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IL-24 Inhibits Invasion and Migration of Human Lung Cancer Cells
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doi:10.1016/j.ymthe.2004.01.019
Ectopic Production of MDA-7/IL-24 Inhibits Invasion and Migration of Human Lung Cancer Cells Rajagopal Ramesh,1,* Isao Ito,1 Began Gopalan,1 Yuji Saito,1 Abner M. Mhashilkar,2 and Sunil Chada2 1
Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA 2 Introgen Therapeutics, Inc., Houston, TX 77030, USA
*To whom correspondence and reprint requests should be addressed at the Department of Thoracic and Cardiovascular Surgery, Unit 445, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Fax: (713) 794-4901. E-mail: [email protected].
We have previously observed the suppression of lung tumor growth in response to overexpression of melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24; approved gene symbol IL24) in vitro and in vivo. MDA-7/IL-24 exerts its tumor-suppressive effects by multiple mechanisms, including the activation of the caspase cascade and the inhibition of angiogenesis. In this study, we used an adenoviral vector (Ad-mda7) to examine the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human non-small-cell lung carcinoma cells. Lung tumor cells (H1299 and A549) treated in vitro with Ad-mda7 migrated and invaded less than cells treated with phosphate-buffered saline (PBS) or Ad-Luc (vector control). MDA-7/IL-24 inhibited migration and invasion by down-regulating the production of phosphatidylinositol 3-kinase/protein kinase B, focal adhesion kinase, and matrix metalloproteinase-2 and -9 relative to PBS and Ad-Luc. Furthermore, tumor cells treated with Ad-mda7 ex vivo or with DOTAP:Chol – mda7 complex in vivo formed significantly fewer tumors in an experimental lung metastasis model. These results show that MDA-7/IL-24 inhibits invasion and migration by lung cancer cells by down-regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis. Key Words: MDA-7, IL-24, gene therapy, MMP, PI3K, FAK, invasion, metastasis, lung, cancer
INTRODUCTION The melanoma differentiation-associated gene-7 (MDA7; approved gene symbol IL24) was identified from melanoma cells that were allowed to differentiate after treatment with interferon-h and mezerin [1]. MDA-7 was reported to be a novel tumor-suppressor gene whose expression was lost during tumorigenesis [1]. Expression of MDA-7 mRNA was reported in normal melanocytes and in melanomas that were allowed to differentiate [1,2]. On the basis of these observations, it was speculated that the MDA-7 protein plays a role in maintaining the normal physiology of melanocytes and that the inhibition of its production results in transformation and progression from a local, nonmetastatic primary tumor to highly metastatic cancer. Support for this concept was provided by a recent study by Ellerhorst et al. [3], who showed that the MDA-7 protein is pro-
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duced in human melanocytes and in primary melanomas but that its production is progressively lost in metastatic melanomas. Strikingly, the authors of this study noted a significant correlation between loss of MDA-7 production and melanoma tumor invasion. They identified specimens in which the superficial layer of tumor was MDA-7 positive, yet MDA-7 production was lost as the tumor invaded the surrounding tissue. Thus, the restoration of MDA-7 production in tumor cells should inhibit tumor growth and tumor invasion and metastasis. Studies by us and several other investigators have shown that the overproduction of MDA-7 selectively inhibited cancer cell growth of diverse origins by inducing apoptosis with minimal toxicity to normal cells both in vitro and in vivo [4 – 14]. However, apart from the findings of Ellerhorst et al. [3], no additional preclinical or clinical
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FIG. 1. MDA-7/IL-24 inhibits tumor cell migration. (A) A549 and (B) H1299 lung tumor cells were treated with PBS, Ad-Luc, or Ad-mda7 and analyzed for cell migration, cell viability, and apoptosis at 24 and 48 h after treatment. Cells treated with Ad-mda7 were significantly ( P = 0.002) less able to migrate than cells treated with PBS or Ad-Luc. That inhibition of cell migration was not due to cytotoxicity was determined by cell viability assay. No significant inhibition of tumor cell proliferation was observed in Ad-mda7-treated cells compared to PBS- and Ad-Luc-treated cells in both cell lines. (C) Analysis for cells in sub-G0 phase of cell cycle, an indicator of apoptotic cells. Bars denote standard error.
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data exist showing a role for MDA-7/IL-24 in regulating tumor metastasis. Furthermore, the underlying mechanism by which MDA-7 might inhibit tumor metastasis is unknown. We therefore investigated the effects of the ectopic production of MDA-7/IL-24 on migration and invasion by human non-small-cell lung carcinoma (NSCLC) cells in vitro and in vivo and attempted to identify the underlying mechanisms of metastasis suppression. In this study, we show for the first time that MDA-7 inhibits the migration and invasion of human lung cancer cells in vitro by down-regulating the protein production of phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and matrix metalloproteinases (MMPs). Furthermore, we confirmed in vivo of the ability of MDA-7 to reduce experimental metastasis. Thus, MDA7/IL-24 may provide multiple benefits as an anti-cancer strategy since it can inhibit primary tumor growth and prevent metastatic spread.
