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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 29, 2001 as doi:10.1096/fj.01-0421fje. |
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,2,2
,,3
Johns Hopkins University School of Medicine, Departments of
* Neurology,
Oncology,
Neuroscience,
Kennedy Krieger Research Institute, Baltimore, Maryland, USA;
University of Iowa College of Medicine, Iowa City, Iowa, USA;
Valentis Inc., Burlingame, California, USA; and
|| Long Island Jewish Medical Center, Albert Einstein College of Medicine, New Hyde Park, New York, USA
3Correspondence: Kennedy Krieger Research Institute, 707 N. Broadway, Baltimore, MD 21205, USA. E-mail: laterra{at}kennedykrieger.org
SPECIFIC AIMS
The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor c-met contribute to the genesis, malignant progression, and chemo/radioresistance of multiple human malignancies, including gliomas. The present study aimed at inhibiting SF/HGF and c-met expression in established human glioblastoma xenografts by in vivo delivery of novel U1snRNA/ribozyme/antisense chimeric transgenes (designated U1/ribozymes) via liposome:DNA complexes and adenoviruses. We studied the effects of anti-SF/HGF and anti-c-met U1/ribozyme delivery on in vivo SF/HGF and c-met expression, receptor activation, tumor growth, apoptosis, angiogenesis, animal survival, and other malignancy parameters.
PRINCIPAL FINDINGS
1. Adenovirus-mediated delivery of U1/ribozymes inhibits SF/HGF and c-met expression and c-met activation
We and others have shown that stable expression of U1/ribozymes can effectively inhibit targeted gene expression. To test the effect of transient U1/ribozyme delivery on SF/HGF and c-met expression, we treated U-87 MG human glioblastoma cells with adenoviruses expressing anti-SF/HGF U1/ribozymes, anti-c-met U1/ribozymes, and controls expressing U1snRNA only (designated Ad-U1/SF, Ad-U1/Met, and Ad-U1, respectively) for 48 h and quantified mRNA and protein levels by Northern and immunoblotting, respectively. Adenovirus-based U1/ribozyme gene delivery led to a reduction in SF/HGF and c-met mRNA levels by 97% and 75% (n=2), respectively. SF/HGF and c-met protein levels were decreased by 47% and 50% (n=2), respectively. To study the effects of transient U1/ribozyme expression on SF/HGF-dependent functional c-met receptor activation, we examined the effect of Ad-U1/SF on c-met tyrosine phosphorylation in U-87 MG cells that express an SF/HGF:c-met autocrine loop. U-87 MG cells were infected with Ad-U1/SF or control Ad-U1 for 48 h, after which total cellular c-met protein was immunoprecipitated and analyzed for c-met tyrosine phosphorylation by immunoblotting. Inhibition of SF/HGF expression by Ad-U1/SF reduced c-met tyrosine phosphorylation by 45% (n=2) relative to total c-met protein.
2. Adenovirus-mediated delivery of U1/ribozymes inhibits tumor cell migration and colony formation in soft agar
To study the effects of transient expression of U1/ribozymes on the malignant phenotype in vitro, we treated U-87 MG cells with Ad-U1/SF, Ad-U1/Met, or Ad-U1 control for 48 h and then assessed them for cell migration and anchorage-independent colony formation. Ad-U1/SF and Ad-U1/Met inhibited cell migration by 60% and 56% (n=24), respectively, and inhibited colony formation in soft agar by 45% and 67% (n=24), respectively. These results indicate that transient expression of anti-SF and anti-c-met U1/ribozymes inhibits parameters of U-87 MG malignancy in vitro.
3. In vivo knockdown of SF/HGF and c-met expression inhibits the growth of established subcutaneous (s.c.) glioblastoma xenografts
We established U-87MG human glioblastoma xenografts s.c. in immunodeficient mice. When the tumors reached a size of 
50 mm3, we injected them twice per week with liposomes complexed with anti-SF/HGF,anti-c-met U1/ribozyme expression plasmids (designated pU1/SF and pU1/Met, respectively), a combination of both, or liposomes complexed with control plasmid (designated pU1). Tumor sizes were measured before each injection for a period of 21 days. Alternatively, we treated animals with established s.c. xenografts every 10 days with intravenous (i.v.) injections of liposome/DNA complexes and monitored tumor sizes for 30 days. We also extracted total RNA from the latter tumors to assess the effect of U1/ribozymes on SF/HGF and c-met mRNA.
Control xenografts directly injected with liposomes only or with pU1 liposomes grew 24- and 23-fold, respectively. In contrast, tumor xenografts injected with pU1/SF and pU1/Met liposomes grew only 3.4- and 2.6-fold, respectively (n=8) (P<0.01). Tumor growth in animals that received i.v. injections of either pU1/SF or pU1/Met liposomes was decreased but not statistically significantly, whereas tumors in animals that received a combination of pU1/SF + pU1/met liposomes were 79% (n=8) smaller than controls (P<0.05).
