Limits...
Angiogenin secretion from hepatoma cells activates hepatic stellate cells to amplify a self-sustained cycle promoting liver cancer.

Bárcena C, Stefanovic M, Tutusaus A, Martinez-Nieto GA, Martinez L, García-Ruiz C, de Mingo A, Caballeria J, Fernandez-Checa JC, Marí M, Morales A - Sci Rep (2015)

Bottom Line: Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules.Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin.These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Death and Proliferation, IIBB-CSIC, IDIBAPS, Barcelona, Spain.

ABSTRACT
Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

Show MeSH

Related in: MedlinePlus

Neomycin administration reduces HCC/HSC tumor growth by blocking angiogenin nuclear translocation and HCC development.A, measurement of tumors from nude mice subcutaneously injected with HepG2/LX2 cells and treated intraperitonealy with saline or neomycin for eight weeks. B, Representative images of tumor cell proliferation by PCNA detection (40×), angiogenin levels (80×) and CD34 (40×) were visualized in tumor samples from mice treated with neomycin or saline. C, Quantification of PCNA positive cells in tumor slides. D, Quantification of CD34 positive areas in tumor slides. E, mRNA quantification of α-SMA and TGF-β in tumors, using β-actin as control. (n ≥ 6). *, p ≤ 0.05, neomycin vs. saline-injected animals. F, RNA quantification of RNA polymerase II (RPII) and 45S ribosomal RNA in tumors, as above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4300499&req=5

f7: Neomycin administration reduces HCC/HSC tumor growth by blocking angiogenin nuclear translocation and HCC development.A, measurement of tumors from nude mice subcutaneously injected with HepG2/LX2 cells and treated intraperitonealy with saline or neomycin for eight weeks. B, Representative images of tumor cell proliferation by PCNA detection (40×), angiogenin levels (80×) and CD34 (40×) were visualized in tumor samples from mice treated with neomycin or saline. C, Quantification of PCNA positive cells in tumor slides. D, Quantification of CD34 positive areas in tumor slides. E, mRNA quantification of α-SMA and TGF-β in tumors, using β-actin as control. (n ≥ 6). *, p ≤ 0.05, neomycin vs. saline-injected animals. F, RNA quantification of RNA polymerase II (RPII) and 45S ribosomal RNA in tumors, as above.

Mentions: To explore the potential therapeutic relevance of neomycin in HCC, we followed tumor growth in the xenograft model with or without in vivo treatment with neomycin. Nude mice were treated daily with neomycin or saline subcutaneously to follow the growth of tumor growth in xenografts injected with HepG2 cells with or without LX2 cells. While no significant changes were observed in tumor multiplicity between groups (7 out of 10 in saline group, vs. 6 out of 10 in neomycin group), tumor growth determined by tumor volume (Fig. 7A) was clearly lower in neomycin-treated mice. Of note, tumors from neomycin-treated animals emerged slower, and tumor development was significantly diminished in equal size tumors (e.g. estimated doubling time for 50 mm3 tumors was 6.0 ± 1.2 days in saline-treated animals vs. 10.1 ± 2.1 in neomycin treated mice). In fact, HCC proliferation was reduced in neomycin-treated animals (Fig. 7B), as quantified in tumor slides by PCNA index measurement (Fig. 7C), while a reduction in tumor vascularization was determined by CD34 immunohistochemistry (Fig. 7B), and the positive areas quantified (Fig. 7D).


Angiogenin secretion from hepatoma cells activates hepatic stellate cells to amplify a self-sustained cycle promoting liver cancer.

Bárcena C, Stefanovic M, Tutusaus A, Martinez-Nieto GA, Martinez L, García-Ruiz C, de Mingo A, Caballeria J, Fernandez-Checa JC, Marí M, Morales A - Sci Rep (2015)

Neomycin administration reduces HCC/HSC tumor growth by blocking angiogenin nuclear translocation and HCC development.A, measurement of tumors from nude mice subcutaneously injected with HepG2/LX2 cells and treated intraperitonealy with saline or neomycin for eight weeks. B, Representative images of tumor cell proliferation by PCNA detection (40×), angiogenin levels (80×) and CD34 (40×) were visualized in tumor samples from mice treated with neomycin or saline. C, Quantification of PCNA positive cells in tumor slides. D, Quantification of CD34 positive areas in tumor slides. E, mRNA quantification of α-SMA and TGF-β in tumors, using β-actin as control. (n ≥ 6). *, p ≤ 0.05, neomycin vs. saline-injected animals. F, RNA quantification of RNA polymerase II (RPII) and 45S ribosomal RNA in tumors, as above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300499&req=5

f7: Neomycin administration reduces HCC/HSC tumor growth by blocking angiogenin nuclear translocation and HCC development.A, measurement of tumors from nude mice subcutaneously injected with HepG2/LX2 cells and treated intraperitonealy with saline or neomycin for eight weeks. B, Representative images of tumor cell proliferation by PCNA detection (40×), angiogenin levels (80×) and CD34 (40×) were visualized in tumor samples from mice treated with neomycin or saline. C, Quantification of PCNA positive cells in tumor slides. D, Quantification of CD34 positive areas in tumor slides. E, mRNA quantification of α-SMA and TGF-β in tumors, using β-actin as control. (n ≥ 6). *, p ≤ 0.05, neomycin vs. saline-injected animals. F, RNA quantification of RNA polymerase II (RPII) and 45S ribosomal RNA in tumors, as above.
Mentions: To explore the potential therapeutic relevance of neomycin in HCC, we followed tumor growth in the xenograft model with or without in vivo treatment with neomycin. Nude mice were treated daily with neomycin or saline subcutaneously to follow the growth of tumor growth in xenografts injected with HepG2 cells with or without LX2 cells. While no significant changes were observed in tumor multiplicity between groups (7 out of 10 in saline group, vs. 6 out of 10 in neomycin group), tumor growth determined by tumor volume (Fig. 7A) was clearly lower in neomycin-treated mice. Of note, tumors from neomycin-treated animals emerged slower, and tumor development was significantly diminished in equal size tumors (e.g. estimated doubling time for 50 mm3 tumors was 6.0 ± 1.2 days in saline-treated animals vs. 10.1 ± 2.1 in neomycin treated mice). In fact, HCC proliferation was reduced in neomycin-treated animals (Fig. 7B), as quantified in tumor slides by PCNA index measurement (Fig. 7C), while a reduction in tumor vascularization was determined by CD34 immunohistochemistry (Fig. 7B), and the positive areas quantified (Fig. 7D).

Bottom Line: Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules.Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin.These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Death and Proliferation, IIBB-CSIC, IDIBAPS, Barcelona, Spain.

ABSTRACT
Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

Show MeSH
Related in: MedlinePlus