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inhibits tumor cell migration and that the inhibitory effect is not due to cytotoxicity. MDA-7 Inhibits Tumor Cell Invasion Tumor cells (A549 and H1299) treated with Ad-mda7 were much less invasive, as indicated by the small number of cells on the outer membrane of the Matrigel invasion assay filter, than cells treated with PBS or AdLuc (Figs. 2A and 2B). In both A549 and H1299 cells, the
RESULTS MDA-7/IL-24 Inhibits Tumor Cell Migration In the present study we tested the ability of Ad-mda7 to inhibit cell migration on two lung cancer cell lines that are wild type for p53 (A549) and null for p53 (H1299). Tumor cells treated with Ad-mda7 were significantly ( P = 0.002) less able to migrate than were cells treated with Ad-Luc or phosphate-buffered saline (PBS) (Figs. 1A and 1B). We also observed inhibition of migration in Ad-luctreated H1299 cells at 48 h compared to PBS-treated cells. However, the degree of inhibition induced by Ad-luc was as not effective as that induced by Ad-mda7. The inhibitory effect on cell migration occurred as early as 24 h in both tumor cell lines (Figs. 1A and 1B). To show that the inhibition of cell migration was not due to MDA-7/IL-24mediated cell death, in a separate but parallel set of experiments, we subjected cells treated with PBS, AdLuc, and Ad-mda7 to a cell viability assay at 24 and 48 h after infection. We observed no significant difference in cell viability at these time points, indicating that the inhibition of migration by MDA-7/IL-24 was not due to cell death (Figs. 1A and 1B). Note that at 24 h posttransduction, all three experimental groups are superimposable, indicating no significant cell death. By 48 h, some cell death is occurring; however, with this vector dose, significant Ad-mda7-mediated death is observed only at 72 and 96 h posttransduction. Additionally, flow cytometric analysis demonstrated no significant increase in the number of cells in sub-G0 phase of the cell cycle in Ad-mda7-treated cells (4 – 6%) compared to Ad-luc-treated cells (4.3 – 6%) at 24 h (Fig. 1C). However, we observed a slight increase in cell number in the sub-G0 phase in Ad-mda7-treated cells (9 – 13%) compared to Ad-luc-treated cells (7 – 10%) at 48 h. The increase in cell number was higher in Ad-mda7-treated H1299 cells than in A549 cells. These results show that MDA-7/IL-24
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FIG. 2. MDA-7/IL-24 inhibits tumor cell invasion. (A) A549 and (B) H1299 tumor cells were treated with PBS, Ad-Luc, Ad-mda7, 10 AM LY294002, or 1 Ag/ml MMP-II inhibitor. After 6 h, cells were harvested, counted, and added into the upper wells of Matrigel-coated wells. Cells were allowed to invade by incubation at 37jC. At 24 and 48 h after treatment, cells were fixed and stained with crystal violet. Cells that migrated to the lower side of the well were observed and counted under a light microscope at 200 magnification. The number of invading cells per treatment was counted in a blind fashion and recorded as the average of three separate experiments. Cells treated with Ad-mda7 showed less invasion ( P = 0.001) than cells treated with PBS or AdLuc. The inhibitory effect mediated by MDA-7 was similar to the inhibitory effect observed with LY294002 and MMP-II inhibitor. Bars denote standard error.
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FIG. 3. MDA-7/IL-24 down-regulates production of proteins associated with migration and invasion. Tumor cells (A549 and H1299) were treated with PBS, Ad-Luc, Ad-mda7, or LY294002. Cells were harvested at 48 h, total cell lysates were prepared, and proteins were separated by SDS – PAGE. Proteins were blotted using antibodies against MDA-7, p85 PI3K, pJNK, p38MAPK, p44/42MAPK, and pFAK. h-Actin was used as a loading control. The relative change in the protein expression was expressed as a ratio with the value for PBS-treated cells arbitrarily set to 1.
number of invading cells was significantly less after treatment with Ad-mda7 ( P = 0.001) than with PBS or Ad-Luc. We observed the inhibitory effect exerted by MDA-7/IL-24 as early as 24 h after Ad-mda7 treatment. Furthermore, MDA-7/IL-24-mediated inhibitory effect was similar to the inhibitory effect observed in cells treated with LY294002, a PI3K inhibitor, or with MMPII inhibitor. A cell viability assay showed that the inhibition was not a result of MDA-7/IL-24-mediated cell death (data not shown). These results show that MDA-7/IL-24 effectively inhibits cell invasion.
MDA-7 Down-regulates the Production of Proteins Associated with Cell Migration and Invasion We next examined the regulation of proteins that are associated with cell migration and invasion signaling pathways by Western blot analysis. We did not observe MDA-7/IL-24 production in PBS- or Ad-Luc-treated tumor cells, but high levels of MDA-7/IL-24 production were found in Ad-mda7-treated cells (Fig. 3). Overproduction of the MDA-7/IL-24 protein in both H1299 and A549 tumor cell lines resulted in decreased production of p85 PI3K and pFAK and increased production of pJNK,
FIG. 4. MDA-7/IL-24 inhibits MMP-2 and -9 production. Tumor cells were treated with PBS, Ad-Luc, or Ad-mda7. At 24 and 48 h after treatment, cell supernatant and cell lysate were collected and analyzed for MMP2 and -9 by zymography and Western blot analysis. (A) Inhibition of MMP-2 and -9 was observed in A549 cells treated with Admda7. Inhibition was shown by both zymography (top) and Western blot analyses (bottom). No change in MMP-2 or -9 was observed in cells treated with PBS or Ad-Luc. (B) Ad-mda7 inhibited MMP-2 but not MMP-9 production in H1299 cells. Inhibition of MMP-2 was observed on both zymography (top) and Western blot analyses (bottom).
