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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5565fjev1 20/11/1904    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyoshi, T. Articles by Ninomiya, Y. Search for Related Content PubMed PubMed Citation Articles by Miyoshi, T. Articles by Ninomiya, Y.

Tumor-specific expression of the RGD-3(IV)NC1 domain suppresses endothelial tube formation and tumor growth in mice

Toru Miyoshi^*,1, Satoshi Hirohata^*,,1, Hiroko Ogawa^*,, Masayuki Doi^*, Masanari Obika^*,, Tomoko Yonezawa, Yoshikazu Sado, Shozo Kusachi, Satoru Kyo^||, Seiji Kondo^¶, Yasushi Shiratori^*, Billy G. Hudson and Yoshifumi Ninomiya^,2

^* Department of Medicine and Medical Science,

Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;

Division of Immunology, Shigei Medical Research Institute, Okayama, Japan;

Department of Medical Technology, Faculty of Health Science, Okayama University Medical School, Okayama, Japan;

^|| Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, Ishikawa, Japan;

^¶ Department of Neurosurgery, University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA; and

Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

²Correspondence: Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2–5-1, Shikata-cho, Okayama 700-8558 Japan. E-mail: yoshinin{at}cc.okayama-u.ac.jp

SPECIFIC AIMS

This study demonstrates that tumor growth inhibition after the transduction of the noncollagenous domain of 3 chain of collagen IV (3(IV)NC1) into tumors inhibits tumor growth using a tumor-targeted gene therapy method. We hypothesized that when using a human telomerase reverse transcriptase (hTERT) promoter-driven, RGD motif-containing 3(IV)NC1 (hTERT/RGD-3(IV)NC1) expressed in telomerase-expressing tumor cells would inhibit tumor growth by its anti-angiogenic property.

PRINCIPAL FINDINGS

1. Expression of hTERT mRNA in tumor and nontumor cell lines and transduction of hTERT/RGD-3(IV)NC1
Tumor cell lines (DU145, HT1080, PC3, and H1299; Fig. 1 a, lanes 1–4) all had a considerable level of hTERT mRNA expression, although the hTERT mRNA expression levels varied. Neither type of nontumor cell (HFK and HSF) examined showed detectable hTERT mRNA expression (Fig. 1a , lanes 5 and 6). As shown in Fig. 1b , a single band at 28 kDa was observed in the conditioned medium from the cells infected with hTERT/RGD-3(IV)NC1 (lane 1), and a band of the same size was detected in cells transfected with pCMV/RGD-3(IV)NC1 serving as a positive control (Fig. 1b , lane 2). RGD-3(IV)NC1 was not detected in conditioned medium from cells infected with control adenovirus CAG/lacZ (Fig. 1b , lane 3). The level of RGD-3(IV)NC1 in conditioned medium from hTERT/RGD-3(IV)NC1-infected cells increased in a time-dependent manner (Fig. 1c ). The secretion of RGD-3(IV)NC1 was detected in conditioned medium from hTERT-expressing cells such as DU145 cells (Fig. 1d , lane 1), PC3 cells (Fig. 1d , lane 2), HT1080 cells (Fig. 1d , lane 3), and H1299 cells (Fig. 1d , lane 4) infected with hTERT/RGD-3(IV)NC1. In contrast, HSF (Fig. 1d , lane 5), HFK (Fig. 1d , lane 6), and HUVEC (data not shown) infected with hTERT/RGD-3(IV)NC1 did not secrete RGD-3(IV)NC1 into conditioned medium.

