J Korean Neurosurg Soc.  2016 Nov;59(6):551-558. 10.3340/jkns.2016.59.6.551.

Metallothinein 1E Enhances Glioma Invasion through Modulation Matrix Metalloproteinases-2 and 9 in U87MG Mouse Brain Tumor Model

• Hur H
• Ryu HH
• Li CH
• Kim IY
• Jang WY
• Jung S

• KMID: 2417335
• DOI: http://doi.org/10.3340/jkns.2016.59.6.551

Abstract

Malignant glioma cells invading surrounding normal brain are inoperable and resistant to radio- and chemotherapy, and eventually lead to tumor regrowth. Identification of genes related to motility is important for understanding the molecular biological behavior of invasive gliomas. According to our previous studies, Metallothionein 1E (MT1E) was identified to enhance migration of human malignant glioma cells. The purpose of this study was to confirm that MT1E could modulate glioma invasion in vivo. Firstly we established 2 cell lines; MTS23, overexpressed by MT1E complementary DNA construct and pV12 as control. The expression of matrix metalloproteinases (MMP)-2, -9 and a disintegrin and metalloproteinase 17 were increased in MTS23 compared with pV12. Furthermore it was confirmed that MT1E could modulate MMPs secretion and translocation of NFkB p50 and B-cell lymphoma-3 through small interfering ribonucleic acid knocked U87MG cells. Then MTS23 and pV12 were injected into intracranial region of 5 week old male nude mouse. After 4 weeks, for brain tissues of these two groups, histological analysis, and immunohistochemical stain of MMP-2, 9 and Nestin were performed. As results, the group injected with MTS23 showed irregular margin and tumor cells infiltrating the surrounding normal brain, while that of pV12 (control) had round and clear margin. And regrowth of tumor cells in MTS23 group was observed in another site apart from tumor cell inoculation. MT1E could enhance tumor proliferation and invasion of malignant glioma through regulation of activation and expression of MMPs.

Keyword

Metallothionein 1E; Malignant glioma; Invasion; Brain tumor model; MMP-2; MMP-9

MeSH Terms

Animals
B-Lymphocytes
Brain Neoplasms*
Brain*
Cell Line
DNA, Complementary
Drug Therapy
Glioma*
Humans
Male
Matrix Metalloproteinases
Metallothionein
Mice*
Mice, Nude
Nestin
RNA
DNA, Complementary
Matrix Metalloproteinases
Metallothionein
RNA

Figure

• 

  Fig. 1 The effect of MT1E on tumor cell invasion using organotypic culture. MT1E could enhance migration and invasion of human malignant glioma cells ex vivo. MT1E : metallothionein 1E.

• 

  Fig. 2 MT1E modulates MMP-2, 9 and ADAM17 in U87MG. A : The stable cell line overexpressing MT1E ('MTS23') was established and endogenous MT1E was detected by RT-PCR (upper) and Western blot (lower). 'MTS23'; U87MG cells transfected with sense MT1E plasmid, 'pV12' (control); U87MG cells transfected with empty vector. B : Western blot in cell lysate. C : Upper; Gelatin zymography, lower : Western blot in supernatant. MT1E : metallothionein 1E, MMP : metalloproteinases, ADAM : a disintegrin and metalloproteinase, GAPDH : glyceraldehyde 3-phosphoate dehydrogenase.

• 

  Fig. 3 MT1E modulated secretion of MMPs through NFkB p50 and Bcl-3. The expression of these proteins was detected by Western blotting. (A) Western blot in nuclear, cytosol fraction and (B) total cell lysate. (C) Western blot in conditioned media. 'siC'; scramble RNA transfected U87MG, 'siMT1E'; siRNA for MT1E transfected U87MG. MT1E : metallothionein 1E, MMP : metalloproteinases, Bcl-3 : B-cell lymphoma-3, RNA : ribonucleic acid.

• 

  Fig. 4 Brain tumor growth patterns of 2 groups inoculating pV12 cells and MTS23. A : pV12 implanted group. B and C : MTS23 implanted group. Tumors of MTS inoculated group showed irregular margin and tumor cells infiltrating the surrounding normal brain (B), while that of pV12 (control) had round and clear margin (A). And regrowth of tumor cells in MTS23 group was observed in another site apart from tumor cell inoculation (C) but in pV12 group was nowhere to be found.

• 

  Fig. 5 The comparison of the survival rate between 2 groups inoculating pV12 cells and MTS23. The survival rate had detected during 70 days after inoculating tumor cells and was calculated by means of Kaplan–Meier survival analysis in orthotopic mouse brain tumor models. Mice transplanted with MTS23 cells showed a longer survival than mice transplanted with pV12 cells (70 days, p<0.048).

• 

  Fig. 6 MMP-2 and -9 were highly expressed on marginal region and tumor cells infiltrated the normal brain (arrow) in MTS23 group, while those in the control group were expressed on tumor, but not on normal region. And tumor cells were identified by immunohistochemistry for Nestin. MMP : metalloproteinases.

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