Skip to main content

Advertisement

SpringerLink
Log in

Lipocalin-2 Induces Apoptosis in Human Hepatocellular Carcinoma Cells Through Activation of Mitochondria Pathways

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

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

  1. Bosch, F. X., Ribes, J., & Borras, J. (1999). Epidemiology of primary liver cancer. Seminars in Liver Disease, 19, 271–285.

    Article  PubMed  CAS  Google Scholar 

  2. Teo, E. K., & Fock, K. M. (2001). Hepatocellular carcinoma: An Asian perspective. Digestive Diseases, 19, 263–268.

    Article  PubMed  CAS  Google Scholar 

  3. Farazi, P. A., & DePinho, R. A. (2006). Hepatocellular carcinoma pathogenesis: From genes to environment. Nature Reviews Cancer, 6, 674–687.

    Article  PubMed  CAS  Google Scholar 

  4. 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.

    Article  PubMed  CAS  Google Scholar 

  5. Kehrer, J. P. (2010). Lipocalin-2: Pro- or Anti-apoptotic? Cell Biology and Toxicology, 26, 83–89.

    Article  PubMed  CAS  Google Scholar 

  6. 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.

    Article  CAS  Google Scholar 

  7. 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.

    Article  PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. 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.

    Article  PubMed  CAS  Google Scholar 

  10. 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.

    Article  PubMed  CAS  Google Scholar 

  11. 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.

    Article  PubMed  CAS  Google Scholar 

  12. 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.

    Article  PubMed  CAS  Google Scholar 

  13. 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.

    Article  PubMed  CAS  Google Scholar 

  14. 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.

    PubMed  CAS  Google Scholar 

  15. Tong, Z., Wu, X., Ovcharenko, D., et al. (2005). Neutrophil gelatinase-associated lipocalin as a survival factor. Biochemical Journal, 391, 441–448.

    Article  PubMed  CAS  Google Scholar 

  16. 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.

    Article  PubMed  CAS  Google Scholar 

  17. 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.

    Article  PubMed  CAS  Google Scholar 

  18. 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.

    Article  Google Scholar 

  19. 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.

    Article  PubMed  CAS  Google Scholar 

  20. 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.

    Article  PubMed  CAS  Google Scholar 

  21. 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.

    PubMed  CAS  Google Scholar 

  22. 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.

    Article  PubMed  CAS  Google Scholar 

  23. 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.

    Article  PubMed  CAS  Google Scholar 

  24. 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.

    CAS  Google Scholar 

  25. 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.

    Article  CAS  Google Scholar 

  26. 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.

    Article  PubMed  Google Scholar 

  27. 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.

    Article  CAS  Google Scholar 

  28. 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.

    Article  PubMed  CAS  Google Scholar 

  29. Yang, J., & Moses, M. A. (2009). Lipocalin 2: A multifaceted modulator of human cancer. Cell Cycle, 8, 2347–2352.

    Article  PubMed  CAS  Google Scholar 

  30. Leng, X., Wu, Y., & Arlinghaus, R. B. (2011). Relationships of lipocalin 2 with breast tumorigenesis and metastasis. Journal of Cellular Physiology, 226, 309–314.

    Article  PubMed  CAS  Google Scholar 

  31. 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.

    Article  CAS  Google Scholar 

  32. Leng, X., Ding, T., Lin, H., et al. (2009). Inhibition of lipocalin 2 impairs breast tumorigenesis and metastasis. Cancer Research, 69, 8579–8584.

    Article  PubMed  CAS  Google Scholar 

  33. 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.

    Article  CAS  Google Scholar 

  34. Crompton, M., Barksby, E., Johnson, N., & Capano, M. (2002). Mitochondrial intermembrane junctional complexes and their involvement in cell death. Biochimie, 84, 143–152.

    Article  PubMed  CAS  Google Scholar 

  35. Green, D. R. (2005). Apoptotic pathways: Ten minutes to dead. Cell, 121, 671–674.

    Article  PubMed  CAS  Google Scholar 

  36. Armstrong, J. S. (2006). The role of the mitochondrial permeability transition in cell death. Mitochondrion, 6, 225–234.

    Article  PubMed  CAS  Google Scholar 

  37. 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.

    Article  PubMed  CAS  Google Scholar 

  38. Cory, S., Huang, D. C., & Adams, J. M. (2003). The bcl-2 family: Roles in cell survival and oncogenesis. Oncogene, 22, 8590–8607.

    Article  PubMed  CAS  Google Scholar 

  39. Salvesen, G. S., & Riedl, S. J. (2008). Caspase mechanisms. Advances in Experimental Medicine and Biology, 615, 13–23.

    Article  PubMed  CAS  Google Scholar 

  40. Peter, M. E., & Krammer, P. H. (2003). The cd95(apo-1/fas) disc and beyond. Cell Death and Differentiation, 10, 26–35.

    Article  PubMed  CAS  Google Scholar 

  41. 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.

    Article  PubMed  CAS  Google Scholar 

Download references

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

  1. Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

    Ming-Hsien Chien

  2. Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

    Ming-Hsien Chien

  3. Department of Obstetrics and Gynecology, School of Medicine, College of Medicine, Chung Shan Medical University, Taichung, Taiwan

    Tsung-Ho Ying

  4. Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung, Taiwan

    Tsung-Ho Ying

  5. Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

    Shun-Fa Yang & Shu-Ching Hsieh

  6. Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan

    Shun-Fa Yang

  7. Division of General Surgery, Department of Surgery, Tungs’ Taichung MetroHarbor Hospital, Taichung, Taiwan

    Ji-Kuen Yu & Chih-Wei Hsu

  8. Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan

    Yi-Hsien Hsieh

  9. Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan

    Yi-Hsien Hsieh

  10. Clinical Laboratory, Chung Shan Medical University Hospital, No. 110, Sect. 1, Chien-Kuo N. Road, Taichung, Taiwan

    Yi-Hsien Hsieh

Authors
  1. Ming-Hsien Chien
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tsung-Ho Ying
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shun-Fa Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ji-Kuen Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chih-Wei Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shu-Ching Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yi-Hsien Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yi-Hsien Hsieh.

Rights and permissions

Reprints 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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-012-9370-1

Keywords

Search

Navigation