View larger version (24K): [in this window] [in a new window]   Figure 1. a) Expression of human telomerase reverse transcriptase (hTERT) mRNA in tumor and normal cells. The hTERT mRNA expression level was measured by quantitative real-time RT-polymerase chain reaction (RT-PCR) analysis. The hTERT mRNA expression level of DU145 cells was taken as 1.0 and the hTERT mRNA expression level of other cell lines was compared with that of DU145 cells (lane 1, DU145 cells; lane 2, HT1080 cells; lane 3, PC3 cells; lane 4, H1299 cells; lane 5, HSF; lane 6, HFK). Each hTERT mRNA expression value was standardized relative to that of the PBGD mRNA level used as an internal control. Each column represents the mean value ± SD from 3 independent experiments. b) Secretion of induced RGD-3(IV)NC1 into conditioned medium (CM). DU145 cells were infected or transfected with hTERT/RGD-3(IV)NC1, pCMV/RGD-3(IV)NC1, or CAG/lacZ. CM from hTERT/RGD-3(IV)NC1-infected cells (lane 1), pCMV/RGD-3(IV)NC1-transfected cells (lane 2; positive control), or CAG/lacZ-infected cells (lane 3; negative control) was collected and subjected to Western blot analysis with an anti-FLAG M2 monoclonal antibody (mAb). RGD-3(IV)NC1 was detected at the size of 28 kDa in CM from DU145 cells infected with hTERT/RGD-3(IV)NC1 at an MOI of 100 for 48 h. c) RGD-3(IV)NC1 secretion increased in a time-dependent manner in hTERT/RGD-3(IV)NC1-infected cells. DU145 cells were infected with hTERT/RGD-3(IV)NC1 and CM was collected at 0 h (lane 1), 24 h (lane 2), 48 h (lane 3), and 72 h (lane 4) and subjected to Western blot analysis with anti-FLAG M2 mAb. d) RGD-3(IV)NC1 was secreted into the CM only from hTERT-positive cells. DU145 cells (lane 1), PC3 cells (lane 2), HT1080 cells (lane 3), H1299 cells (lane 4), HSF (lane 5), and HFK (lane 6) were infected with hTERT/RGD-3(IV)NC1 at an MOI of 100. After 48 h of infection, each type of CM was collected and subjected to Western blot analysis. Note that RGD-3(IV)NC1 was not detected in CM from HSF or HFK.

2. RGD-3(IV)NC1 produced by hTERT/RGD-3(IV)NC1 showed inhibitory effects on proliferation and tube formation in endothelial cells but not in tumor cell lines
The proliferation of tumor cells and somatic cells treated with conditioned medium-RGD-3(IV)NC1 was examined. The proliferation of HUVEC treated with conditioned medium-RGD-3(IV)NC1 was significantly reduced to 67% of the level of proliferation of HUVEC treated with conditioned medium-lacZ (P=0.022). In addition, the proliferation of HUVEC was not inhibited when conditioned medium-RGD-3(IV)NC1FLAG [RGD-3(IV)NC1 had been depleted by immunoprecipitation with anti-FLAG antibody (Ab)], indicating that the inhibitory effect on proliferation was due to the secreted RGD-3(IV)NC1. This inhibitory effect on proliferation appeared to be specific to endothelial cells, because conditioned medium-RGD-3(IV)NC1 did not affect the growth rate of DU145 cells when compared with the effect of conditioned medium-lacZ. We also evaluated the effect on cell viability using the MTT assay. Conditioned medium-RGD-3(IV)NC1 significantly decreased the cell viability of HUVEC by 51% compared with that of HUVEC treated with conditioned medium-lacZ (P=0.004), whereas conditioned medium-RGD-3(IV)NC1FLAG did not show a significant inhibitory effect on HUVEC. Neither conditioned medium-RGD-3(IV)NC1 nor conditioned medium-lacZ had any effect on the viability of DU145 cells. Conditioned medium from cells infected with hTERT/RGD-3(IV)NC1 exerts effects only on endothelial cells. The effects of RGD-3(IV)NC1 on tube formation of endothelial cells were examined by a tube formation assay performed using HUVEC on Matrigel. HUVEC formed tubes on Matrigel-coated plates supplemented with conditioned medium-lacZ. Conditioned medium-RGD-3(IV)NC1 strongly reduced the formation of tube-like structures. Tube formation was not reduced when HUVEC were incubated with conditioned medium-RGD-3(IV)NC1FLAG. Quantitative analysis of the tube formation showed significant inhibition by conditioned medium-RGD-3(IV)NC1 compared with conditioned medium-lacZ or conditioned medium-RGD-3(IV)NC1FLAG (P=0.002 and 0.013, respectively).

