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Reoxygenation from chronic hypoxia promotes metastatic processes in pancreatic cancer through the Hedgehog signaling.

Morifuji Y, Onishi H, Iwasaki H, Imaizumi A, Nakano K, Tanaka M, Katano M - Cancer Sci. (2014)

Bottom Line: A Hedgehog (Hh) signaling component, Gli1, was significantly increased by reoxygenation.Gli1 knockdown inhibited reoxygenation-induced increases in proliferation and tumorigenicity and decreased invasiveness through suppression of matrix metalloproteinase (MMP) 2 and MMP9.These results suggest that metastatic processes in PDAC are induced through activation of the Hh signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Therapy and Research, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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Reoxygenation enhances Gli1 and MMP expression. (a) Gli1 mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (b) Representative images of GLI1 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of GLI1 positive cells (right panel). (c) MMP2 (upper panels) and MMP9 (lower panels) mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (d) Representative images of MMP2 and MMP9 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of MMP2 or MMP9 positive cells, respectively (right panels). The graph shows mean ± SD. *P < 0.05.
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fig02: Reoxygenation enhances Gli1 and MMP expression. (a) Gli1 mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (b) Representative images of GLI1 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of GLI1 positive cells (right panel). (c) MMP2 (upper panels) and MMP9 (lower panels) mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (d) Representative images of MMP2 and MMP9 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of MMP2 or MMP9 positive cells, respectively (right panels). The graph shows mean ± SD. *P < 0.05.

Mentions: To analyze the mechanism of reoxygenation effects, we investigated the Hh signaling pathway because we previously reported it to be correlated with the augmentation of PDAC cell proliferation and invasiveness under acute hypoxia.10 In this study, Gli1 expression was considered a valuable marker of Hh signal activation. First, we evaluated Gli1 mRNA expression; expression of Gli1 mRNA was significantly higher in Ch-H-R cells than in Ac-H cells, and also significantly higher in reoxygenated Ch-H-R cells than in Ch-H-R cells (Fig.2a). The amount of Gli1 positive cells in reoxygenated Ch-H-R cells was significantly higher than in Ch-H-R cells (Figs2b,S6), and the GLI1 protein expression was also confirmed by western blotting analysis (Fig. S7). Next, we evaluated two matrix metalloproteinases, MMP2 and MMP9, as factors in reoxygenation-enhanced invasion. As expected, MMP2 and MMP9 mRNA expression and the ratios of positive cells were significantly increased after reoxygenation (Figs2c,d,S8), and MMP2 and MMP9 protein expression were also confirmed by western blotting analysis and gelatin zymography (Fig. S9a,b).


Reoxygenation from chronic hypoxia promotes metastatic processes in pancreatic cancer through the Hedgehog signaling.

Morifuji Y, Onishi H, Iwasaki H, Imaizumi A, Nakano K, Tanaka M, Katano M - Cancer Sci. (2014)

Reoxygenation enhances Gli1 and MMP expression. (a) Gli1 mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (b) Representative images of GLI1 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of GLI1 positive cells (right panel). (c) MMP2 (upper panels) and MMP9 (lower panels) mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (d) Representative images of MMP2 and MMP9 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of MMP2 or MMP9 positive cells, respectively (right panels). The graph shows mean ± SD. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Reoxygenation enhances Gli1 and MMP expression. (a) Gli1 mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (b) Representative images of GLI1 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of GLI1 positive cells (right panel). (c) MMP2 (upper panels) and MMP9 (lower panels) mRNA expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells were analyzed by real-time RT-PCR (n = 3). (d) Representative images of MMP2 and MMP9 expression in wild type cells, Ac-H cells, Ch-H-R cells and reoxygenated Ch-H-R cells derived from AsPC-1 cells by immunofluorescence (left panels) (×400). The graph shows the relative ratio of MMP2 or MMP9 positive cells, respectively (right panels). The graph shows mean ± SD. *P < 0.05.
Mentions: To analyze the mechanism of reoxygenation effects, we investigated the Hh signaling pathway because we previously reported it to be correlated with the augmentation of PDAC cell proliferation and invasiveness under acute hypoxia.10 In this study, Gli1 expression was considered a valuable marker of Hh signal activation. First, we evaluated Gli1 mRNA expression; expression of Gli1 mRNA was significantly higher in Ch-H-R cells than in Ac-H cells, and also significantly higher in reoxygenated Ch-H-R cells than in Ch-H-R cells (Fig.2a). The amount of Gli1 positive cells in reoxygenated Ch-H-R cells was significantly higher than in Ch-H-R cells (Figs2b,S6), and the GLI1 protein expression was also confirmed by western blotting analysis (Fig. S7). Next, we evaluated two matrix metalloproteinases, MMP2 and MMP9, as factors in reoxygenation-enhanced invasion. As expected, MMP2 and MMP9 mRNA expression and the ratios of positive cells were significantly increased after reoxygenation (Figs2c,d,S8), and MMP2 and MMP9 protein expression were also confirmed by western blotting analysis and gelatin zymography (Fig. S9a,b).

Bottom Line: A Hedgehog (Hh) signaling component, Gli1, was significantly increased by reoxygenation.Gli1 knockdown inhibited reoxygenation-induced increases in proliferation and tumorigenicity and decreased invasiveness through suppression of matrix metalloproteinase (MMP) 2 and MMP9.These results suggest that metastatic processes in PDAC are induced through activation of the Hh signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Therapy and Research, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Show MeSH
Related in: MedlinePlus