Abstract
Lipocalin 2 (LCN2) is a secreted, iron-binding glycoprotein that is abnormally expressed in some malignant human cancers. However, the roles of LCN2 in hepatocellular carcinoma (HCC) cells are unknown. In this study, we suggested the LCN2 and LCN2R were weak detected in the HCC cell lines, LCN2 and LCN2R were found to be down-regulated in tumor tissues in 16 HCC patients. MTT, DAPI, TUNEL, and flow cytometry analyses revealed that LCN2 overexpression dramatically inhibited cell viability, induced apoptosis features of cell-cycle arrest in sub-G1 phase, in DNA fragmentation, and in condensation of chromatin in Huh-7 and SK-Hep-1 cells. Western blots were used to detect the activation of caspase, pro-apoptosis, and anti-apoptosis protein expression in overexpress-LCN2 HCC cells. LCN2-induced apoptosis was characterized by cleavage of caspase-9, -8, -3, and PARP protein, and a reduction in the mitochondrial membrane potential (MMP). Furthermore, LCN2 also enhanced the down-regulated Bcl-2 and up-regulated the expression of Bax. In addition, our experiments with caspase inhibitors LEHD-FMK and IETD-FMK prevent LCN2-induced apoptosis. We also demonstrated that treatment of overexpress-LCN2 HCC cells with the LCN2 neutralized antibody also significantly attenuated LCN2-induced cell apoptosis. These findings indicate that LCN2 overexpression can effectively induce apoptosis of HCC cells and may be used as a potent therapy against human HCC.
Similar content being viewed by others
Abbreviations
- LCN2:
-
Lipocalin 2
- LCN2R:
-
Lipocalin 2 receptor
- PARP:
-
Poly (ADP-ribose) polymerase
- HCC:
-
Human hepatocellular carcinoma
- MMP:
-
Mitochondrial membrane potential
References
Bosch, F. X., Ribes, J., & Borras, J. (1999). Epidemiology of primary liver cancer. Seminars in Liver Disease, 19, 271–285.
Teo, E. K., & Fock, K. M. (2001). Hepatocellular carcinoma: An Asian perspective. Digestive Diseases, 19, 263–268.
Farazi, P. A., & DePinho, R. A. (2006). Hepatocellular carcinoma pathogenesis: From genes to environment. Nature Reviews Cancer, 6, 674–687.
Flower, D. R., North, A. C., & Sansom, C. E. (2000). The lipocalin protein family: Structural and sequence overview. Biochimica et Biophysica Acta, 1482, 9–24.
Kehrer, J. P. (2010). Lipocalin-2: Pro- or Anti-apoptotic? Cell Biology and Toxicology, 26, 83–89.
Stoesz, S. P., Friedl, A., Haag, J. D., Lindstrom, M. J., et al. (1998). Heterogeneous expression of the lipocalin NGAL in primary breast cancers. International Journal of Cancer, 79, 565–572.
Zhang, H., Xu, L., Xiao, D., et al. (2007). Upregulation of neutrophil gelatinase-associated lipocalin in oesophageal squamous cell carcinoma: Significant correlation with cell differentiation and tumour invasion. Journal of Clinical Pathology, 60, 555–561.
Friedl, A., Stoesz, S. P., Buckley, P., & Gould, M. N. (1999). Neutrophil gelatinase-associated lipocalin in normal and neoplastic human tissues. Cell type-specific pattern of expression. Histochemical Journal, 31, 433–441.
Nielsen, B. S., Borregaard, N., Bundgaard, J. R., et al. (1996). Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases. Gut, 38, 414–420.
Patil, M. A., Chua, M. S., Pan, K. H., et al. (2005). An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma. Oncogene, 24, 3737–3747.
Cho, H., & Kim, J. H. (2009). Lipocalin2 expressions correlate significantly with tumor differentiation in epithelial ovarian cancer. Journal of Histochemistry and Cytochemistry, 57, 513–521.
Alpizar-Alpizar, W., Laerum, O. D., Illemann, M., et al. (2009). Neutrophil gelatinase-associated lipocalin (NGAL/LCN2) is upregulated in gastric mucosa infected with helicobacter pylori. Virchows Archiv, 455, 225–233.
Tong, Z., Kunnumakkara, A. B., Wang, H., et al. (2008). Neutrophil gelatinase-associated lipocalin: A novel suppressor of invasion and angiogenesis in pancreatic cancer. Cancer Research, 68, 6100–6108.
Miharada, K., Hiroyama, T., Sudo, K., et al. (2005). Lipocalin 2 functions as a negative regulator of red blood cell production in an autocrine fashion. FASEB J, 19, 1881–1883.
Tong, Z., Wu, X., Ovcharenko, D., et al. (2005). Neutrophil gelatinase-associated lipocalin as a survival factor. Biochemical Journal, 391, 441–448.
Nelson, A. M., Zhao, W., Gilliland, K. L., et al. (2008). Neutrophil gelatinase-associated lipocalin mediates 13-cis retinoic acid-induced apoptosis of human sebaceous gland cells. J Clin Investig, 118, 1468–1478.
Wu, T. T., Hsieh, Y. H., Hsieh, Y. S., & Liu, J. Y. (2008). Reduction of PKC alpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma. Journal of Cellular Biochemistry, 103, 9–20.
