TGF-beta signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures

A. M. Lustri, S. Di Matteo, A. Fraveto, D. Costantini, A. Cantafora, C. Napoletano, M. C. Bragazzi, F. Giuliante, A. M. De Rose, P. B. Berloco, G. L. Grazi, G. Carpino, D. Alvaro

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-beta signaling is upregulated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and mixed-intrahepatic CCA. In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-beta1-induced EMT; and (ii) LY2157299, a TGF-beta receptor I kinase inhibitor. We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. RESULTS: at a dose of 10 muM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30muM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA). Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels. The effects of CX4945 on viability and apoptosis were associated with an increased number of gamma-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 muM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and LY2157299 impairing cell migration. These results suggest that targeting the TGF-beta signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.
Original languageEnglish
Pages (from-to)e0183932
JournalPLoS One
Volume12
Issue number9
DOIs
Publication statusPublished - Sep 5 2017

Fingerprint

Primary Cell Culture
Cholangiocarcinoma
mucins
transforming growth factor beta
LY-2157299
Cell culture
Transforming Growth Factor beta
cell culture
apoptosis
Cells
cell viability
Apoptosis
Mucins
Casein Kinase II
Survival
cell movement
Epithelial-Mesenchymal Transition
Chrysanthemum morifolium
cell lines
tissue repair

Keywords

  • Apoptosis
  • Cell Line, Tumor
  • Cell Movement
  • Cell Survival
  • Cholangiocarcinoma/metabolism/pathology
  • Drug Resistance, Neoplasm
  • Epithelial-Mesenchymal Transition
  • Humans
  • Naphthyridines/chemistry
  • Neoplastic Stem Cells/cytology
  • Primary Cell Culture
  • Pyrazoles/chemistry
  • Quinolines/chemistry
  • Signal Transduction
  • Transforming Growth Factor beta/metabolism
  • Wound Healing

Cite this

TGF-beta signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures. / Lustri, A. M.; Matteo, S. Di; Fraveto, A.; Costantini, D.; Cantafora, A.; Napoletano, C.; Bragazzi, M. C.; Giuliante, F.; Rose, A. M. De; Berloco, P. B.; Grazi, G. L.; Carpino, G.; Alvaro, D.

In: PLoS One, Vol. 12, No. 9, 05.09.2017, p. e0183932.

Research output: Contribution to journalArticle

Lustri, AM, Matteo, SD, Fraveto, A, Costantini, D, Cantafora, A, Napoletano, C, Bragazzi, MC, Giuliante, F, Rose, AMD, Berloco, PB, Grazi, GL, Carpino, G & Alvaro, D 2017, 'TGF-beta signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures', PLoS One, vol. 12, no. 9, pp. e0183932. https://doi.org/10.1371/journal.pone.0183932 [doi]
Lustri, A. M. ; Matteo, S. Di ; Fraveto, A. ; Costantini, D. ; Cantafora, A. ; Napoletano, C. ; Bragazzi, M. C. ; Giuliante, F. ; Rose, A. M. De ; Berloco, P. B. ; Grazi, G. L. ; Carpino, G. ; Alvaro, D. / TGF-beta signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures. In: PLoS One. 2017 ; Vol. 12, No. 9. pp. e0183932.
@article{d40cb4053cc04c0abdf2bf21a930cd08,
title = "TGF-beta signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures",
abstract = "Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-beta signaling is upregulated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and mixed-intrahepatic CCA. In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-beta1-induced EMT; and (ii) LY2157299, a TGF-beta receptor I kinase inhibitor. We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. RESULTS: at a dose of 10 muM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30muM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA). Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels. The effects of CX4945 on viability and apoptosis were associated with an increased number of gamma-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 muM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and LY2157299 impairing cell migration. These results suggest that targeting the TGF-beta signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.",
keywords = "Apoptosis, Cell Line, Tumor, Cell Movement, Cell Survival, Cholangiocarcinoma/metabolism/pathology, Drug Resistance, Neoplasm, Epithelial-Mesenchymal Transition, Humans, Naphthyridines/chemistry, Neoplastic Stem Cells/cytology, Primary Cell Culture, Pyrazoles/chemistry, Quinolines/chemistry, Signal Transduction, Transforming Growth Factor beta/metabolism, Wound Healing",
author = "Lustri, {A. M.} and Matteo, {S. Di} and A. Fraveto and D. Costantini and A. Cantafora and C. Napoletano and Bragazzi, {M. C.} and F. Giuliante and Rose, {A. M. De} and Berloco, {P. B.} and Grazi, {G. L.} and G. Carpino and D. Alvaro",
note = "LR: 20171025; JID: 101285081; 0 (5-(3-chlorophenylamino)benzo(c)(2,6)naphthyridine-8-carboxylic acid); 0 (Naphthyridines); 0 (Pyrazoles); 0 (Quinolines); 0 (Transforming Growth Factor beta); 700874-72-2 (LY-2157299); 2017/02/16 00:00 [received]; 2017/08/14 00:00 [accepted]; 2017/09/06 06:00 [entrez]; 2017/09/06 06:00 [pubmed]; 2017/10/27 06:00 [medline]; epublish",
year = "2017",
month = "9",
day = "5",
doi = "10.1371/journal.pone.0183932 [doi]",
language = "English",
volume = "12",
pages = "e0183932",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "9",

