Antisense inhibition of laminin-8 expression reduces invasion of human gliomas in vitro.
Using gene array technology, we recently observed for the first time an up-regulation of laminin alpha4 chain in human gliomas. The data were validated by semiquantitative reverse transcription-PCR for RNA expression and immunohistochemistry for protein expression. Moreover, increase of the alpha4 chain-containing laminin-8 correlated with poor prognosis for patients with brain gliomas. Therefore, we hypothesized that inhibition of laminin-8 expression by a new generation of highly specific and stable antisense oligonucleotides (Morpholino) against chains of laminin-8 could slow or stop the spread of glioma and its recurrence and thus might be a promising approach for glioma therapy. We next sought to establish an in vitro model to test the feasibility of this approach and to optimize conditions for Morpholino treatment. To develop a model, we used human glioblastoma multiforme cell lines M059K and U-87MG cocultured with normal human brain microvascular endothelial cells (HBMVEC). Using Western blot analysis and immunohistochemistry, we confirmed that antisense treatment effectively blocked laminin-8 protein synthesis. Antisense oligonucleotides against both alpha4 and beta1 chains of laminin-8 were able to block significantly the invasion of cocultures through Matrigel. On average, the invasion was blocked by 62% in cocultures of U-87MG with HBMVEC and by 53% in cocultures of M059K with HBMVEC. The results show that laminin-8 may contribute to glioma progression and recurrence not only as part of the neovascularization process but also by directly increasing the invasive potential of tumor cells.
Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
Natalya M
NM
Khazenzon
Alexander V
AV
Ljubimov
Alexander J
AJ
Lakhter
Manabu
M
Fujita
Hironobu
H
Fujiwara
Kiyotoshi
K
Sekiguchi
Lydia M
LM
Sorokin
Noora
N
Petäjäniemi
Ismo
I
Virtanen
Keith L
KL
Black
Julia Y
JY
Ljubimova
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen / Q000494:pharmacology; D018450:Disease Progression; D004338:Drug Combinations; D005910:Glioma / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D007797:Laminin / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009361:Neoplasm Invasiveness; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011509:Proteoglycans / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D004338:Drug Combinations; C501369:LAMA4 protein, human; C478221:LAMB1 protein, human; D007797:Laminin; D016376:Oligonucleotides, Antisense; D011509:Proteoglycans; D012333:RNA, Messenger; C110308:laminin 8; C059485:matrigel; D003094:Collagen