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p38MAPK, and p44/42MAPK compared to PBS- or Ad-Luctreated cells (Fig. 3). In H1299 cells, treatment with AdLuc resulted in increased expression of p85 PI3K, pJNK, and pFAK compared to PBS-treated cells. We also observed the inhibition of p85 PI3K and pFAK in cells treated with the PI3K inhibitor LY294002, although the inhibition differed between the two cell lines. The decrease in pFAK expression in cell lines overproducing MDA-7 was greater than that observed with LY294004. Furthermore, LY294002 treatment resulted in increased production of p38MAPK and p44/42MAPK in H1299 cells. In A549 cells,
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LY294004 increased the production of pJNK and p44/ 42MAPK. These results indicate that MDA-7, like LY294002, effectively inhibits PI3K and pFAK while increasing the expression of other signaling proteins. Inhibition of Matrix Metalloproteinase Production in Tumor Cells by MDA-7/IL-24 We next examined tumor cells overproducing MDA-7 for MMP regulation by zymography and Western blot analysis. Zymography and Western blot analysis showed that production of the MMP-2 and -9 proteins was decreased
FIG. 5. MDA-7/IL-24 inhibits lung metastases. (A) A549 lung tumor cells were treated with PBS, Ad-Luc, and Ad-mda7 ex vivo. After 6 h, cells were harvested, washed, resuspended in PBS, and injected into female nude mice via the tail vein. Three weeks after the injection of the tumor cells, the animals were euthanized by CO2 inhalation, and the presence of lung tumors was determined by histopathologic examination (top; representative tumor indicated by circles) and the number of tumors determined by counting tumor nodules under a dissecting microscope (bottom). Significantly ( P = 0.01) fewer lung tumors were observed in animals injected with tumor cells treated with Ad-mda7 than in animals injected with tumor cells treated with PBS or Ad-Luc. Results are the means of two separate experiments. Bars denote standard error. (B) Mice bearing experimental A549 lung metastases were either untreated (control) or treated with DOTAP:Chol – CAT or DOTAP:Chol – mda7 complex. Animals were treated daily for six doses via tail vein injection. Three weeks after the last treatment animals were euthanized and the number of lung tumors was counted. Animals treated with DOTAP:Chol – mda7 complex demonstrated significant inhibition of lung metastasis compared to animals that were untreated or treated with DOTAP:Chol – CAT complex ( P = 0.001). Bars denote standard error.
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in A549 tumor cells treated with Ad-mda7, compared with cells that were treated with PBS or Ad-Luc (Fig. 4A). In H1299 cells treated with Ad-mda7, we observed a decrease in MMP-2 but not in MMP-9 compared with cells treated with PBS or Ad-Luc (Fig. 4B). Results of zymography analysis correlated with the results of the Western blot analysis (Figs. 4A and 4B). Thus Ad-mda7 can modulate MMP expression and activity in both p53 wild-type and p53-null NSCLC lines. MDA-7/IL-24 Inhibits Experimental Lung Metastases In an experimental lung metastasis model using A549 human lung cancer cells in nude mice, significantly ( P = 0.01) fewer lung tumor nodules formed in mice injected with tumor cells treated with Ad-mda7 than in mice injected with tumor cells treated with PBS or Ad-Luc (Fig. 5A). Inhibition of experimental metastasis correlated with histological tissue staining that demonstrated smaller number of tumors. To confirm further that inhibition of experimental metastasis was not due solely to cell death, we carried out additional in vivo experiments. Treatment of lung tumor-bearing animals with DOTAP:cholesterol (DOTAP:Chol) – mda7 resulted in significant inhibition ( P = 0.001) of experimental metastasis compared to mice that were untreated or treated with DOTAP:Chol – CAT complex (Fig. 5B). The ability to inhibit experimental metastasis is considered an indirect correlate of inhibition of migration and invasion of tumor cells. These results demonstrate that MDA-7 inhibits tumor cell migration and invasion in vivo and is in agreement with our observed in vitro results.
DISCUSSION In this study, we show for the first time that MDA-7 can inhibit the migration and invasion of human NSCLC cells in vitro and in vivo. In vitro, tumor cells treated with Ad-mda7 were significantly inhibited from growth factorinduced migration. Protein production analysis showed that the inhibition of cell migration was due to MDA-7. The ability of MDA-7 to inhibit tumor cell migration is not surprising and is consistent with our recent findings in which inhibition of normal endothelial cell migration by recombinant MDA-7 (rMDA-7) protein was demonstrated [15]. It could be argued that the observed inhibition of cell migration was due to the cytotoxicity of Admda7 as opposed to rMDA-7; however, that was ruled out by a cell proliferation assay. Note that at the doses and time frames employed in this study, more than 90% of the cells were viable. The ability of Ad-mda7 to inhibit cell migration of lung tumor cells supports the findings of Chen et al. [19], who recently reported the inhibition of rat smooth muscle cell (SMC) migration by Ad-mda7. Furthermore, in that study, the inhibition of cell migration was found to be independent of the antitumor
