|Abstract: ||Abstract:In addition to the known function in the glycolytic pathway, phosphoglycerate kinase 1 (PGK-1) promotes reduction of plasmin disulfide bonds leading to angiostatin formation and inhibition of tumor angiogenesis. In this study, the effects of PGK-1 on anti- tumor immunity against lung cancer were evaluated using the Tet-Off control of PGK-1 expression in the Lewis lung carcinoma (LLC-1). There was no significant difference in cell proliferation between parental LLC-1 and LLC-1 transduced with PGK-1 (PGK-LLC-1). However, expression of PGK-1 was found to limit tumor growth in mice subcutaneously injected with the cell lines and tumor growth was restored after doxycycline treatment. In addition, the cell invasion ability of PGK-LLC-1 became weaker than that of LLC-1. Expressions of COX-2, TGF-β1 and PGE2 were all found to be down-regulated in PGK-LLC-1. PGK-LLC-1 cells treated with doxycycline recovered their COX-2 protein expression. In the presence of conditioned medium from PGK-LLC-1, the endothelial cell migration was reduced. Moreover, PGK-LLC-1 also stimulated T lymphocytes to express higher levels of Th1 cytokine (IFN-γ) and lower levels of IL-10 in comparison with parental LLC-1. PGK-LLC-1 cells restored the growth rate in immunodeficient mice when compared with the growth rate in normal mice. In the tissue sections, reduced COX-2 expressions and marked infiltrated CD3 T lymphocytes were observed in the PGK-LLC-1 injected group. These findings indicate that overexpression of PGK-1 in LLC-1 reduces the COX-2 expression, and, in turn, affect PGE2, cell invasion, angiogenesis, and the immune functions, and finally inhibit the tumor progression. © 2008 Wiley-Liss, Inc.
Lung cancer is the leading cause of cancer-related death. This disease has two major clinical types: the small cell lung carcinoma and non-small cell lung carcinoma (NSCLC).1 In addition to surgery, various combined-modality therapies including chemotherapy and radiation therapy have been evaluated to be unsatisfactory for the treatment of NSCLC and 5-year survival was accomplished in only 15% of the patients.2
Recently, effective therapeutic strategies targeting NSCLC have been designed based on novel immunological findings and newly discovered molecules in tumorigenesis.3 CD4+ Th cells, especially IFN-γ-producing Th1 cells, have been considered to be essential for tumor eradication, since they play a critical role in the activation and maintenance of cytotoxic T cells (CTL).4 IL-12 may induce the differentiation of Th1 to produce IL-2, IFN-γ, and effective cell-mediated anti-tumor responses.5 In addition, IL-10 may induce tumor-mediated immune suppression by limiting Th1 cytokine production,6 antigen presentation, and antigen-specific T cell proliferation.7 Therefore, alterations on the balance control of IL-10 and IL-12 may restore the anti-tumor reactivity of the host.8
Phosphoglycerate kinase 1 (PGK-1) is the enzyme required in the sixth step of mammalian glycolytic pathway. It catalyzes the transfer of phosphate between Position 1 of 1,3-bisphosphoglycerate and γ-phosphate of Mg-ATP.2–9 However, accumulating evidences show that this enzyme is indeed a multifunctional molecules. PGK-1 can affect DNA replication and repair in the mammalian nucleus10 and stimulate viral mRNA synthesis in the cytosol.11 This enzyme may also act as a mRNA binding protein and involve in post-transcriptional regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA.12 Overexpression of PGK-1 has been reported to reduce the expression of uPAR in human lung cancer cells H157 and inhibit migration in H157 cells.13 Moreover, PGK-1 may play a role in inhibiting tumor angiogenesis by promoting an extracellular formation of angiostatin from plasmin.14 Peptides of PGK-1 may also enhance the cytotoxicity of the infiltrated T lymphocytes isolated from human colon cancer by stimulating their IFN-γ expression.15 Therefore, PGK-1 can be considered as a candidate target molecule for specific cancer immunotherapy.
In this study, we established a murine PGK-overexpressing NSCLC cell line to investigate the role of PGK-1 in tumor eradication. The Tet-Off system16 was applied to control PGK-1 expression in LLC-1 cells. Using this system, PGK-1 gene expression was turned off under the addition of doxycycline (Dox). Effects of PGK-1 on tumor cells can be clarified in the presence or absence of Dox. The results indicate that overexpression of PGK-1 in lung cancer cells reduces the COX-2 expression as well as promote the Th1 immune function, and finally inhibit the tumor progression in vivo.