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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.

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HSCs are activated by conditioned medium (CM) from HepG2 and Hep3B cells.Representative western blot showing α-SMA activation primary murine HSCs (day 7), previously exposed to conditioned medium from HepG2 (A), Hep3B (B) or LX2 cells (C) for 0–5 days, using β-actin levels as a control. D, Microscopic images of morphological changes in HSCs preteated with CM from HepG2 or LX2 during 0, 1 or 2 days. E and F, mRNA quantification of TGF-β and COL1A1 in 7-days-old HSCs after previous CM-HepG2 addition for the indicated periods of time. (n = 3). *, p ≤ 0.05, specific time vs. 0 time point.
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f1: HSCs are activated by conditioned medium (CM) from HepG2 and Hep3B cells.Representative western blot showing α-SMA activation primary murine HSCs (day 7), previously exposed to conditioned medium from HepG2 (A), Hep3B (B) or LX2 cells (C) for 0–5 days, using β-actin levels as a control. D, Microscopic images of morphological changes in HSCs preteated with CM from HepG2 or LX2 during 0, 1 or 2 days. E and F, mRNA quantification of TGF-β and COL1A1 in 7-days-old HSCs after previous CM-HepG2 addition for the indicated periods of time. (n = 3). *, p ≤ 0.05, specific time vs. 0 time point.

Mentions: 7-day old primary mouse HSCs were collected after being exposed for up to 5 days to conditioned medium from human cell lines HepG2 (CM-HepG2) or Hep3B (CM-Hep3B) to verify the capacity of hepatoma-derived CM to induce HSC activation27. Compared to 7-day old non-treated HSCs, exposure to CM-HepG2 induced significant changes in α-SMA expression (Fig. 1A), while exposure to CM-Hep3B augmented also α-SMA expression but to a lesser extent (Fig. 1B). In fact, changes in HSC morphology, indicative of HSC transformation, were observed in HSCs as soon as 2 days after exposure to CM-HepG2 (Fig. 1D). However, addition of medium from LX2 cells (CM-LX2), a cell line of human activated stellate cells, induced no visible changes in HSC appearance (Fig. 1D) or α-SMA expression (Fig. 1C). Moreover, COL1A1 mRNA levels displayed increased expression in HSCs within hours of CM-HepG2 exposure (Fig. 1E), showing significant increases as soon as 4 h after CM addition, before the overexpression of other profibrogenic genes such as TGF-β. In addition, enhanced mRNA expression of HSC-activated genes, such as TGF-β and COL1A1, was exhibited for several days (Fig. 1F).


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)

HSCs are activated by conditioned medium (CM) from HepG2 and Hep3B cells.Representative western blot showing α-SMA activation primary murine HSCs (day 7), previously exposed to conditioned medium from HepG2 (A), Hep3B (B) or LX2 cells (C) for 0–5 days, using β-actin levels as a control. D, Microscopic images of morphological changes in HSCs preteated with CM from HepG2 or LX2 during 0, 1 or 2 days. E and F, mRNA quantification of TGF-β and COL1A1 in 7-days-old HSCs after previous CM-HepG2 addition for the indicated periods of time. (n = 3). *, p ≤ 0.05, specific time vs. 0 time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: HSCs are activated by conditioned medium (CM) from HepG2 and Hep3B cells.Representative western blot showing α-SMA activation primary murine HSCs (day 7), previously exposed to conditioned medium from HepG2 (A), Hep3B (B) or LX2 cells (C) for 0–5 days, using β-actin levels as a control. D, Microscopic images of morphological changes in HSCs preteated with CM from HepG2 or LX2 during 0, 1 or 2 days. E and F, mRNA quantification of TGF-β and COL1A1 in 7-days-old HSCs after previous CM-HepG2 addition for the indicated periods of time. (n = 3). *, p ≤ 0.05, specific time vs. 0 time point.
Mentions: 7-day old primary mouse HSCs were collected after being exposed for up to 5 days to conditioned medium from human cell lines HepG2 (CM-HepG2) or Hep3B (CM-Hep3B) to verify the capacity of hepatoma-derived CM to induce HSC activation27. Compared to 7-day old non-treated HSCs, exposure to CM-HepG2 induced significant changes in α-SMA expression (Fig. 1A), while exposure to CM-Hep3B augmented also α-SMA expression but to a lesser extent (Fig. 1B). In fact, changes in HSC morphology, indicative of HSC transformation, were observed in HSCs as soon as 2 days after exposure to CM-HepG2 (Fig. 1D). However, addition of medium from LX2 cells (CM-LX2), a cell line of human activated stellate cells, induced no visible changes in HSC appearance (Fig. 1D) or α-SMA expression (Fig. 1C). Moreover, COL1A1 mRNA levels displayed increased expression in HSCs within hours of CM-HepG2 exposure (Fig. 1E), showing significant increases as soon as 4 h after CM addition, before the overexpression of other profibrogenic genes such as TGF-β. In addition, enhanced mRNA expression of HSC-activated genes, such as TGF-β and COL1A1, was exhibited for several days (Fig. 1F).

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