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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 inhibits tumor growth and invasion in ovo and enhances gemcitabine efficacy(A) PANC-1 cells were transplanted into a plastic ring placed on the CAM of 9-day-old chick embryos. Double-distilled water (CO, 40 μL), aspirin (ASA, 30 μL, 15 mM), gemcitabine (GEM, 10 μL, 100 nM) or aspirin and gemcitabine together (A + G) were placed on Whatman filter papers (0.5 cm2) directly adjacent to the tumor xenografts. Aspirin was applied at days 11, 13, 15 and 17 and gemcitabine at days 11 and 15. The tumor xenografts were resected at day 17, and the tumor volumes were measured with calipers. Representative images of each group are shown. The diagram shows single measurements and the means ± SD (**P < 0.01, *P < 0.05). (B) Genomic DNA was isolated from CAM tissue (n = 6) directly adjacent to the tumor xenografts, and a PCR with primers for human Alu sequences was performed. Double-distilled water served as a negative control (Neg CO), and genomic DNA isolated from a tumor xenograft served as a positive control (Pos CO). The DNA marker is shown in the first lane (Marker), and the table on the right summarizes the number of positive bands per group (No. Alu+ CAM). (C) Tumor tissue sections from xenografts were evaluated by immunohistochemistry for the expression of c-Met, CD133, Ki67, cleaved active fragment of caspase-3, c-Rel and p65. Representative images at 400× magnification are shown. The bar indicates 50 μm. Positive cells are colored red to dark-red. For evaluation of the expression levels, a semi-quantitative scoring system was used based on the percentage of positive cells: very high (++++), high (+++), medium (++), low (+) and absent (−).
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Figure 5: Aspirin inhibits tumor growth and invasion in ovo and enhances gemcitabine efficacy(A) PANC-1 cells were transplanted into a plastic ring placed on the CAM of 9-day-old chick embryos. Double-distilled water (CO, 40 μL), aspirin (ASA, 30 μL, 15 mM), gemcitabine (GEM, 10 μL, 100 nM) or aspirin and gemcitabine together (A + G) were placed on Whatman filter papers (0.5 cm2) directly adjacent to the tumor xenografts. Aspirin was applied at days 11, 13, 15 and 17 and gemcitabine at days 11 and 15. The tumor xenografts were resected at day 17, and the tumor volumes were measured with calipers. Representative images of each group are shown. The diagram shows single measurements and the means ± SD (**P < 0.01, *P < 0.05). (B) Genomic DNA was isolated from CAM tissue (n = 6) directly adjacent to the tumor xenografts, and a PCR with primers for human Alu sequences was performed. Double-distilled water served as a negative control (Neg CO), and genomic DNA isolated from a tumor xenograft served as a positive control (Pos CO). The DNA marker is shown in the first lane (Marker), and the table on the right summarizes the number of positive bands per group (No. Alu+ CAM). (C) Tumor tissue sections from xenografts were evaluated by immunohistochemistry for the expression of c-Met, CD133, Ki67, cleaved active fragment of caspase-3, c-Rel and p65. Representative images at 400× magnification are shown. The bar indicates 50 μm. Positive cells are colored red to dark-red. For evaluation of the expression levels, a semi-quantitative scoring system was used based on the percentage of positive cells: very high (++++), high (+++), medium (++), low (+) and absent (−).

Mentions: The in vivo relevance of these data was examined by xenotransplantation of PANC-1 cells onto the CAM of fertilized chick eggs on developmental day 11 followed by in ovo treatment with aspirin on days 11, 13, 15 and 17 and gemcitabine treatment on days 11 and 15. The xenografts were resected on day 18. Whereas aspirin and gemcitabine alone significantly reduced the tumor volume, treatment with both in combination nearly completely prevented tumor formation (Figure 5A). To evaluate the invasion potential, genomic DNA was prepared from the CAM surrounding the tumor xenograft and from liver and lung tissue. Human Alu sequences, reflecting the presence of invading and metastasizing human cells, were detected by PCR. Whereas 4 of 6 CAM tissues from untreated eggs were Alu-positive, none of the tissues from the aspirin- or gemcitabine-treated groups were positive (Figure 5B). Alu sequences were not detectable in the liver or lung in any group, indicating that the xenografts did not spread to other organs (Figure S4A). In addition, we observed neither significant change in body weight of the embryos nor liver necrosis or developmental effects, indicating that the treatment was well tolerated (Figure S4B, S4C). Immunohistochemical staining for c-Met, CD133, SOX2, Ki67, cleaved fragment of caspase-3, p65 and c-Rel indicated that both single treatments reduced the proliferation and the expression of CSC markers and NF-κB subunits and induced apoptosis, although the combination treatment was most effective (Figure 5C); these findings were confirmed by immunofluorescence-double staining of the CSC markers EpCAM and Ki67 (Figure S4D).