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activity of Ad-mda7. These results show that Ad-mda7 can inhibit tumor cell migration in vitro. Although the ability of Ad-mda7 to inhibit SMC migration has been shown previously [19], the mechanism by which MDA-7 inhibits cell migration was not studied. Cell migration is an important element in the physiology of a normal cell and of a cell in pathologic states. For example, the migration of inflammatory cells to a site of injury is a requisite for wound healing [20,21]. Similarly, the migration of tumor cells to distant organ sites plays a pivotal role in metastasis [22,23]. Cell migration has previously been shown to be regulated by numerous molecules, including PI3K, p38MAPK, p44/42MAPK, pJNK, and FAK [24 – 28]. Inhibition of PI3K and MAPK activity using specific inhibitors impaired epidermal growth factor (EGF)-stimulated cell migration of ovarian tumor cells [29]. In our study, we found that MDA-7 inhibited cell migration by downregulating the production of the p85, PI3K, and FAK proteins. Furthermore, the inhibitory effect exerted by MDA-7 was equivalent to that observed with the PI3K inhibitor LY294002. In support of these findings, Mhashilkar et al. [30] recently reported the inhibition of p85 PI3K by MDA-7 in human lung and breast cancer cells. Although LY294002 inhibited p85 PI3K and pFAK expression in A549 and H1299 cells, the inhibition of these molecules varied between the two cell lines. The cause for the observed difference is not clear. One possibility is that the concentration of LY294002 required to inhibit the expression of the two molecules may vary among the cell types. Another possibility is that the expression of pFAK in H1299 cells is regulated by additional mechanisms heretofore unrecognized. We are currently examining these possibilities in our laboratory. Examination of the regulation of additional signaling molecules, such p38MAPK, p44/42MAPK, and JNK, that have previously been shown to participate in cell migration demonstrated increased production of these proteins upon MDA-7 expression. Increased expression of these molecules was also observed when cells were treated with LY294002. These observations are somewhat surprising and suggest that these signaling pathways are uncoupled from of the PI3K pathway. Thus, MDA-7, like LY294002, selectively inhibits the PI3K pathway without affecting other signaling pathways, resulting in reduced migration. In addition to migration, tumor cells need to invade to establish metastasis successfully at a distant site. Tumor cell invasion involves the degradation of the extracellular matrix, and this destruction has been attributed to the activity of proteolytic enzymes [31,32]. Among the proteases implicated in tumor cell dissemination are MMPs [33], which play an important role in tissue remodeling in normal cells. However, the production of MMPs has been observed in many invasive tumor cell lines and during tumor growth [34]. In fact,
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the MMP levels appear to be prognostically significant during tumor progression in the breast, lung, and colon [35 – 37]. Evidence that MMPs are involved in invasion and angiogenesis in lung cancer comes from the observation that MMP-2 and -9 have been found in several lung cancer cell lines and surgical specimens [38], that MMP-2 and -9 proteins are localized to the tumor neovasculature [39], and that the extent of MMP overproduction correlates with prognosis [40]. On the basis of these reports, we next investigated the effect of Ad-mda7 on tumor cell invasion. In this study, we found that MDA-7 inhibited tumor cell invasion in a manner similar to that of LY294002 and MMP-II inhibitor. Furthermore, MDA-7-mediated inhibition of invasion occurred via the down-regulation of MMP-2 and -9. However, the exact mechanism by which MDA-7 inhibits MMP-2 and -9 is not clear. Microarray analyses performed on H1299 cells indicated that MMP-2 and -9 were down-regulated by Ad-mda7 (data not shown). Thus MDA-7 production may activate a signaling cascade resulting in regulation of transcription of MMPs. However, further studies are warranted to explore these possibilities. Whatever the underlying mechanism, the ability of Ad-mda7 to inhibit tumor cell invasion has been clearly shown in this study. Finally, the inhibition of cell migration and invasion by MDA-7 was investigated in an experimental lung metastasis model. Intravenous injection of tumor cells transfected with Ad-mda7 ex vivo into nude mice demonstrated a significantly reduced number of tumor metastases than injection of tumor cells transfected with AdLuc. The inhibition of tumor metastasis was also observed when tumor-bearing animals were treated with a liposomal vector carrying the MDA-7 plasmid DNA. Although the results of the present study establish a role for MDA-7 in inhibiting tumor progression and metastasis, the inhibitory effect observed in vivo may still not be sufficient to inhibit metastasis completely. Thus, it is envisioned that a combination therapy of Ad-mda7 with a PI3K or MMP inhibitor may produce a synergistic inhibitory effect on tumor cell metastasis. These studies are currently in progress in our laboratory. In conclusion, we have shown for the first time that MDA-7 inhibits cell migration and invasion by human lung tumor cells both in vitro and in vivo, resulting in reduced experimental metastasis. Thus, MDA-7 genebased drugs may provide a novel therapeutic strategy that can inhibit tumor growth directly via induction of apoptosis and also prevent tumor invasion and ultimately reduce metastasis.
MATERIALS AND METHODS Cell culture. Human NSCLC cell line A549 (adenocarcinoma) was purchased from the American Type Culture Collection (Rockville, MD, USA). Human large-cell lung carcinoma cell line H1299 was a gift from Drs. A.
