Cloning And Expression Of RKIP And Preliminary Study Of Its Biological Properties In Apoptosis Detection And Tumor Metastasis

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding protein (PEBP) family, which is a widely expressed protein in a variety of different species. RKIP plays a pivotal modulatory role in several intracellular signaling cascades. RKIP plays a critical role in several physiological processes including membrane biosynthesis, spermatogenesis, neural development and apoptosis. It also contributes to pathophysiological processes including Alzheimer’s disease and diabetic nephropathy. Recent researches show RKIP is a novel metastasis suppressor.Metastasis is a complex and multi-step process, a process of interaction between cancer cells and the host. Tumor cells escape from the original tumor lesions into the circulatory system, and invade into a distant tissue. The cells grow to a visible tumor at the target site. The gene which inhibits tumor transfer process is called metastasis suppressor gene (MSG). Metastasis suppressor gene is expected to become a new target for diagnosis and treatment.Aberrant RKIP expression plays a critical role in cancer. Recently, researcher has identified a novel anti-metastasis function for RKIP in prostate cancer. We compared levels of RKIP expression in non-metastatic prostate cancer cell lines and metastatic prostate cancer cell lines. The metastatic prostate cancer cells had much less RKIP expression than non-metastatic prostate cancer cells. Immunochemistry of tissues from patients indicated moderate to high RKIP protein expression in normal prostate and primary prostate cancers; however, its expression was down-regulated or undetectable in prostate cancer metastases. Restoration of RKIP expression in metastatic prostate cancer cell lines was associated with decreased in vitro invasiveness of the cells. Furthermore, RKIP restoration in metastatic prostate cancer cells reduced spontaneous lung metastasis, but not primary tumor growth rate, in mice injected with tumor cells orthotopically. These observations define RKIP as a metastasis suppressor gene which, by definition, suppress metastasis without affecting tumor igenicity. The mechanisms through which RKIP suppresses metastasis are not known. However, increased RKIP expression was associated with decreased vascular invasion and decreased ability to form new blood vessels in the primary tumors in the rodent model suggesting that RKIP may act at the early angioinvasive stages of metastasis. Taken together, these data demonstrate that RKIP is a clinically relevant prostate cancer metastasis suppressor gene that regulates the Raf/MEK/ERK pathway. RKIP could be a promising molecular target for compounds designed for cancer treatment.RKIP has the molecular weight of 23 kDa, contains 187 amino acid residues in one single-chain. Immunohistochemistry confirmed that RKIP presents in the cytoplasm and plasma membrane. The crystal structure shows that RKIP in different species has a similar topology:the central is a large 6 layers of p fold and the N-terminal is three layers of smaller P fold, the C-terminal has two a helix. In this structure, there is a highly conserved phosphate binding pocket (phosphate-binding pocket), which is very important for the function of PEBP.When apoptosis occurs, phosphatidyl serine (phosphatidylserine, PS) flips from the inner side of membrane to the lateral. Fluorescence or radioactivity marked Annexin V specifically combines PS, which can be used to detect apoptosis together with chromatin fluorescent dye propidium iodide (PI). When apoptosis occurs, cell membrane keeps integrity and PI can not enter cell, but labeled Annexin V has membrane-bound characteristics, which is different from the double positive results during cell necrosis.The RKIP protein’s function outside the cell has not been investigated, this study use the prokaryotic expression system to express and purify RKIP protein and GFP-RKIP fusion protein with fluorescence. RKIP protein has been used to observe its influence on tumor cell metastasis and the ability of apoptotic detection of EGFP-RKIP protein. Two variants of RKIP, one mutant removed the N-terminal β fold and the other removed the two a helices at C-terminal, have been construected to study the structure-function relationship of RKIP.