Abstract
A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1 % O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.
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Abbreviations
- HIF-1α:
-
Hypoxia inducible factor-1 alpha
- IL-8:
-
Interleukin-8
- shRNA:
-
Small hairpin RNA
- TUNEL:
-
Terminal deoxynucleotidyl transferase dUTP nick end labeling
- shRNA:
-
Small hairpin RNA
- IF:
-
Immunofluorescence
- PI:
-
Propidium iodide
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Ad-shRNA:
-
Adenovirus mediated shRNA
- piLenti-siRNA:
-
Plasmid lentiviral vector mediated siRNA
- TRAIL:
-
TNF-related apoptosis-inducing ligand
- TNF-α:
-
Tumor necrosis factor-α
- FasL:
-
Fas ligand
References
Llovet JM, Bruix J (2008) Novel advancements in the management of hepatocellular carcinoma in 2008. J Hepatol 48(Suppl 1):S20–S37
Fabregat I, Roncero C, Fernandez M (2007) Survival and apoptosis: a dysregulated balance in liver cancer. Liver Int 27(2):155–162
Fabregat I (2009) Dysregulation of apoptosis in hepatocellular carcinoma cells. World J Gastroenterol 15(5):513–520
Sutter CH, Laughner E, Semenza GL (2000) Hypoxia-inducible factor 1alpha protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations. Proc Natl Acad Sci USA 97(9):4748–4753
Wang GL, Semenza GL (1993) General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci USA 90(9):4304–4308
Choi SH, Kwon OJ, Park JY, Kim DY, Ahn SH, Kim SU, Ro SW, Kim KS, Park JH, Kim S et al (2013) Inhibition of tumour angiogenesis and growth by small hairpin HIF-1alpha and IL-8 in hepatocellular carcinoma. Liver Int 34:632–642
Dong Z, Wang J (2004) Hypoxia selection of death-resistant cells. A role for Bcl-X(L). J Biol Chem 279(10):9215–9221
Li A, Dubey S, Varney ML, Dave BJ, Singh RK (2003) IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol 170(6):3369–3376
Nor JE, Christensen J, Mooney DJ, Polverini PJ (1999) Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression. Am J Pathol 154(2):375–384
Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM (1992) Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 258(5089):1798–1801
Kountouras J, Zavos C, Chatzopoulos D (2003) Apoptosis in hepatocellular carcinoma. Hepatogastroenterology 50(49):242–249
Rust C, Gores GJ (2000) Apoptosis and liver disease. Am J Med 108(7):567–574
Schattenberg JM, Galle PR, Schuchmann M (2006) Apoptosis in liver disease. Liver Int 26(8):904–911
Chen N, Chen X, Huang R, Zeng H, Gong J, Meng W, Lu Y, Zhao F, Wang L, Zhou Q (2009) BCL-xL is a target gene regulated by hypoxia-inducible factor-1{alpha}. J Biol Chem 284(15):10004–10012
Kudo M (2011) Molecular targeted therapy for hepatocellular carcinoma: bench to bedside. Dig Dis 29(3):273–277
Muntane J (2011) Targeting cell death and survival receptors in hepatocellular carcinoma. Anti-Cancer Agents Med Chem 11(6):576–584
Spangenberg HC, Thimme R, Blum HE (2009) Targeted therapy for hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 6(7):423–432
Kaseb AO, Hanbali A, Cotant M, Hassan MM, Wollner I, Philip PA (2009) Vascular endothelial growth factor in the management of hepatocellular carcinoma: a review of literature. Cancer 115(21):4895–4906
Zhu AX, Duda DG, Sahani DV, Jain RK (2011) HCC and angiogenesis: possible targets and future directions. Nat Rev Clin Oncol 8(5):292–301
Sampat KR, O’Neil B (2013) Antiangiogenic therapies for advanced hepatocellular carcinoma. Oncologist 18(4):430–438
Liu QY, Rubin MA, Omene C, Lederman S, Stein CA (1998) Fas ligand is constitutively secreted by prostate cancer cells in vitro. Clin Cancer Res 4(7):1803–1811
Dittel BN, Merchant RM, Janeway CA Jr (1999) Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis. J Immunol 162(11):6392–6400
Kubo F, Ueno S, Hiwatashi K, Sakoda M, Kawaida K, Nuruki K, Aikou T (2005) Interleukin 8 in human hepatocellular carcinoma correlates with cancer cell invasion of vessels but not with tumor angiogenesis. Ann Surg Oncol 12(10):800–807
Welling TH, Fu S, Wan S, Zou W, Marrero JA (2012) Elevated serum IL-8 is associated with the presence of hepatocellular carcinoma and independently predicts survival. Cancer Invest 30(10):689–697
Park SY, Han J, Kim JB, Yang MG, Kim YJ, Lim HJ, An SY, Kim JH (2013) Interleukin-8 is related to poor chemotherapeutic response and tumourigenicity in hepatocellular carcinoma. Eur J Cancer 50:341–350
Acknowledgments
We are grateful to Dong-Su Jang (Medical Illustrator, Medical Research Support Section, Yonsei University College of Medicine, Seoul, Korea) for his help with the figures.
Financial support
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (NRF-2011-0014537).
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10495_2015_1185_MOESM1_ESM.tif
Supplement 1. Compared hypoxia and normoxia in FADD expression. HUVECs were cultured under hypoxic and normoxic conditions using conditioned media prepared from the Huh7 cell line. Under the hypoxic condition, combination knockdown resulted in a higher rate of apoptosis than under the normoxic condition. Supplementary material 1 (TIFF 1939 kb)
10495_2015_1185_MOESM2_ESM.tif
Supplement 2. Comparison of death signal ligand in Hep3B cell line. In Hep3B cells, expressions of FasL, TNF-α, and TRAIL, an apoptosis inducer, were investigated over time using ELISA. With 6 h of hypoxic stimulation, there was an increase compared to the control group, though there were no significant differences. After 24 h of stimulation, FasL expression manifested remarkable differences compared to the control group. However, TNF-α and TRAIL remain unchanged. Supplementary material 2 (TIFF 1391 kb)
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Choi, S.H., Park, J.Y., Kang, W. et al. Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells. Apoptosis 21, 85–95 (2016). https://doi.org/10.1007/s10495-015-1185-2
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DOI: https://doi.org/10.1007/s10495-015-1185-2