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Sublethal dose of irradiation enhances invasion of malignant glioma cells through p53-MMP 2 pathway in U87MG mouse brain tumor model.

Pei J, Park IH, Ryu HH, Li SY, Li CH, Lim SH, Wen M, Jang WY, Jung S - Radiat Oncol (2015)

Bottom Line: TIMP-2 expression did not increase in U87MG cells.MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group.Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Chonnam National University Hwasun Hospital and Mediacal School, 322 Seoyang-ro, Hwasun-eup, Hwasun-gun, Jeonnam, 519-763, Republic of Korea.

ABSTRACT

Background: Glioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments.

Methods: The U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2-6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography.

Results: MMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

Conclusion: Radiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent "U87-Fluc"was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemistry for MMP-2 expression and in situ zymography for gelatinolytic activity in brain tissues with either radiation exposure or not. Autofluorescence of mice in the in situ zymography experiment was minimized by NaBH4. a Hematoxylin-Eosin stain; b Immunohistochemistry for MMP-2; c, d In situ zymography, green: gelatinolytic activity, blue: nucleus
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Fig7: Immunohistochemistry for MMP-2 expression and in situ zymography for gelatinolytic activity in brain tissues with either radiation exposure or not. Autofluorescence of mice in the in situ zymography experiment was minimized by NaBH4. a Hematoxylin-Eosin stain; b Immunohistochemistry for MMP-2; c, d In situ zymography, green: gelatinolytic activity, blue: nucleus

Mentions: In situ zymography was performed in radiation-exposed and unexposed nude mouse brains. This is a unique technique that enables localization of gelatinolytic activities in histological sections. Notably, autofluorescence during in situ zymography was minimized by NaBH4. Green fluorescence as a signal for gelatinolytic activity was found around the tumor margin area in the radiated group but not in non-radiated group (Fig. 7c, d). However, MMP-2 expression showed no difference between the two groups (Fig. 7b).Fig. 7


Sublethal dose of irradiation enhances invasion of malignant glioma cells through p53-MMP 2 pathway in U87MG mouse brain tumor model.

Pei J, Park IH, Ryu HH, Li SY, Li CH, Lim SH, Wen M, Jang WY, Jung S - Radiat Oncol (2015)

Immunohistochemistry for MMP-2 expression and in situ zymography for gelatinolytic activity in brain tissues with either radiation exposure or not. Autofluorescence of mice in the in situ zymography experiment was minimized by NaBH4. a Hematoxylin-Eosin stain; b Immunohistochemistry for MMP-2; c, d In situ zymography, green: gelatinolytic activity, blue: nucleus
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4554349&req=5

Fig7: Immunohistochemistry for MMP-2 expression and in situ zymography for gelatinolytic activity in brain tissues with either radiation exposure or not. Autofluorescence of mice in the in situ zymography experiment was minimized by NaBH4. a Hematoxylin-Eosin stain; b Immunohistochemistry for MMP-2; c, d In situ zymography, green: gelatinolytic activity, blue: nucleus
Mentions: In situ zymography was performed in radiation-exposed and unexposed nude mouse brains. This is a unique technique that enables localization of gelatinolytic activities in histological sections. Notably, autofluorescence during in situ zymography was minimized by NaBH4. Green fluorescence as a signal for gelatinolytic activity was found around the tumor margin area in the radiated group but not in non-radiated group (Fig. 7c, d). However, MMP-2 expression showed no difference between the two groups (Fig. 7b).Fig. 7

Bottom Line: TIMP-2 expression did not increase in U87MG cells.MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group.Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Chonnam National University Hwasun Hospital and Mediacal School, 322 Seoyang-ro, Hwasun-eup, Hwasun-gun, Jeonnam, 519-763, Republic of Korea.

ABSTRACT

Background: Glioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments.

Methods: The U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2-6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography.

Results: MMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells.

Conclusion: Radiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent "U87-Fluc"was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.

No MeSH data available.


Related in: MedlinePlus