Tsai, H. T., Su, P. H., Lee, T. H., et al. (2001). Significant elevation and correlation of plasma neutrophil gelatinase associated lipocalin and its complex with matrix metalloproteinase-9 in patients with pelvic inflammatory disease. Clinica Chimica Acta, 412, 1252–1256.
Jung, J. I., Lim, S. S., Choi, H. J., et al. (2006). Isoliquiritigenin induces apoptosis by depolarizing mitochondrial membranes in prostate cancer cells. Journal of Nutritional Biochemistry, 17, 689–696.
Hsieh, Y. H., Wu, T. T., Huang, C. Y., et al. (2007). p38 mitogen-activated protein kinase pathway is involved in protein kinase Calpha-regulated invasion in human hepatocellular carcinoma cells. Cancer Research, 67, 4320–4327.
Lee, E. K., Kim, H. J., Lee, K. J., et al. (2011). Inhibition of the proliferation and invasion of hepatocellular carcinoma cells by lipocalin 2 through blockade of JNK and PI3K/AkT signaling. International Journal of Oncology, 38, 325–333.
Devireddy, L. R., Teodoro, J. G., Richard, F. A., & Green, M. R. (2001). Induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by IL-3 deprivation. Science, 293, 829–834.
Lin, H. H., Li, W. W., Lee, Y. C., & Chu, S. T. (2007). Apoptosis induced by uterine 24p3 protein in endometrial carcinoma cell line. Toxicology, 234, 203–215.
Lee, S., Lee, J., Kim, S., et al. (2007). A dual role of lipocalin 2 in the apoptosis and deramification of activated microglia. Journal of Immunology, 179, 3231–3241.
Berger, T., Togawa, A., Duncan, G. S., et al. (2006). Lipocalin 2-deficient mice exhibit increased sensitivity to escherichia coli infection but not to ischemia-reperfusion injury. Proceedings of the National Academy of Sciences USA, 103, 1834–1839.
Mannelqvist, M., Stefansson, I. M., Wik, E., et al. (2012). Lipocalin 2 expression is associated with aggressive features of endometrial cancer. BMC Cancer, 12, 169.
Iannetti, A., Pacifico, F., Acquaviva, R., et al. (2008). The neutrophil gelatinase associated lipocalin (NGAL), a NF-kappaB-regulated gene, is a survival factor for thyroid neoplastic cells. Proceedings of the National Academy of Sciences USA, 105, 14058–14063.
Bauer, M., Eickhoff, J. C., Gould, M. N., Mundhenke, C., et al. (2008). Neutrophil gelatinase-associated lipocalin (NGAL) is a predictor of poor prognosis in human primary breast cancer. Breast Cancer Research and Treatment, 108, 389–397.
Yang, J., & Moses, M. A. (2009). Lipocalin 2: A multifaceted modulator of human cancer. Cell Cycle, 8, 2347–2352.
Leng, X., Wu, Y., & Arlinghaus, R. B. (2011). Relationships of lipocalin 2 with breast tumorigenesis and metastasis. Journal of Cellular Physiology, 226, 309–314.
Yang, J., Bielenberg, D. R., Rodig, S. J., et al. (2009). Lipocalin 2 promotes breast cancer progression. Proceedings of the National Academy of Sciences USA, 106, 3913–3918.
Leng, X., Ding, T., Lin, H., et al. (2009). Inhibition of lipocalin 2 impairs breast tumorigenesis and metastasis. Cancer Research, 69, 8579–8584.
Lee, H. J., Lee, E. K., Lee, K. J., et al. (2006). Ectopic expression of neutrophil gelatinase associated lipocalin suppresses the invasion and liver metastasis of colon cancer cells. International Journal of Cancer, 118, 2490–2497.
Crompton, M., Barksby, E., Johnson, N., & Capano, M. (2002). Mitochondrial intermembrane junctional complexes and their involvement in cell death. Biochimie, 84, 143–152.
Green, D. R. (2005). Apoptotic pathways: Ten minutes to dead. Cell, 121, 671–674.
Armstrong, J. S. (2006). The role of the mitochondrial permeability transition in cell death. Mitochondrion, 6, 225–234.
Radogna, F., Cristofanon, S., Paternoster, L., et al. (2008). Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of bcl-2. Journal of Pineal Research, 44, 316–325.
Cory, S., Huang, D. C., & Adams, J. M. (2003). The bcl-2 family: Roles in cell survival and oncogenesis. Oncogene, 22, 8590–8607.
Salvesen, G. S., & Riedl, S. J. (2008). Caspase mechanisms. Advances in Experimental Medicine and Biology, 615, 13–23.
Peter, M. E., & Krammer, P. H. (2003). The cd95(apo-1/fas) disc and beyond. Cell Death and Differentiation, 10, 26–35.
Li, F., Srinivasan, A., Wang, Y., et al. (1997). Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xl has activity independent of cytochrome c release. Journal of Biological Chemistry, 272, 30299–30305.
Acknowledgments
This work was supported by grants from National Science Council, Taiwan (NSC 100-2313-B-040-001) and Chung Shan Medical Hospital, Taichung, Taiwan (CSH-99-C-018).
Conflict of interest
The authors declare no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chien, MH., Ying, TH., Yang, SF. et al. Lipocalin-2 Induces Apoptosis in Human Hepatocellular Carcinoma Cells Through Activation of Mitochondria Pathways. Cell Biochem Biophys 64, 177–186 (2012). https://doi.org/10.1007/s12013-012-9370-1
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12013-012-9370-1