Genetic down-regulation of phosphoinositide 3-kinase by bikunin correlates with suppression of invasion and metastasis in human ovarian cancer HRA cells.
Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.
Kobayashi
Hiroshi
H
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka, 431-3192, Japan. hirokoba@hama-med.ac.jp
Suzuki
Mika
M
Kanayama
Naohiro
N
Terao
Toshihiko
T
D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000378:metabolism; D003720:Densitometry; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010051:Ovarian Neoplasms / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism*; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D016376:Oligonucleotides, Antisense; C109148:SPINT2 protein, human; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014360:Trypsin Inhibitor, Kunitz Soybean; D019869:Phosphatidylinositol 3-Kinases; D014568:Urokinase-Type Plasminogen Activator