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Leptin, a cytokine synthesized in adipose tissue, may provide an explanation for why obesity is a risk factor for postmenopausal breast cancer (BC). Serum leptin levels increase in proportion to body weight/fat and when added to human BC cell lines leptin increases proliferation. In the present study effects of leptin concentration on expression of proteins involved in signal transduction and apoptosis were determined for estrogen-receptor (ER) positive (+) MCF-7 and T-47D and the ER negative (-) MDA-MB-231 human BC cell lines. Cells were cultured in 6-well plates in serum free medium for 18 hr and then leptin was added at 0, 5, 10, 25, 50 and 100 ng/ml for 24 or 48 hr and proteins extracted. Extracts were probed for the leptin receptor OB-R and for the specific leptin receptor isoform OB-Rb that is responsible for intracellular leptin signaling. JAK2, Stat3, phospho(P)-Stat3, PCNA and Bcl-2 were measured as representative of leptin signaling, cell proliferation and apoptosis. In MCF-7 cells 24 hr of leptin exposure increased expression of most proteins in a dose-dependent manner up to 50 ng/ml. Bcl-2 and PCNA were not affected. However, after 48 hr only PCNA was increased and Bcl-2 levels declined at higher leptin concentrations. For T-47D cells leptin had little impact at 24 hr but at 48 hr OB-R was increased at lower concentrations and expression of JAK2, Stat3, P-Stat3 and Bcl-2 increased in concert with higher leptin concentrations. No consistent changes were detected in MDA-MB-231 cells for OB-R, OB-Rb, JAK2 and Stat3 in response to increasing levels of leptin, however, P-Stat3 demonstrated a concentration dependent increase at 48 hr. PCNA expression increased over the range 5-50 ng/ml leptin at 24 hr but was unaffected at 48 hr. Corresponding MTT assays indicated that leptin had little effect on MCF-7 cell proliferation at either 24 or 48 hr, but there was a dose related increase in proliferation of T-47D cells at both time points. For MDA-MB-231 cells leptin increased cell proliferation at 24 but not 48 hr. Clearly these breast cancer cell lines respond differently to the concentration and length of leptin exposure. While both ER+ cell lines are leptin sensitive, the nature of the response was different. MCF-7 cells initially were sensitive to low leptin concentrations but this was not sustained. In contrast, T-47D cells had a more robust response to leptin including a possible anti-apoptotic effect and the MTT assay clearly indicated increased cell proliferation. ER- MDA-MB-231 cells exhibited an inconsistent response to leptin with respect to all proteins. Thus, leptin may impact BC development dependent upon characteristics of specific cells types. Ongoing studies will expand these results to provide comprehensive evaluation of the response of BC cells to leptin. This approach will lead to a better understanding of the role of body weight in BC development.Support: Breast Cancer Research Foundation & Hormel Foundation

[Proc Amer Assoc Cancer Res, Volume 46, 2005]