The Lcn2-engineered HEK-293 cells show senescence under stressful condition

Document Type : Original Article

Authors

1 Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran

2 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

3 Anatomical Sciences Department, Medicine Faculty, Hamadan University of Medical Sciences, Hamadan, Iran

Abstract

Objective(s): Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely.  Materials and Material and Methods: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by β-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR.
Results: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress.  This decrease was further found to be attributed to senescence.
Conclusion: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence.

Keywords

References

1. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp cell Res 1961; 25:585-621.
2. Rodier F, Campisi J. Four Faces of Cellular Senescence. J Cell Biol 2011; 192:547-556.
3. Ben-Porath I, Weinberg RA. The signals and pathways activating cellular senescence. Int J Biochem Cell Biol 2005; 37:961-976.
4. Erenpreisa J, Cragg MS. Three steps to the immortality of cancer cells: senescence, polyploidy and self-renewal. Cancer cell Int 2013; 13:13-92.
5. Kuilman T, Michaloglou C, Mooi WJ, Peeper DS. The essence of senescence. Genes Dev 2011; 24:2463-2479.
6. Santin AD, Zhan F, Bellone S, Palmieri M, Cane S, Bignotti E, et al. Gene expression profiles in primary ovarian serous papillary tumors and normal ovarian epithelium: identification of candidate molecular mmarkers for ovarian cancer diagnosis and therapy. Int J Cancer 2004; 112:14-25.
7. Missiaglia E, Blaveri E, Terris B, Wang YH, Costello E, Neoptolemos P, et al. Analysis of gene expression in cancer cell lines identifies candidate markers for pancreatic tumorigenesis and metastasis. Int J Cancer 2004; 112:100-112.
8. Liu Q, Nilsen-Hamilton M. Identification of a new acute phase protein. J Biol Chem 1995; 270:22565-22570.
9. Nielsen BS, Borregaard N, Bundgaard JR, Timshel S,Sehested M, Kjeldsen L. Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases. Gut 1996; 38:414-420.
10. Mishra J, Mori K, Ma Q, Kelly C, Barasch J, Devarajan P. Neutrophil gelatinase-associated lipocalin: a novel early urinary biomarker for cisplatin nephrotoxicity. Am J Nephrol 2004; 24:307-315.
11. Hemdahl AL, Gabrielsen A, Zhu C, Eriksson P, Hedin U, Kastrup J, et al. Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction. Arterioscler Thromb Vasc Biol 2006; 26:136-142.
12. Tong Z, Wu X, Ovcharenko D, Zhu J, Chen CS, Kehrer JP. Neutrophil gelatinase-associated lipocalin as a survival factor. Biochem J 2005; 391:441-448.
13. Berger T, Togawa A, Duncan GS, Elia AJ, You-Ten A, Wakeham A, et al. Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury. Proc Natl Acad Sci USA 2006; 103:1834-1839.
14. Rodvold JJ, Mahadevan NR, Zanetti M. Lipocalin2 in cancer: when good immunity goes bad. Cancers Lett 2012; 316:132-138.
15. Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005; 365:1231-1238.
16. Viau  A, El Karoui K, Laouari D, Burtin M, Nguyen C, Mori K, et al. Lipocalin 2 is essential for chronic kidney disease progression in mice and humans. J Clin Invest 2010; 120:4065-4076.
17. Zhao P, Stephens JM.  STAT1, NF-jB and ERKs play a role in the induction of lipocalin-2 expression in adipocytes. Mol Metab 2013; 2:161-170.
18. Bahmani B, Roudkenar MH, Halabian R, Jahanian-Najafabadi A, Amiri F, Jalili MA. Lipocalin 2 decreases senescence of bone marrow-derived mesenchymal stem cells under sub-lethal doses of oxidative stress. Cell Stress Chaperones 2014; 19:685-693.
19. Roudkenar MH, Halabian R, Roushandeh AM, Nourani MR, Masroori N, Ebrahimi M, et al. Lipocalin 2 regulation by thermal stresses: Protective role of Lcn2/NGAL against cold and heat stresses. Exp Cell Res 2009; 315:3140-3151.
20. Lafferty-Whyte K, Bilsland A, Cairney CJ, Hanley L, Jamieson NB, Zaffaroni N, et al. Scoring of senescence signalling in multiple human tumour gene expression datasets, identification of a correlation between senescence score and drug toxicity in the NCI60 panel and a pro-inflammatory signature correlating with survival advantage in peritoneal mesothelioma. BMC Genomics 2010; 11:532.
21. Kasper G, Mao L, Geissler S, Draycheva A, Trippens J, Kühnisch J, et al. Insights into mesenchymal stem cell aging: involvement of antioxidant defense and actin cytoskeleton. Stem cells 2009; 27:1288-1297.
22. Kim JS, Kim EJ, Kim HJ, Yang JY, Hwang GS, Kim CW. Proteomic and metabolomic analysis of H2O2-induced premature senescent human mesenchymal stem cells. Exp Gerontol 2011; 46:500-510.

Files

Share

How to cite

Statistics