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Gazdar and J. D. Minna (The University of Texas Southwestern Medical Center, Dallas, TX, USA). Tumor cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS; GIBCO BRL, Grand Island, NY, USA), antibiotics (GIBCO), and L-glutamine. Before the experiments were started, the absence of mycoplasma from the cells was verified. Cells were used in the log phase of growth. Recombinant adenoviral vector. Construction of the replication-deficient human type 5 adenoviral vector carrying the MDA-7 gene (Ad-mda7) has been previously described [6,7]. Viruses were propagated in human embryonic kidney 293 cells and purified by chromatography. Cell migration assay. Tumor cells (H1299 and A549) were seeded at a density of 5 105 cells/well in six-well tissue culture plates. The next day, cells were infected with Ad-mda7 or Ad-Luc at a multiplicity of infection (m.o.i.) of 2500 viral particles (vp)/cell. At 6 h after infection, the cells were trypsinized, washed in PBS, and resuspended in serumfree RPMI 1640 medium. A cell migration assay was performed in a 24well Transwell unit (Millipore, Cambridge, MA, USA), as described previously [15]. Briefly, polycarbonate filters with 8-Am pores were used. The lower chambers of the Transwell units were filled with serum-free medium, and the upper chambers were seeded with 1 104 cells from each treatment group in triplicate wells. After 24- and 48-h incubations, the cells that had passed through the filter into the lower wells were counted, and the number was expressed as a percentage of the sum of the cells in the upper and lower wells. The experiments were performed four times, and the results were recorded as the mean of these experiments. In a parallel set of experiments, tumor cells subjected to various treatments as described above were subjected to cell viability assays at 24 and 48 h, as described previously [6,7]. These experiments were performed to exclude the possibility that the inhibition of cell migration by MDA-7/IL-24 was a result of cytotoxicity. Cell invasion assay. Tumor cells (H1299 and A549) were seeded at 5 105 cells/well in 6-well tissue culture plates. The next day, cells were infected with Ad-mda7 or Ad-Luc at an m.o.i. of 2500 vp/cell or treated with 10 AM LY294002 (Cell Signaling, Beverly, MA, USA) or 1 Ag/ml MMP-II inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After transfection, cultures were replenished with complete medium. At 6 h after treatment, cells were trypsinized, washed in PBS, and resuspended in serum-free RPMI 1640 medium. A cell invasion assay was performed in a 24-well Transwell unit coated with Matrigel (Becton – Dickinson, Franklin Lakes, NJ, USA), as described previously [16]. Briefly, the lower chambers of the Matrigel-coated Transwell units were filled with serumfree medium, and the upper chambers were seeded with 1 104 cells from each treatment in triplicate wells. After 24- and 48-h incubations, the cells that had passed through the Matrigel-coated filter membrane into the lower well were counted as a measure of invasion. The invading cells were counted for each treatment and expressed as a percentage of the sum of the cells in the upper and lower wells. Experiments were performed at least three times, and the results were recorded as the mean of these experiments. Gelatin zymography analysis. To determine the effect of Ad-mda7 treatment on MMP production, a gelatin zymography assay was performed, as described previously [17]. Briefly, tumor cells (H1299 and A549) grown in low-serum (1% FBS) medium were seeded at 5 105 cells/well in six-well tissue culture plates and infected with Ad-mda7 or Ad-Luc at an m.o.i. of 2500 vp/cell. Cells treated with PBS served as a negative control in these experiments. At 6 h after infection, the culture medium was removed and replaced with fresh medium containing 1% FBS. At 24 and 48 h after infection, cell culture supernatants were collected, clarified by centrifugation, and subjected to electrophoresis in sodium dodecyl sulfate (SDS) – polyacrylamide gels copolymerized with gelatin (Sigma Chemicals, St. Louis, MO, USA). The gels were then washed and incubated with reaction buffer (50 mM Tris – HCl (pH 7.4), 0.02% NaN3, and 10 mM CaCl2) with constant shaking for 16 h at 37jC, stained, and destained. The protein concentration in the culture supernatant was measured to confirm that equal amounts were used for the assays. Relative
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activities of MMP-2 and -9 were quantified using ImageQuant software (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Immunoblotting. Immunoblotting using various antibodies was performed as described previously [6]. Briefly, cells were harvested by trypsinization and resuspended in lysis buffer (62.5 mM Tris – HCl, 2% SDS, 10% glycerol, and 4 M urea). Protein samples (50 Ag each) were diluted into a 20-Al solution of lysis buffer and 5% 2-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA, USA) and heated in a water bath at 95jC for 5 min. Then, protein extracts were separated by 10% SDS – polyacrylamide gel electrophoresis (SDS-PAGE) in a vertical-slab gel electrophoresis cell (Bio-Rad). Next, the separated proteins were transferred from the gel to a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech) and then blocked in a blocking solution (5% dry milk and 0.3% Tween 20 in PBS) for 1 h. Then, the membranes were incubated with the primary antibodies for MMP-2, MMP-9, and p85 PI3K (Santa Cruz Biotechnology), phosphorylated FAK (Pharmingen, San Diego, CA, USA), MDA-7 (Introgen Therapeutics, Inc., Houston, TX, USA), and h-actin (Sigma Chemicals). The membranes were then incubated with horseradish peroxidaselabeled secondary antibodies (Amersham). Finally, the proteins were visualized on enhanced chemiluminescence film (Hyperfilm; Amersham) using Amersham’s Enhanced Chemiluminescence Western Blotting Detection System. The relative changes in the protein expression levels after various treatments were quantified using ImageQuant software (Amersham Pharmacia Biotech) and expressed as a ratio with 1 being the value for PBS-treated cells. Experimental lung metastasis model. To determine whether MDA-7/IL24 production can inhibit metastasis, animal experiments were performed using an experimental lung metastasis model [18]. Briefly, A549 tumor cells (5 106) seeded in 150-mm tissue culture plates were treated with PBS, Ad-Luc, or Ad-mda7 (2500 vp/cell). At 6 h after infection, cells were harvested, washed, and resuspended to a final volume of 1 ml in sterile PBS (1 106 cells/100 Al). Cells were injected into female nude mice intravenously via the tail vein. There were seven animals in each treatment group. Three weeks after the injection of the cells, the animals were euthanized by CO2 inhalation. Lungs from five mice from the three groups were injected intratracheally with India ink and fixed in Fekete’s solution, as described previously [18]. The effect of MDA-7/IL-24 on tumor metastasis was determined by counting the number of metastatic tumors in each lung under a dissecting microscope. Lungs from the remaining two mice in each group were subjected to hematoxylin and eosin staining and examined for tumors. Experiments were performed two times, and the results were recorded as the mean of the two experiments. To eliminate the possibility that inhibition of tumor growth by Admda7 was due to cell killing, additional in vivo experiments were performed using an expression plasmid carrying the MDA-7 or chloramphenicol acetyl transferase (CAT) gene and a cationic DOTAP:Chol liposome vector. Experimental lung metastasis was established in nude mice by injecting 1 106 A549 cells via the tail vein. Ten days later, animals were divided into three groups and treated as follows: group 1 was not treated, group 2 was treated with DOTAP:Chol – CAT complex, and group 3 was treated with DOTAP:Chol – mda7 complex. Preparation of complex was carried out as previously described [18]. Animals were treated daily for a total of six doses (50 Ag/dose) via the tail vein. Animals were euthanized and examined for lung metastasis as described above [18]. Ability to inhibit lung metastasis by DOTAP:Chol – mda7 complex was considered a measure of inhibition of tumor cell migration and invasion. Statistical analysis. The statistical significance of the experimental results was calculated using ANOVA and the Mann – Whitney rank-sum test. The differences among groups were interpreted as statistically significant if the P value was less than 0.05.
