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Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Zhang Y, Liu L, Fan P, Bauer N, Gladkich J, Ryschich E, Bazhin AV, Giese NA, Strobel O, Hackert T, Hinz U, Gross W, Fortunato F, Herr I - Oncotarget (2015)

Bottom Line: Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling.Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine.Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice.

View Article: PubMed Central - PubMed

Affiliation: Molecular OncoSurgery, University of Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

No MeSH data available.


Related in: MedlinePlus

Aspirin overcomes gemcitabine resistance and alters the expression of reprogramming factors(A) The human established PDA cell lines BxPc-3, PANC-1, BxPC-3/GEM and AsPC-1 were left untreated (CO) or were treated with 2, 5, or 10 mM aspirin (ASA), 50 nM gemcitabine (GEM), or both together, and the cellular viability was measured 72 h later with an MTT assay. The gemcitabine resistance of the cells is indicated by a triangle above the diagrams. The morphology of AsPC-1 cells 72 h after treatment was documented by microscopy at 200× magnification. Representative pictures are shown, and the bar indicates 50 μm. (B) The primary, low-passage human PDA cell lines PaCaDD183, PaCaDD159 and PaCaDD135 or (C) human MSCs and non-malignant, immortalized human pancreatic ductal cells (CRL-4023) were treated and analyzed as described above. (D) BxPc-3, PANC-1 and BxPc-3/GEM cells were treated as described above, and after 48 h, expression of the aspirin target and pro-inflammatory factor TNF-α and expression of the re-programming factors Oct-4, Nanog and SOX2 were measured by Western blot analysis. β-Actin served as a control. The data are presented as means ± SD (**P < 0.01, *P < 0.05).
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Figure 1: Aspirin overcomes gemcitabine resistance and alters the expression of reprogramming factors(A) The human established PDA cell lines BxPc-3, PANC-1, BxPC-3/GEM and AsPC-1 were left untreated (CO) or were treated with 2, 5, or 10 mM aspirin (ASA), 50 nM gemcitabine (GEM), or both together, and the cellular viability was measured 72 h later with an MTT assay. The gemcitabine resistance of the cells is indicated by a triangle above the diagrams. The morphology of AsPC-1 cells 72 h after treatment was documented by microscopy at 200× magnification. Representative pictures are shown, and the bar indicates 50 μm. (B) The primary, low-passage human PDA cell lines PaCaDD183, PaCaDD159 and PaCaDD135 or (C) human MSCs and non-malignant, immortalized human pancreatic ductal cells (CRL-4023) were treated and analyzed as described above. (D) BxPc-3, PANC-1 and BxPc-3/GEM cells were treated as described above, and after 48 h, expression of the aspirin target and pro-inflammatory factor TNF-α and expression of the re-programming factors Oct-4, Nanog and SOX2 were measured by Western blot analysis. β-Actin served as a control. The data are presented as means ± SD (**P < 0.01, *P < 0.05).

Mentions: To evaluate the effect of aspirin on gemcitabine responsiveness, we examined 4 established PDA cell lines with different grades of aggressiveness [11, 22] and gemcitabine resistance (Figure 1A). The cells were treated with gemcitabine or 3 different aspirin concentrations, or a combination thereof. Viability, cell morphology and apoptosis were determined 72 h after treatment according to the MTT assay, microscopy, and staining with AnnexinV-FITC followed by flow cytometry, respectively (Figure 1A, Figure S1A, S1B). Whereas gemcitabine was effective in sensitive BxPc-3 cells, as expected, it was minimally effective in median resistant PANC-1 cells and non-effective in the totally resistant BxPc-3/GEM and AsPC-1 cells. In contrast, aspirin at doses of 5 and 10 mM was effective in all cell lines, and aspirin treatment significantly increased the efficacy of gemcitabine upon combination. These results were confirmed in the primary, low-passage, human PDA cell lines PaCaDD135 (gemcitabine-sensitive), PaCaDD159 (median gemcitabine-resistant) and PaCaDD183 (totally gemcitabine-resistant), which were in passage 5 (Figure 1B). Most importantly, aspirin did not significantly affect the viability of non-malignant human primary MSCs or immortalized human pancreatic ductal CRL-4023 cells, in contrast to gemcitabine, which significantly reduced the viability of MSCs and CRL-4023 cells (Figure 1C).


Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Zhang Y, Liu L, Fan P, Bauer N, Gladkich J, Ryschich E, Bazhin AV, Giese NA, Strobel O, Hackert T, Hinz U, Gross W, Fortunato F, Herr I - Oncotarget (2015)

Aspirin overcomes gemcitabine resistance and alters the expression of reprogramming factors(A) The human established PDA cell lines BxPc-3, PANC-1, BxPC-3/GEM and AsPC-1 were left untreated (CO) or were treated with 2, 5, or 10 mM aspirin (ASA), 50 nM gemcitabine (GEM), or both together, and the cellular viability was measured 72 h later with an MTT assay. The gemcitabine resistance of the cells is indicated by a triangle above the diagrams. The morphology of AsPC-1 cells 72 h after treatment was documented by microscopy at 200× magnification. Representative pictures are shown, and the bar indicates 50 μm. (B) The primary, low-passage human PDA cell lines PaCaDD183, PaCaDD159 and PaCaDD135 or (C) human MSCs and non-malignant, immortalized human pancreatic ductal cells (CRL-4023) were treated and analyzed as described above. (D) BxPc-3, PANC-1 and BxPc-3/GEM cells were treated as described above, and after 48 h, expression of the aspirin target and pro-inflammatory factor TNF-α and expression of the re-programming factors Oct-4, Nanog and SOX2 were measured by Western blot analysis. β-Actin served as a control. The data are presented as means ± SD (**P < 0.01, *P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496413&req=5

Figure 1: Aspirin overcomes gemcitabine resistance and alters the expression of reprogramming factors(A) The human established PDA cell lines BxPc-3, PANC-1, BxPC-3/GEM and AsPC-1 were left untreated (CO) or were treated with 2, 5, or 10 mM aspirin (ASA), 50 nM gemcitabine (GEM), or both together, and the cellular viability was measured 72 h later with an MTT assay. The gemcitabine resistance of the cells is indicated by a triangle above the diagrams. The morphology of AsPC-1 cells 72 h after treatment was documented by microscopy at 200× magnification. Representative pictures are shown, and the bar indicates 50 μm. (B) The primary, low-passage human PDA cell lines PaCaDD183, PaCaDD159 and PaCaDD135 or (C) human MSCs and non-malignant, immortalized human pancreatic ductal cells (CRL-4023) were treated and analyzed as described above. (D) BxPc-3, PANC-1 and BxPc-3/GEM cells were treated as described above, and after 48 h, expression of the aspirin target and pro-inflammatory factor TNF-α and expression of the re-programming factors Oct-4, Nanog and SOX2 were measured by Western blot analysis. β-Actin served as a control. The data are presented as means ± SD (**P < 0.01, *P < 0.05).
Mentions: To evaluate the effect of aspirin on gemcitabine responsiveness, we examined 4 established PDA cell lines with different grades of aggressiveness [11, 22] and gemcitabine resistance (Figure 1A). The cells were treated with gemcitabine or 3 different aspirin concentrations, or a combination thereof. Viability, cell morphology and apoptosis were determined 72 h after treatment according to the MTT assay, microscopy, and staining with AnnexinV-FITC followed by flow cytometry, respectively (Figure 1A, Figure S1A, S1B). Whereas gemcitabine was effective in sensitive BxPc-3 cells, as expected, it was minimally effective in median resistant PANC-1 cells and non-effective in the totally resistant BxPc-3/GEM and AsPC-1 cells. In contrast, aspirin at doses of 5 and 10 mM was effective in all cell lines, and aspirin treatment significantly increased the efficacy of gemcitabine upon combination. These results were confirmed in the primary, low-passage, human PDA cell lines PaCaDD135 (gemcitabine-sensitive), PaCaDD159 (median gemcitabine-resistant) and PaCaDD183 (totally gemcitabine-resistant), which were in passage 5 (Figure 1B). Most importantly, aspirin did not significantly affect the viability of non-malignant human primary MSCs or immortalized human pancreatic ductal CRL-4023 cells, in contrast to gemcitabine, which significantly reduced the viability of MSCs and CRL-4023 cells (Figure 1C).

Bottom Line: Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling.Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine.Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice.

View Article: PubMed Central - PubMed

Affiliation: Molecular OncoSurgery, University of Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany.

ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

No MeSH data available.


Related in: MedlinePlus