To assess in vivo knockdown of SF/HGF and c-met expression, we treated animals bearing established s.c. glioma xenografts with two i.v. injections of either pU1/SF + pU1/Met liposomes or pU1 control liposomes. Analysis of total tumor RNA by quantitative Light Cycler RT-PCR showed that in vivo delivery of pU1/SF and pU1/Met liposomes inhibited SF/HGF and c-met expression based on a shift in Light-Cycler PCR product amplification curves by 3–10 cycles (n=5) for SF/HGF and 2 cycles (n=4) for c-met when compared with control-injected animals. GAPDH mRNA levels, measured in the same tumors and by the same method, were unchanged.
4. In vivo delivery of U1/ribozymes via adenoviruses or liposome/DNA complexes inhibits the growth of established intracranial glioblastoma xenografts and promotes animal survival
We implanted U-87MG human glioblastoma cells within the caudate/putamen of nude mice and 4 days later began either intratumoral or i.v. treatment with control or anti-SF + anti-c-met U1/ribozyme expression vectors. We performed direct intratumoral stereotactic injections of Ad-U1/SF + Ad-U1/Met or control Ad/U1 (n=20) every 5 days, and animals were killed on postimplantation day 18 for histological assessment. Alternatively, we administered liposomes complexed with either pU1/SF + pU1/Met or control pU1 i.v. every 10 days (n=10); animals were killed on postimplantation day 18. We found significant anti-tumor responses regardless of route of administration or expression vector used. Tumors were 3-fold smaller (P<0.05) in response to direct intratumoral U1/ribozyme delivery and 23-fold smaller (P<0.01) in response to i.v. delivery (Fig. 1
A, B). Only 10 of 20 animals receiving intratumoral U1/ribozymes had his-tologically detectable tumor vs. 17 of 20 controls. Seven of 10 animals receiving i.v. U1/ribozymes had detectable tumors vs. 10 of 10 controls. Thus, various potential strategies exist for delivering U1/ribozymes for brain tumor gene targeting.
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We implanted human glioblastoma cells intracranially and 4 days later treated animals i.v. with liposome:U1/ribozyme complexes, as described above. Animal survival was then examined. Animals treated with pU1/SF + pU1/met liposomes showed a 27% increase in median survival compared with controls (n=10; P<0.01) (Fig. 1C
). One animal in the pU1/SF + pU1/met liposome group survived more than 60 days, after which it was killed. These results indicate that in vivo targeting of the SF/HGF:c-met signaling pathway can prolong survival of glioma-bearing animals.
5. In vivo inhibition of SF/HGF and c-met expression promotes apoptosis and inhibits tumor angiogenesis
To dissect the mechanisms by which anti-SF and anti-c-met U1/ribozymes inhibit in vivo tumor growth, we treated animals bearing s.c. tumor xenografts i.v. with U1/ribozymes and analyzed the tumors for changes in apoptosis and angiogenesis, factors known to be affected by SF/HGF:c-met signaling. Hematoxylin and eosin (H&E) stained histological sections revealed zones of necrosis and fibrosis and an increased number of apoptotic bodies in tumors treated with pU1/SF or pU1/Met liposomes. Immunohistochemistry showed that the number of cells expressing activated caspase-3,a marker of apoptosis, was increased by 5.3-fold and 6-fold (P<0.001) in tumors treated with pU1/SF and pU1/Met liposomes, respectively (Fig. 2
). Tumor angiogenesis based on blood vessel density was inhibited by 43% and 38% (P<0.001) in tumors treated with pU1/SF and pU1/Met liposomes, respectively (Fig. 2)
.
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CONCLUSIONS
Innovative strategies for inhibiting cellular/molecular pathways of malignancy are desperately needed. We demonstrate for the first time that in vivo inhibition of SF/HGF and c-met expression can inhibit the growth of human glioma xenografts that express an intact autocrine SF/HGF:c-met signaling pathway (Fig. 3
). We also show for the first time that chimeric U1snRNA/ribozymes can be used in vivo for proto-oncogene targeting in systemic as well as intracranial neoplasms.
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We identified at least two potential mechanisms for the anti-tumor response to the anti-SF/HGF and anti-Met U1/ribozymes. SF/HGF is a potent angiogenic factor with expression levels correlating with angiogenic indices in human gliomas. Tumors treated with U1/ribozymes contained 40% fewer blood vessels than controls, consistent with an anti-angiogenic tumor response. Most interesting were our findings that U1/ribozyme-treated tumors contained zones of necrosis and fibrosis, with a dramatic elevation in tumor cell apoptosis. Our results strongly suggest that in vivo anti-SF/HGF and anti-c-met U1/ribozyme therapy can provide a biological window of opportunity for enhancing tumor responses to radio/chemotherapy similar to our earlier findings in cultured glioma cells. Human malignant gliomas are highly infiltrative neoplasms not subject to cure using even the most aggressive local therapies available. Thus, novel molecular/biological therapeutics deliverable broadly to brain regions known to contain microinvasive glioma cells will likely have the highest therapeutic effect. Furthermore, i.v. efficacy suggests that anti-SF/HGF and anti-c-met U1/ribozymes may be developed against common systemic malignancies whose metastatic potential is associated with aberrant c-met signaling.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0421fje; to cite this article, use FASEB J. (November 29, 2001) 10.1096/fj.01-0421fje 
2 These authors contributed equally to this work.
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