ACKNOWLEDGMENTS The authors thank David Galloway for editorial assistance. This study was partially supported by a Texas Higher Education Coordinating Board ATP/ARP
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grant (003657-0078-2001), by an M. D. Anderson Cancer Center Institutional Research Grant, by M. D. Anderson Cancer Center Support Grant CA16672, by the W. M. Keck Foundation Fund for Human Cancer Gene Prevention and Therapy, by NCI Grant R43 CA86587, and by a sponsored research agreement with Introgen Therapeutics, Inc. RECEIVED FOR PUBLICATION AUGUST 14, 2003; ACCEPTED JANUARY 27, 2004.
REFERENCES 1. Jiang, H., et al. (1995). Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11: 2477 – 2486. 2. Jiang, H., et al. (1996). The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc. Natl. Acad. Sci. USA 93: 9160 – 9165. 3. Ellerhorst, J. A., et al. (2001). Loss of MDA-7 expression with progression of melanoma. J. Clin. Oncol. 15: 1069 – 1074. 4. Su, Z. Z., et al. (1998). The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc. Natl. Acad. Sci. USA 95: 14400 – 14405. 5. Madireddi, M. T., et al. (2000). Mda-7, a novel melanoma differentiation associated gene with promise for cancer gene therapy. In Cancer Gene Therapy: Past Achievements and Future Challenges (N. Habib, Ed.), pp. 239-261. Kluwer Academic/Plenum, New York. 6. Saeki, T., et al. (2000). Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther. 7: 2051 – 2057. 7. Mhashilkar, A., et al. (2001). Melanoma-differentiation associated gene-7 (Mda-7): A novel anti-tumor gene for cancer gene therapy. Mol. Med. 7: 271 – 282. 8. Saeki, T., et al. (2002). Inhibition of lung cancer growth following adenovirus mediated mda7 gene expression in vivo. Oncogene 21: 4558 – 4566. 9. Lebedeva, I. V., et al. (2002). The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene 21: 708 – 718. 10. Sarkar, D., et al. (2002). mda-7/IL-24 mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38MAPK. Proc. Natl. Acad. Sci. USA 99: 10054 – 10059. 11. Began, G., et al. (2003). Gene therapeutic approach for treatment of human ovarian cancer using mda-7/IL-24. Proc. Am. Assoc. Cancer Res. 44: 2382. 12. Saito, Y., et al. (2003). Selective induction of apoptosis and cell cycle arrest in prostate cancer cells by adenovirus-mediated mda-7 gene. Proc. Am. Assoc. Cancer Res. 44: 247. 13. Yacoub, A., et al. (2003). Mda-7 (IL-24) inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling. Cancer Biol. Ther. 2: 347 – 353. 14. Cao, X. X., et al. (2002). Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Mol. Med. 8: 869 – 876. 15. Ramesh, R., et al. (2003). The melanoma differentiation associated-7 gene (mda-7)/ interleukin-24 (IL-24) is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res. 63: 5105 – 5113. 16. Stewart, A., et al. (2002). PI-3 kinase blockade by Ad-PTEN inhibits invasion and induces apoptosis in RPG and metastatic melanoma cells. Mol. Med. 8: 451 – 461. 17. Zhang, Y., et al. (2002). A role for focal adhesion kinase in hyaluronan-dependent MMP-2 secretion in a human small-cell lung carcinoma cell line, QG90. Biochem. Biophys. Res. Commun. 290: 1123 – 1127. 18. Ramesh, R., et al. (2001). Successful treatment of primary and disseminated human lung cancer by systemic delivery of tumor suppressor genes using an improved liposome vector. Mol. Ther. 3: 337 – 350. 19. Chen, J., Chada, S., Mhashilkar, A., and Miano, J. M. (2003). Tumor suppressor MDA-7/ IL-24 selectively inhibits vascular smooth muscle cell growth and migration. Mol. Ther. 8: 207 – 219. 20. Deregibus, M. C., Buttiglieri, S., Russo, S., Bussolati, B., and Camussi, G. (2003). CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis. J. Biol. Chem. 278: 18008 – 18014. 21. Fan, J., and Malik, A. B. (2003). Toll-like receptor-4 (TLR4) signaling augments chemokine-induced neutrophil migration by modulating cell surface expression of chemokine receptors. Nat. Med. 9: 315 – 321. 22. Berens, M. E., et al. (1994). The role of extracellular matrix in human astrocytoma migration and proliferation studied in a microliter scale assay. Clin. Exp. Metastasis 12: 405 – 415. 23. Giese, A., et al. (1999). Thromboxane synthase regulates the migratory phenotype of human glioma cells. Neuro-Oncology 1: 3 – 13. 24. Wang, F., Nohara, K., Olivera, A., Thompson, E. W., and Speigel, S. (1999). Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphinosine 1-phosphate. Exp. Cell Res. 247: 17 – 28. 25. Neudauer, C. L., and McCarthy, J. B. (2003). Insulin-like growth factor I-stimulated
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ARTICLE
26.
