The Relativity Of Peroxiredoxin Ⅱ And Prostate Cancer

Background and ObjectivePeroxiredoxins (Prxs), initially characterized in yeast, constitute a family of antioxidant enzymes with no homology with conventional antioxidant proteins. More than a hundred homologues have been described from bacteria to mammals, sharing highly conserved protein domains. Prxs play a key role in several cellular functions including protein and lipid protection against oxidative injury, cell proliferation, differentiation and intracellular signaling pathways regulating apoptosis. Increased cellular levels of antioxidant enzymes confer protection against apoptosis. Prxs as reported to be one of major antioxidants in RBCs, and was induced at early stages of erythroid differentiation. The Prx family consists of a kind of peroxidase that is able to remove both peroxide and hydroxyl radical– using reactive cysteine.Six Prx isoforms have been identified which, based on the amino acid sequences, are generally divided into two subfamilies; groups I, II, III, and IV with two conserved cysteines and groups V and VI with one conserved cysteine. Prx I and Prx II proteins are known to be located in cytoplasm. Prx isotypes (I-VI) are associated with the cellular proliferation, differentiation, and antiapoptotic effect, and their expression is relevant to cancer. Prx II was identified as a thiol-specific antioxidant in the K-562 erythroleukemia cell line and erythrocytes, respectively. Prx over-expression is frequently observed in certain types of cancer tissues. Three types of Prx (I, II, and III) have been shown to be over-expressed in the case of human breast cancer, and it has been suggested that their over-expressions are related to cancer development or progression. The increased expression of Prx I is also detected in lung cancer, thyroid tumors and oral cancer, and is suggested to constitute a potential tumor marker.Prx II is a human homologue of yeast thioredoxin peroxidase (TPX), also named thioredoxin-dependent peroxide reductase 1 (TDPX1) or natural killer-enhancing factor-B (NEKF-B). It is located on chromosome 13q12 and is expressed in human neurons but not in glial cells.Prx II is an antioxidant enzyme that reduces H2O2 and other reactive oxygen species using thioredoxin as the immediate electron donor, and its peroxidase activity prevents cells from reactive oxygen species insult. Prx II is also involved in the cellular signaling pathways of growth factors and tumor necrosis factor-a, by virtue of its regulation of intracellular H2O2.Several properties of Prx II, such as thioredoxin- dependent peroxidase activity, inhibition of apoptosis, characteristic binding to membrane, and correlation with Ca2+-activated potassium transport in erythrocytes, have been reported. In previous studies, we found that at least 3 isotypes of Prx have been identified in the skin, and Prx II was identified at the epidermal cells and epidermal appendage structures (hair follicle, eccrine gland, and sebaceous gland) of the dermis. We also found that Prx II was expressed in vascular endothelial cells in normal skin. Prx II has been reported to be present in the cytoplasm of RBCs and human umbilical endothelial cells. In a previous study, Prx II is expressed in endothelial cells of benign and malignant vascular tumors in human skin in vivo. In addition to biologic availability of Prx II as an endothelial marker, it has an advantage in detecting vascular spaces in tissue specimens because it is strongly expressed in intravascular erythrocytes.Prx II is known not only to protect cells from oxidative damage caused by hydrogen peroxide (H2O2), but also to endow cancer cells with resistance to both H2O2 and cisplatin and to grant them radioresistance. Prx II is up-regulated by H2O2 and cisplatin treatment, and its increased expression inhibits the apoptosis induced by cisplatin, thyrotropin, serum deprivation, ceramide, or etoposide, thereby rendering tumor cells resistant to some chemotherapeutic agents. We demonstrated earlier that Prx II was involved in radioresistance.In previous studies, we found that Prx II expression was notably down-regulated in tumor cell lines PC-3M-1E8 with high metastasis potentials compared with lowly metastatic cancer cell lines PC-3M-2B4 through two-dimensional gel electrophoresis (2-DE) and mass spectrometry analyses。