Identification of proteins secreted from leptin stimulated MCF-7 breast cancer cells: a dual proteomic approach.
Leptin is an adipocyte-derived hormone that regulates energy expenditure and food intake. A significant role for leptin in breast cancer has also been indicated by the resistance of leptin knockout mice in development of mammary tumors. In vitro, leptin induces proliferation of MCF-7 cells by activating cellular signaling pathways (1, 11, 12, 16, 17, 56). As leptin is emerging as an important factor for tumor growth, and hormones can exert their actions via autocrine/paracrine mechanisms, we hypothesized leptin may act by regulating epithelial-derived proteins. To test this hypothesis, leptin-regulated proteins secreted from MCF-7 mammary tumor cells were identified using proteomics techniques. Treatment of MCF-7 cells with 500 ng/ml leptin for 24 hours resulted in a 40% increase in cell number and a 5-fold increase in protein secretion as compared to controls. Establishing the significance of leptin-induced secreted factors, the addition of conditioned media from leptin-treated MCF-7 cells to synchronized MCF-7 cells resulted in 40% increase in cell number. Identification of leptin-regulated secreted proteins was done by 2D gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteins identified using Pro Found software and NCBI database included KF10 Collagen Precursor, Serologically Defined Breast Cancer Antigen NY-BR-62 and Cortactin Isoform a. A Human Cytokine Antibody Array system was used to identify low abundant proteins in the media of control and 500 ng/ml leptin-stimulated MCF-7 cells. In leptin treated cells, levels of FGF-9 were increased while IGFBP-3 and TGF-beta3 levels were decreased. Many previous studies have focused on the regulation of distinct cellular proteins by leptin during mammary tumor cell proliferation. However, ours is the first study to identify leptin-regulated secreted proteins, many of which are known to play important roles in cancer. Our data support that leptin can influence mammary tumor growth and progression through regulation of autocrine/paracrine factors and by modulating the extracellular matrix composition.
Perera
Candida N
CN
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Spalding
Heather S
HS
Mohammed
Sulma I
SI
Camarillo
Ignacio G
IG
D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020738:Leptin / Q000378:metabolism*; D013058:Mass Spectrometry; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017077:Culture Media, Conditioned; D016207:Cytokines; D020738:Leptin; D009363:Neoplasm Proteins; D011506:Proteins; D012333:RNA, Messenger