27.
28.
29.
30.
31. 32.
melanoma cell migration requires phosphoinositide 3-kinase but not extracellular-regulated kinase activation. Exp. Cell Res. 286: 128 – 137. Goncharova, E. A., et al. (2002). PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 283: L354 – L363. Kadri, Z., et al. (2000). Erythropoietin induction of tissue inhibitors of metalloproteinase-1 expression and secretion is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. Cell Growth Differ. 11: 573 – 580. Lakka, S. S., et al. (2000). Regulation of MMP-9 (type IV collagenase) production and invasiveness in gliomas by the extracellular signal-regulated kinase and jun aminoterminal kinase signaling cascades. Clin. Exp. Metastasis 18: 245 – 252. Ellerbroek, S. M., et al. (2001). Phosphatidylinositol 3-kinase activity in epidermal growth factor-stimulated matrix metalloproteinase-9 production and cell surface association. Cancer Res. 61: 1855 – 1861. Mhashilkar, A., et al. (2003). MDA-7 regulates tumor suppressor gene activation and oncogene inactivation via h-catenin and PI3K pathways in breast and lung tumor cells. Mol. Ther. 8: 220 – 229. Goldfarb, J. R., and Liotta, L. A. (1986). Proteolytic enzymes in cancer invasion and metastasis. Semin. Thromb. Hemostasis 12: 294 – 307. Liotta, L. A. (1986). Tumor invasion and metastases—role of the extracellular matrix: Rhoads Memorial Award Lecture. Cancer Res. 46: 1 – 7.
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33. Matrisian, L. M. (1992). The matrix degrading metalloproteinases. Bioessays 14: 455 – 463. 34. Shapiro, S. D. (1998). Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr. Opin. Cell Biol. 10: 602 – 608. 35. Pritchard, S. C., et al. (2001). Expression of matrix metalloproteinases 1, 2, 9 and their tissue inhibitors in stage II non-small cell lung cancer: implications for MMP inhibition therapy. Oncol. Rep. 8: 421 – 424. 36. Nakopoulou, L., et al. (2003). MMP-2 protein in invasive breast cancer and the impact of MMP-2/TIMP-2 phenotype on overall survival. Breast Cancer Res. Treat. 77: 145 – 155. 37. Oberg, A., Hoyhtya, M., Tavelin, B., Stenling, R., and Lindmark, G. (2000). Limited value of preoperative serum analyses of matrix metalloproteinases (MMP-2, MMP-9) and tissue inhibitors of matrix metalloproteinases (TIMP-1, TIMP-2) in colorectal cancer. Anticancer Res. 20: 1085 – 1091. 38. Ohbayashi, H. (2002). Matrix metalloproteinases in lung diseases. Curr. Protein Pept. Sci. 3: 409 – 421. 39. Sienel, W., et al. (2003). Prognostic impact of matrix metalloproteinase-9 in operable non-small cell lung cancer. Int. J. Cancer 103: 647 – 651. 40. Herbst, R., et al. (2000). Differential expression of E-cadherin and type IV collagenase genes predicts outcome in patients with stage I non-small cell lung carcinoma. Clin. Cancer Res. 6: 790 – 797.
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Ectopic Production of MDA-7/IL-24 Inhibits Invasion and Migration of Human Lung Cancer Cells Rajagopal Ramesh,1,* Isao Ito,1 Began Gopalan,1 Yuji Saito,1 Abner M. Mhashilkar,2 and Sunil Chada2 1
Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA 2 Introgen Therapeutics, Inc., Houston, TX 77030, USA
*To whom correspondence and reprint requests should be addressed at the Department of Thoracic and Cardiovascular Surgery, Unit 445, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Fax: (713) 794-4901. E-mail: [email protected].
We have previously observed the suppression of lung tumor growth in response to overexpression of melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24; approved gene symbol IL24) in vitro and in vivo. MDA-7/IL-24 exerts its tumor-suppressive effects by multiple mechanisms, including the activation of the caspase cascade and the inhibition of angiogenesis. In this study, we used an adenoviral vector (Ad-mda7) to examine the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human non-small-cell lung carcinoma cells. Lung tumor cells (H1299 and A549) treated in vitro with Ad-mda7 migrated and invaded less than cells treated with phosphate-buffered saline (PBS) or Ad-Luc (vector control). MDA-7/IL-24 inhibited migration and invasion by down-regulating the production of phosphatidylinositol 3-kinase/protein kinase B, focal adhesion kinase, and matrix metalloproteinase-2 and -9 relative to PBS and Ad-Luc. Furthermore, tumor cells treated with Ad-mda7 ex vivo or with DOTAP:Chol – mda7 complex in vivo formed significantly fewer tumors in an experimental lung metastasis model. These results show that MDA-7/IL-24 inhibits invasion and migration by lung cancer cells by down-regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis. Key Words: MDA-7, IL-24, gene therapy, MMP, PI3K, FAK, invasion, metastasis, lung, cancer
INTRODUCTION The melanoma differentiation-associated gene-7 (MDA7; approved gene symbol IL24) was identified from melanoma cells that were allowed to differentiate after treatment with interferon-h and mezerin [1]. MDA-7 was reported to be a novel tumor-suppressor gene whose expression was lost during tumorigenesis [1]. Expression of MDA-7 mRNA was reported in normal melanocytes and in melanomas that were allowed to differentiate [1,2]. On the basis of these observations, it was speculated that the MDA-7 protein plays a role in maintaining the normal physiology of melanocytes and that the inhibition of its production results in transformation and progression from a local, nonmetastatic primary tumor to highly metastatic cancer. Support for this concept was provided by a recent study by Ellerhorst et al. [3], who showed that the MDA-7 protein is pro-
