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.
Khazenzon
Natalya M
NM
Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
Ljubimov
Alexander V
AV
Lakhter
Alexander J
AJ
Fujita
Manabu
M
Fujiwara
Hironobu
H
Sekiguchi
Kiyotoshi
K
Sorokin
Lydia M
LM
Petäjäniemi
Noora
N
Virtanen
Ismo
I
Black
Keith L
KL
Ljubimova
Julia Y
JY
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