To better understand the molecular mechanisms of prostate cancer and demonstrate the relativity of Prx II and prostate cancer, the function of Prx II gene was further investigated. Based on the prevalence of altered Prx II expression and function in different deseases, it was of interest to consider the therapeutic potential of controlling Prx II function or their regulated pathways through pharmacologic or genetic modulation.MethodsPrx II mRNA and protein expression of matched-pair of prostate cancer cell lines with high and low metastasis potentials were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry and western blotting . Prx II full-length eukaryotic expression vectors were constructed using the technique of gene engineering; the prostate cancer cell clones stable expressing Prx II were screened by G418. After transfection, the changes of cell proliferation and motility were detected by CFSE-flow cytometry and monolayer wound healing assay, respectively. The changes of AKT,P-AKT,E-cadherin,β-catenin and Bcl-X/L expression in 1E8/Prx II and 1E8/pcDNA3.1 cell clones were detected by RT-PCR and western blotting. The capabilities of cell growth, cell motility, in vitro invasion and cell adhesion in 1E8/Prx II and 1E8/pcDNA3.1 cell clones were performed by monolayer wound healing assay, Transwell migration assay and cell adhesion assay, respectively. In addition, To construct and identify a yeast two hybrid system bait vector for screening of homo sapiens PrxⅡbinding proteins in prostate cancer, full fragment of ORF of PrxⅡcDNA was amplified using PCR and directly ligated to the pGBKT7 vector. Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay.Results1. In contrast, Prx II mRNA and protein expression was notably down-regulated in prostate tumor cell line PC-3M-1E8 with high metastasis potentials compared with lowly metastatic cancer cell line PC-3M-2B4; These results indicated that abnormal-expression of Prx II gene closely correlated with neoplasm metastasis. Morever, its over-expression inhibited cancer metastasis in prostate cancer;2. Constructed Prx II full-length eukaryotic expression vector successfully, and obtained prostate cancer cell clone(1E8/Prx II) which stably expressed Prx II. At the same time, we obtained control cell clone 1E8/pcDNA3.1.3. After transferring Prx II gene into human prostate cancer cell line PC-3M-1E8, Prx II decreased the abilities of cell proliferation and motility of PC-3M-1E8 cells, Prx II is up-regulated by H2O2(TBHP) treatment, and its over-expression increases the apoptosis induced by TBHP. These functions might be regulated by activating PI4K/AKT intracellular signaling pathway4. Prx II gene over-expression inhibited AKT phosphorylated at Ser473 site and regulated the recycling of internalized E-cadherin. Because of changing the function of E-cadherin /catenin complex retaining cell polarity and tissue structure, Prx II gene over-expression reversed the malignant phenotypes of PC-3M-1E8 cell lines.5. The fragments of PrxⅡprotein were successfully obtained. The recombinant pGBKT7-PrxⅡplasmids and empty pGBKT7 vector could grow white colonies on SD/-Trp/X-α-gal plates and none could survive on SD/-His /-Trp /X-α-gal, SD/-Ade/-Trp/X-α-gal plates. The bait plasmid pGBKT7-PrxⅡconstructed expresses correctly, and can not activate the transcription of reporter gene alone. ConclusionIdentification of PrxⅡmRNA and protein expression in matched-pairs of human prostate cancer cell lines with high and low metastasis potentials showed that abnormal-expression of PrxⅡgene closely correlated with neoplasm metastasis, moreover, in prostate cancer PrxⅡover-expression inhibited cancer metastasis and decreased the abilities of cell proliferation and motility of PC-3M-1E8 cell with high metastasis potentials. Prx II is upregulated by H2O2(TBHP) treatment, and its over-expression increases the apoptosis induced by TBHP.To further investigate the molecular mechanism of PrxⅡin prostate cancer invasion and metastasis, the present studies showed that forced PrxⅡexpression inhibited the capabilities of cell growth and motility of Skov-3 cells through down-regulating the PI4K/AKT signaling; Over-expression of that gene inhibited AKT phosphorylated at Ser473 site and regulated the recycling of internalized E-cadherin. Because of changing the function of E-cadherin/catenin complex retaining cell polarity and tissue structure, Prx II gene over-expression reversed the malignant phenotypes of PC-3M-1E8 cell lines. Moreover, the bait plasmid pGBKT7-PrxⅡconstructed expresses correctly, and can not activate the transcription of reporter gene alone. The yeast two hybrid GAL4 system3 can be utilized to fish PrxⅡr egion interacting protein.