and Ob-Ra was found to be more abundant than Ob-Rb in these cells. Moreover, the expressions of both isoforms were inversely correlated with histoprognostic grading. We also showed that leptin stimulated cell proliferation and induced activations of signal transducers and activators of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK1/2), AKT, and cyclooxygenase (COX)-2 in endometrial cancer cells dose-dependently by [(3)H] thymidine incorporation assay and western blotting. Leptin-stimulation resulted in increased expression of COX-2 mRNA and prostaglandin E2 (PGE2) production of endometrial cancer cells by reverse transcription-polymerase chain reaction and enzyme immunoassay, respectively, which was effectively blocked by pharmacological inhibitors of Janus tyrosine kinase 2 (JAK2), AG490; of mitogen-activated protein kinase (MAPK) kinase, U0126; of phosphatidylinositol 3-kinase (PI3K), LY294002; and of COX-2, NS398. These results suggest that leptin promotes cell proliferation of endometrial cancer cells via the aforementioned multiple signal-transduction pathways. Leptin-induced functional activation of COX-2 is JAK2/STAT3-, MAPK/ERK-, and PI3K/AKT-dependent, indicating that COX-2 may be a critical factor of endometrial carcinogenesis in obesity.

Figure 1

Expression of the leptin receptor and effect of leptin on the proliferation of endometrial cancer cells. (a) Western blot analysis of leptin receptor expression, long form (Ob‐Rb, 120 kDa) and short form (Ob‐Ra, 90 kDa), in endometrial cancer cells. Equal loading and transfer were shown by repeat probing with β‐actin. (b) Serum‐starved Ishikawa cells were treated for 24 h with increasing concentrations of leptin and (c) six lines of serum‐starved endometrial cancer cells were treated with a 100 ng/mL dose of leptin for 24 h. DNA syntheses were determined by [³H] thymidine incorporation assay. *P < 0.01 and **P < 0.05 compared with untreated control cells, respectively.

Figure 2

Multiple intracellular pathways involved in the growth‐stimulatory effect of leptin on endometrial cancer cells. Serum‐starved Ishikawa cells were treated with leptin (100 ng/mL) for various intervals of time (a) and treated for 10 min with increasing concentrations of leptin (b). Protein expressions of cyclooxygenase (COX)‐2, total and phosphorylated forms of signal transducers and activators of transcription 3 (STAT3), extracellular signal‐regulated kinase (ERK1/2), as well as AKT were detected by western blot analysis. Equal loading and transfer were shown by repeat probing with β‐actin.

Figure 3

Leptin induced cycooxygenase (COX)‐2 mRNA/protein expression and prostaglandin E2 (PGE2) secretion in endometrial cancer cells. Six lines of serum‐starved endometrial cancer cells were stimulated for 4 or 24 h. (a) COX‐2 mRNA expression was detected by reverse transcription–polymerase chain reaction and glyceraldehydes‐3‐phosphate dehydrogenase (GAPDH) was used as an internal control. (b) COX‐2 protein expression and (c) PGE2 production were measured by western blot analysis and enzyme immunoassay, respectively. Results expressed as PGE2 production relative to untreated control cells. *P < 0.01 compared with untreated cells.

Figure 4

Leptin‐induced proliferation and functionally active cyclooxygenase (COX)‐2 of endometrial cancer cells were abolished in the presence of signal transducers and activators of transcription 3 (STAT3), extracellular signal‐related kinase (ERK1/2), AKT, and COX‐2 inhibitors. Six lines of serum‐starved endometrial cancer cells were pretreated with various inhibitors for 1 h followed by combining with/without stimulation of a 100 ng/mL dose of leptin for 24 h. (a) DNA syntheses. (b) Protein expressions of COX‐2, total and phosphorylated forms of STAT3 and ERK1/2, as well as AKT, and (c) prostaglandin E2 (PGE2) production were determined by [³H] thymidine incorporation assay, western blot analysis and enzyme immunoassay, respectively. Equal loading and transfer were shown by repeat probing with β‐actin. *P < 0.01 compared with untreated cells and #P < 0.01 compared with leptin treatment.