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 inhibits tumor growth and invasion in ovo and enhances gemcitabine efficacy(A) PANC-1 cells were transplanted into a plastic ring placed on the CAM of 9-day-old chick embryos. Double-distilled water (CO, 40 μL), aspirin (ASA, 30 μL, 15 mM), gemcitabine (GEM, 10 μL, 100 nM) or aspirin and gemcitabine together (A + G) were placed on Whatman filter papers (0.5 cm2) directly adjacent to the tumor xenografts. Aspirin was applied at days 11, 13, 15 and 17 and gemcitabine at days 11 and 15. The tumor xenografts were resected at day 17, and the tumor volumes were measured with calipers. Representative images of each group are shown. The diagram shows single measurements and the means ± SD (**P < 0.01, *P < 0.05). (B) Genomic DNA was isolated from CAM tissue (n = 6) directly adjacent to the tumor xenografts, and a PCR with primers for human Alu sequences was performed. Double-distilled water served as a negative control (Neg CO), and genomic DNA isolated from a tumor xenograft served as a positive control (Pos CO). The DNA marker is shown in the first lane (Marker), and the table on the right summarizes the number of positive bands per group (No. Alu+ CAM). (C) Tumor tissue sections from xenografts were evaluated by immunohistochemistry for the expression of c-Met, CD133, Ki67, cleaved active fragment of caspase-3, c-Rel and p65. Representative images at 400× magnification are shown. The bar indicates 50 μm. Positive cells are colored red to dark-red. For evaluation of the expression levels, a semi-quantitative scoring system was used based on the percentage of positive cells: very high (++++), high (+++), medium (++), low (+) and absent (−).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496413&req=5

Figure 5: Aspirin inhibits tumor growth and invasion in ovo and enhances gemcitabine efficacy(A) PANC-1 cells were transplanted into a plastic ring placed on the CAM of 9-day-old chick embryos. Double-distilled water (CO, 40 μL), aspirin (ASA, 30 μL, 15 mM), gemcitabine (GEM, 10 μL, 100 nM) or aspirin and gemcitabine together (A + G) were placed on Whatman filter papers (0.5 cm2) directly adjacent to the tumor xenografts. Aspirin was applied at days 11, 13, 15 and 17 and gemcitabine at days 11 and 15. The tumor xenografts were resected at day 17, and the tumor volumes were measured with calipers. Representative images of each group are shown. The diagram shows single measurements and the means ± SD (**P < 0.01, *P < 0.05). (B) Genomic DNA was isolated from CAM tissue (n = 6) directly adjacent to the tumor xenografts, and a PCR with primers for human Alu sequences was performed. Double-distilled water served as a negative control (Neg CO), and genomic DNA isolated from a tumor xenograft served as a positive control (Pos CO). The DNA marker is shown in the first lane (Marker), and the table on the right summarizes the number of positive bands per group (No. Alu+ CAM). (C) Tumor tissue sections from xenografts were evaluated by immunohistochemistry for the expression of c-Met, CD133, Ki67, cleaved active fragment of caspase-3, c-Rel and p65. Representative images at 400× magnification are shown. The bar indicates 50 μm. Positive cells are colored red to dark-red. For evaluation of the expression levels, a semi-quantitative scoring system was used based on the percentage of positive cells: very high (++++), high (+++), medium (++), low (+) and absent (−).
Mentions: The in vivo relevance of these data was examined by xenotransplantation of PANC-1 cells onto the CAM of fertilized chick eggs on developmental day 11 followed by in ovo treatment with aspirin on days 11, 13, 15 and 17 and gemcitabine treatment on days 11 and 15. The xenografts were resected on day 18. Whereas aspirin and gemcitabine alone significantly reduced the tumor volume, treatment with both in combination nearly completely prevented tumor formation (Figure 5A). To evaluate the invasion potential, genomic DNA was prepared from the CAM surrounding the tumor xenograft and from liver and lung tissue. Human Alu sequences, reflecting the presence of invading and metastasizing human cells, were detected by PCR. Whereas 4 of 6 CAM tissues from untreated eggs were Alu-positive, none of the tissues from the aspirin- or gemcitabine-treated groups were positive (Figure 5B). Alu sequences were not detectable in the liver or lung in any group, indicating that the xenografts did not spread to other organs (Figure S4A). In addition, we observed neither significant change in body weight of the embryos nor liver necrosis or developmental effects, indicating that the treatment was well tolerated (Figure S4B, S4C). Immunohistochemical staining for c-Met, CD133, SOX2, Ki67, cleaved fragment of caspase-3, p65 and c-Rel indicated that both single treatments reduced the proliferation and the expression of CSC markers and NF-κB subunits and induced apoptosis, although the combination treatment was most effective (Figure 5C); these findings were confirmed by immunofluorescence-double staining of the CSC markers EpCAM and Ki67 (Figure S4D).

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