Leptin stimulation of cell cycle and inhibition of apoptosis gene and protein expression in OVCAR-3 ovarian cancer cells

Anna Ptak,Elzbieta Kolaczkowska,Ewa L Gregoraszczuk

doi:10.1007/s12020-012-9788-7

Abstract

The OVCAR-3 cell line expressing the long (ObRb) and short (ObRt) isoforms of leptin receptor mRNA was used to analyze the effect of leptin on the expression of selected genes and proteins involved in the cell cycle and apoptosis. OVCAR-3 cells were exposed to 2, 20, 40, and 100 ng/ml of leptin. Cell proliferation was determined using the alamarBlue cell viability test and flow cytometry. Apoptosis was measured using a cellular DNA fragmentation ELISA kit. The expression of selected cell cycle and apoptosis genes was evaluated by real-time PCR and confirmed by western blot. The stimulatory action of leptin on cell proliferation was observed as an increase in cells in the S and G2/M phases. Up-regulation of genes responsible for inducing cell proliferation and suppression of genes responsible for inhibition of proliferation were noted. Western blots revealed increased expression of cyclins D and A and inhibition of p21WAF1/CIP1 protein expression by leptin. Inhibition of DNA fragmentation was observed under all leptin doses. Suppression of genes involved in the extrinsic and intrinsic apoptotic pathway was observed. Western blots illustrated decreased Bad, TNFR1, and caspase 6 protein expression in response to leptin treatment. Leptin promotes ovarian cancer cell line growth by up-regulating genes and proteins responsible for inducing cell proliferation as well as down-regulating pro-apoptotic genes and proteins in apoptotic pathways. Results of this study warrant examining the relationship between the risk of ovarian cancer and elevated leptin levels in obese women.

Highlights

Leptin is a multifunctional peptide hormone with wideranging biologic activities, including appetite regulation, bone formation, reproductive function, and angiogenesis
The cells were seeded in 24-well culture plates at 1 9 105 cell/well in RPMI 1640 supplemented with 5 % FBS or without serum as the control medium, or with leptin at 40 ng/ml for 24 and 48 h
We evaluated the effects of leptin on cell proliferation and apoptosis in the human ovarian cancer cell line OVCAR-3

Summary

Introduction

Leptin is a multifunctional peptide hormone with wideranging biologic activities, including appetite regulation, bone formation, reproductive function, and angiogenesis. Circulating levels of leptin are strongly correlated to body fat content and are markedly elevated in obese compared with normal-weight individuals [1, 2]. Plasma leptin levels have been reported to be higher in overweight and obese women (37.7 ng/ml) than in normal-weight women (3.92–16.9 ng/ml) [2,3,4]. Recent studies have demonstrated that leptin stimulates growth, migration, invasion, and angiogenesis in tumor cell models, suggesting that it can promote an aggressive cancer phenotype [6, 7]. Several studies have addressed the possible role of leptin, the product of the obesity gene (Ob), in ovarian cancer development and progression [11,12,13]. In vitro studies have demonstrated that leptin induces proliferation in the ovarian cancer cell line BG-1 [11, 12], and it has been

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Conclusion