Identification and molecular characterization of a novel splice variant of human 5- lipoxygenase gene.

5-Lipoxygenase (5-LO) is one of the members of Lipoxygenase family. It breaks down arachidonic acid to pro-inflammatory compounds like leukotrienes. Leukotriene plays a major role in the inflammatory process. In this study, while cloning full length 5-LO, a novel splice variant of 5-LO (t5-LO) was found to be expressed in HepG2 cell line. The complete ORF of t5-LO is 420 bp long, expressing 139 amino acid long proteins from N-terminal. The splice variant of 5-LO was cloned, expressed, purified in bacterial system and characterized by MS/MS and western blot experiments. The full length 5-LO is 674 amino acids long encoded by 2,025 bp long ORF. RT-PCR and western blot revealed that t5-LO is extensively expressed in HepG2 cell line.

Structure based design and synthesis of peptide inhibitor of human LOX-12: in vitro and in vivo analysis of a novel therapeutic agent for breast cancer.

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (K(D)) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39 × 10(-8) M and 8.6 × 10(-8) M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45 × 10(-7) M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC(50)) of 75 µM and 400 µM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 µM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.