3. In vivo expression of RGD-3(IV)NC1 with adenoviral hTERT/RGD-3(IV)NC1 and the inhibition of angiogenesis by hTERT/RGD-3(IV)NC1 in tumor-bearing mice
Transfection with plasmid vector encoding hTERT/RGD-3(IV)NC1 (phTERT/RGD-3(IV)NC1) significantly inhibited HT1080 tumor growth compared with the extent of tumor growth shown in animals transfected with the control plasmid (phTERT/lacZ). For adenoviral gene therapy, DU145 cells were inoculated subcutaneously (s.c.) into the right flank (1x10⁶ cells) of 6- to 8-wk-old female BALB/c nude mice. Mice received an intratumor injection of hTERT/RGD-3(IV)NC1 or CAG/lacZ at a dose of 1 x 10⁹ pfu or PBS only on day 0 (five mice/group). The expression of RGD-3(IV)NC1 in hTERT/RGD-3(IV)NC1-treated mice was examined by immunofluorescent staining. RGD-3(IV)NC1 was expressed restrictively in tumors and not in organs such as the kidney. Treatment with CAG/lacZ instead of hTERT/RGD-3(IV)NC1 did not induce RGD-3(IV)NC1 signals. In hTERT/RGD-3(IV)NC1-treated mice there were no clear signs of inflammation, as indicated by cell infiltration. We then examined the effect of RGD-3(IV)NC1 on angiogenesis in vivo in tumors by immunostaining with CD31. Tumors of each group of mice were removed 10 days after the initial treatment with hTERT/RGD-3(IV)NC1. Tumors from animals receiving PBS (Fig. 2 a) or CAG/lacZ (Fig. 2b ) showed intense signals for CD31 staining, indicating the presence of extensive angiogenesis in the tumors in these mice. However, tumors from hTERT/RGD-3(IV)NC1-treated animals showed a marked reduction in microvessel density (Fig. 2c ). Quantitative analysis demonstrated a significant reduction of the intratumor microvessel density in hTERT/RGD-3(IV)NC1-treated animals compared with that in control animals receiving PBS or CAG/lacZ (P=0.027 and 0.022, respectively) (Fig. 2d ).

View larger version (95K): [in this window] [in a new window]   Figure 2. Inhibition of tumor angiogenesis by treatment with hTERT/RGD-3(IV)NC1. Frozen tumor sections were stained with anti-CD31 Ab as described in Material and Methods. Representative light micrographs showing microvessels in tumors from animals that received PBS (a), CAG/lacZ (b), or hTERT/RGD-3(IV)NC1 (c). d) Quantitative analysis of microvessel density was performed by counting the positively stained cells in five different fields (x200). Significantly fewer blood vessels were observed in the tumor sections of the hTERT/RGD-3(IV)NC1-treated group. #P < 0.05.

4. Inhibition of tumor growth by hTERT/RGD-3(IV)NC1 in tumor-bearing mice
Treatment with a single injection of hTERT/RGD-3(IV)NC1 resulted in an inhibition of tumor growth compared with the extent of tumor growth shown in animals treated with PBS or control adenovirus CAG/lacZ. The tumor size at day 20 was significantly smaller than that in animals from the control groups (i.e., PBS or CAG/lacZ; P=0.005 and 0.016, respectively). In the case of weekly injections, mice were killed when tumor volumes exceeded 1000 mm³. All animals treated with vehicle were killed by day 60 and only one mouse in the CAG/lacZ group survived until day 60. In contrast, all mice in the hTERT/RGD-3(IV)NC1-treated group survived past 60 days. The maximal tumor volume in the hTERT/RGD-3(IV)NC1-treated group was <1000 mm³ at 60 days after the initial treatment.

CONCLUSIONS AND SIGNIFICANCE

Figure 3 provides a summary of the tumor-targeted anti-angiogenic gene therapy system. The present work demonstrated that hTERT promoter-driven RGD-3(IV)NC1 (hTERT/RGD-3(IV)NC1) limited RGD-3(IV)NC1 expression in tumor cells (i.e., hTERT-positive cells). Secreted RGD-3(IV)NC1 showed the inhibitory effect of proliferation and tube formation on endothelial cells, and our previous finding of angiogenesis inhibition by RGD-3(IV)NC1 was confirmed by this study. Adenoviral hTERT/RGD-3(IV)NC1 gene therapy was able to inhibit tumor growth by inhibiting angiogenesis in tumor-bearing nude mice. This is the first report showing that intermittent administration of anti-angiogenic molecule (i.e., RGD-3(IV)NC1) achieved tumor dormancy for >60 days. However, it is not yet conclusive that hTERT is an appropriate promoter for tumor-targeted anti-angiogenic gene therapy because hTERT expression has recently been reported in nontumor cells such as hair follicles and stem cells. In conclusion, we examined the effect of tumor-targeted anti-angiogenic gene therapy utilizing hTERT promoter in tumor-bearing mice, which resulted in the promotion of tumor dormancy.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic representation showing tumor targeted anti-angiogenic gene therapy. Our system, hTERT/RGD-3(IV)NC1, promoted RGD-3(IV)NC1 expression in tumor cells. In contrast, hTERT/RGD-3(IV)NC1 did not induce RGD-3(IV)NC1 expression in normal cells (i.e., hTERT-negative cells). RGD-3(IV)NC1 showed an anti-angiogenic property that inhibited tumor progression.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5565fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5565fjev1 20/11/1904    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyoshi, T. Articles by Ninomiya, Y. Search for Related Content PubMed PubMed Citation Articles by Miyoshi, T. Articles by Ninomiya, Y.