alpha1-antitrypsin and its C-terminal fragment attenuate effects of degranulated neutrophil-conditioned medium on lung cancer HCC cells, in vitro - PubMed (original) (raw)
alpha1-antitrypsin and its C-terminal fragment attenuate effects of degranulated neutrophil-conditioned medium on lung cancer HCC cells, in vitro
Inga Zelvyte et al. Cancer Cell Int. 2004.
Abstract
BACKGROUND: Tumor microenvironment, which is largely affected by inflammatory cells, is a crucial participant in the neoplastic process through promotion of cell proliferation, survival and migration. We measured the effects of polymorphonuclear neutrophil (PMN) conditioned medium alone, and supplemented with serine proteinase inhibitor alpha-1 antitrypsin (AAT) or its C-terminal fragment (C-36 peptide), on cultured lung cancer cells. METHODS: Lung cancer HCC cells were grown in a regular medium or in a PMN-conditioned medium in the presence or absence of AAT (0.5 mg/ml) or its C-36 peptide (0.06 mg/ml) for 24 h. Cell proliferation, invasiveness and release of IL-8 and VEGF were analyzed by [3H]-thymidine incorporation, Matrigel invasion and ELISA methods, respectively. RESULTS: Cells exposed to PMN-conditioned medium show decreased proliferation and IL-8 release by 3.9-fold, p < 0.001 and 1.3-fold, p < 0.05, respectively, and increased invasiveness by 2-fold (p < 0.001) compared to non-treated controls. In the presence of AAT, PMN-conditioned medium loses its effects on cell proliferation, invasiveness and IL-8 release, whereas VEGF is up-regulated by 3.7-fold (p < 0.001) compared to controls. Similarly, C-36 peptide abolishes the effects of PMN-conditioned medium on cell invasiveness, but does not alter its effects on cell proliferation, IL-8 and VEGF release. Direct HCC cell exposure to AAT enhances VEGF, but inhibits IL-8 release by 1.7-fold (p < 0.001) and 1.4-fold (p < 0.01) respectively, and reduces proliferation 2.5-fold (p < 0.01). In contrast, C-36 peptide alone did not affect these parameters, but inhibited cell invasiveness by 51.4% (p < 0.001), when compared with non-treated controls. CONCLUSIONS: Our data provide evidence that neutrophil derived factors decrease lung cancer HCC cell proliferation and IL-8 release, but increase cell invasiveness. These effects were found to be modulated by exogenously present serine proteinase inhibitor, AAT, and its C-terminal fragment, which points to a complexity of the relationships between tumor cell biological activities and local microenvironment.
Figures
Figure 1
[3H]Thymidine incorporation assay. The HCC cells were exposed to AAT (0.5 mg/ml) or its C-36 peptide (0.06 mg/ml) in a regular medium or in a PMN-conditioned medium for 20 h following addition of [3H] thymidine for 4 h. Each bar represents mean ± standard deviation from three separate experiments with three repeats in each. *** p < 0.001, ** p < 0.01.
Figure 2
HCC cell invasiveness after exposure to PMN-conditioned media, AAT or C-36 peptide separately and in combinations. HCC cells cultured in a regular medium (I) and in a PMN-conditioned medium (II) alone or supplemented with AAT or C-36 peptide (this figure represents one of three independent experiments).
Figure 3
VEGF release from HCC cells alone or exposed to PMN-conditioned medium with and without addition of AAT. Each bar represents the mean ± standard deviation of six repeats from two separate experiments with three repeats in each. *** p< 0.001
Figure 4
IL-8 release from HCC cells alone or exposed to PMN-conditioned medium with and without addition of AAT. Each bar represents the mean ± standard deviation of six repeats from two separate experiments with three repeats in each. ** p < 0.01, * p < 0.05.
Figure 5
Effects of PMN-conditioned medium alone and supplemented with AAT or C-36 peptide on HCC cell gelatinolytic activity. Lane 1-PMN-conditioned medium alone; lanes 2 and 3, PMN-conditioned medium supplemented with C-36 peptide and AAT, respectively; and incubated with cancer cells for 24 h, lane 4-PMN-conditioned medium incubated with HCC cells alone; lanes 5 and 6 – HCC cells exposed to C-36 peptide and AAT, respectively, in a regular medium; 7-HCC cells alone. This figure represents 1 of 3 separate experiments performed under the same experimental conditions.
Figure 6
Effects of PMN-conditioned medium alone and supplemented with AAT or C-36 peptide on HCC cell caseinolytic activity. Lane 1-PMN-conditioned medium alone; lanes 2-PMN-conditioned medium supplemented with C-36 peptide and incubated with HCC cells for 24 h, line 3-HCC cells exposed to C-36; lane 4-; PMN-conditioned medium supplemented with AAT and incubated with HCC cells for 24 h, lane 5-HCC cells exposed to AAT; lane 6-PMN-conditioned medium incubated with HCC cells; lane 7-HCC cells alone. This figure represents 1 of 3 separate experiments performed under the same experimental conditions.
Figure 7
HCC cell culture medium alone and in the presence of AAT studied by Western blot analysis. Cell culture supernatants were applied to 10% SDS-PAGE and immunoblotted with polyclonal antibody against AAT. M, molecular size markers (myosin-205 000, β-galactosidase-123 000, bovine serum albumin-79 000, carbonic anhydrase-45 700), lane 1-HCC cells alone, lane 2-HCC cells stimulated with AAT, lanes 3 and 4-HCC cells cultured in PMN-conditioned medium alone and supplemented with AAT, respectively lane 5-ATT done. The arrow indicates complexed AAT. The AAT profile shown in this figure represents one of three similar experiments.
References
- Carney DN. The biology of lung cancer. Curr Opin Pulm Med. 1995;1:271–277. - PubMed
- van Kessel KP, van Strijp JA, van Kats-Renaud HJ, Miltenburg LA, Fluit AC, Verhoef J. Uncoupling of oxidative and non-oxidative mechanisms in human granulocyte-mediumted cytotoxicity: use of cytoplasts and cells from chronic granulomatous disease patient. J Leukoc Biol. 1990;48:359–366. - PubMed
- Simpson-Haidaris PJ, Rybarczyk B. Tumors and fibrinogen. The role of fibrinogen as an extracellular matrix protein. Ann N Y Acad Sci. 2001;936:406–425. - PubMed
LinkOut - more resources
Full Text Sources
Research Materials
Miscellaneous