}

TY - JOUR

T1 - TGF-beta signaling is an effective target to impair survival and induce apoptosis of human cholangiocarcinoma cells: A study on human primary cell cultures

AU - Lustri, A. M.

AU - Matteo, S. Di

AU - Fraveto, A.

AU - Costantini, D.

AU - Cantafora, A.

AU - Napoletano, C.

AU - Bragazzi, M. C.

AU - Giuliante, F.

AU - Rose, A. M. De

AU - Berloco, P. B.

AU - Grazi, G. L.

AU - Carpino, G.

AU - Alvaro, D.

N1 - LR: 20171025; JID: 101285081; 0 (5-(3-chlorophenylamino)benzo(c)(2,6)naphthyridine-8-carboxylic acid); 0 (Naphthyridines); 0 (Pyrazoles); 0 (Quinolines); 0 (Transforming Growth Factor beta); 700874-72-2 (LY-2157299); 2017/02/16 00:00 [received]; 2017/08/14 00:00 [accepted]; 2017/09/06 06:00 [entrez]; 2017/09/06 06:00 [pubmed]; 2017/10/27 06:00 [medline]; epublish

PY - 2017/9/5

Y1 - 2017/9/5

N2 - Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-beta signaling is upregulated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and mixed-intrahepatic CCA. In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-beta1-induced EMT; and (ii) LY2157299, a TGF-beta receptor I kinase inhibitor. We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. RESULTS: at a dose of 10 muM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30muM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA). Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels. The effects of CX4945 on viability and apoptosis were associated with an increased number of gamma-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 muM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and LY2157299 impairing cell migration. These results suggest that targeting the TGF-beta signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.

AB - Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-beta signaling is upregulated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and mixed-intrahepatic CCA. In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-beta1-induced EMT; and (ii) LY2157299, a TGF-beta receptor I kinase inhibitor. We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. RESULTS: at a dose of 10 muM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30muM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA). Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels. The effects of CX4945 on viability and apoptosis were associated with an increased number of gamma-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 muM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and LY2157299 impairing cell migration. These results suggest that targeting the TGF-beta signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.

KW - Apoptosis

KW - Cell Line, Tumor

KW - Cell Movement

KW - Cell Survival

KW - Cholangiocarcinoma/metabolism/pathology

KW - Drug Resistance, Neoplasm

KW - Epithelial-Mesenchymal Transition

KW - Humans

KW - Naphthyridines/chemistry

KW - Neoplastic Stem Cells/cytology

KW - Primary Cell Culture

KW - Pyrazoles/chemistry

KW - Quinolines/chemistry

KW - Signal Transduction

KW - Transforming Growth Factor beta/metabolism

KW - Wound Healing

U2 - 10.1371/journal.pone.0183932 [doi]

DO - 10.1371/journal.pone.0183932 [doi]

M3 - Article

VL - 12

SP - e0183932

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 9

ER -