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duced in human melanocytes and in primary melanomas but that its production is progressively lost in metastatic melanomas. Strikingly, the authors of this study noted a significant correlation between loss of MDA-7 production and melanoma tumor invasion. They identified specimens in which the superficial layer of tumor was MDA-7 positive, yet MDA-7 production was lost as the tumor invaded the surrounding tissue. Thus, the restoration of MDA-7 production in tumor cells should inhibit tumor growth and tumor invasion and metastasis. Studies by us and several other investigators have shown that the overproduction of MDA-7 selectively inhibited cancer cell growth of diverse origins by inducing apoptosis with minimal toxicity to normal cells both in vitro and in vivo [4 – 14]. However, apart from the findings of Ellerhorst et al. [3], no additional preclinical or clinical
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FIG. 1. MDA-7/IL-24 inhibits tumor cell migration. (A) A549 and (B) H1299 lung tumor cells were treated with PBS, Ad-Luc, or Ad-mda7 and analyzed for cell migration, cell viability, and apoptosis at 24 and 48 h after treatment. Cells treated with Ad-mda7 were significantly ( P = 0.002) less able to migrate than cells treated with PBS or Ad-Luc. That inhibition of cell migration was not due to cytotoxicity was determined by cell viability assay. No significant inhibition of tumor cell proliferation was observed in Ad-mda7-treated cells compared to PBS- and Ad-Luc-treated cells in both cell lines. (C) Analysis for cells in sub-G0 phase of cell cycle, an indicator of apoptotic cells. Bars denote standard error.
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data exist showing a role for MDA-7/IL-24 in regulating tumor metastasis. Furthermore, the underlying mechanism by which MDA-7 might inhibit tumor metastasis is unknown. We therefore investigated the effects of the ectopic production of MDA-7/IL-24 on migration and invasion by human non-small-cell lung carcinoma (NSCLC) cells in vitro and in vivo and attempted to identify the underlying mechanisms of metastasis suppression. In this study, we show for the first time that MDA-7 inhibits the migration and invasion of human lung cancer cells in vitro by down-regulating the protein production of phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and matrix metalloproteinases (MMPs). Furthermore, we confirmed in vivo of the ability of MDA-7 to reduce experimental metastasis. Thus, MDA7/IL-24 may provide multiple benefits as an anti-cancer strategy since it can inhibit primary tumor growth and prevent metastatic spread.
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inhibits tumor cell migration and that the inhibitory effect is not due to cytotoxicity. MDA-7 Inhibits Tumor Cell Invasion Tumor cells (A549 and H1299) treated with Ad-mda7 were much less invasive, as indicated by the small number of cells on the outer membrane of the Matrigel invasion assay filter, than cells treated with PBS or AdLuc (Figs. 2A and 2B). In both A549 and H1299 cells, the
RESULTS MDA-7/IL-24 Inhibits Tumor Cell Migration In the present study we tested the ability of Ad-mda7 to inhibit cell migration on two lung cancer cell lines that are wild type for p53 (A549) and null for p53 (H1299). Tumor cells treated with Ad-mda7 were significantly ( P = 0.002) less able to migrate than were cells treated with Ad-Luc or phosphate-buffered saline (PBS) (Figs. 1A and 1B). We also observed inhibition of migration in Ad-luctreated H1299 cells at 48 h compared to PBS-treated cells. However, the degree of inhibition induced by Ad-luc was as not effective as that induced by Ad-mda7. The inhibitory effect on cell migration occurred as early as 24 h in both tumor cell lines (Figs. 1A and 1B). To show that the inhibition of cell migration was not due to MDA-7/IL-24mediated cell death, in a separate but parallel set of experiments, we subjected cells treated with PBS, AdLuc, and Ad-mda7 to a cell viability assay at 24 and 48 h after infection. We observed no significant difference in cell viability at these time points, indicating that the inhibition of migration by MDA-7/IL-24 was not due to cell death (Figs. 1A and 1B). Note that at 24 h posttransduction, all three experimental groups are superimposable, indicating no significant cell death. By 48 h, some cell death is occurring; however, with this vector dose, significant Ad-mda7-mediated death is observed only at 72 and 96 h posttransduction. Additionally, flow cytometric analysis demonstrated no significant increase in the number of cells in sub-G0 phase of the cell cycle in Ad-mda7-treated cells (4 – 6%) compared to Ad-luc-treated cells (4.3 – 6%) at 24 h (Fig. 1C). However, we observed a slight increase in cell number in the sub-G0 phase in Ad-mda7-treated cells (9 – 13%) compared to Ad-luc-treated cells (7 – 10%) at 48 h. The increase in cell number was higher in Ad-mda7-treated H1299 cells than in A549 cells. These results show that MDA-7/IL-24
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FIG. 2. MDA-7/IL-24 inhibits tumor cell invasion. (A) A549 and (B) H1299 tumor cells were treated with PBS, Ad-Luc, Ad-mda7, 10 AM LY294002, or 1 Ag/ml MMP-II inhibitor. After 6 h, cells were harvested, counted, and added into the upper wells of Matrigel-coated wells. Cells were allowed to invade by incubation at 37jC. At 24 and 48 h after treatment, cells were fixed and stained with crystal violet. Cells that migrated to the lower side of the well were observed and counted under a light microscope at 200 magnification. The number of invading cells per treatment was counted in a blind fashion and recorded as the average of three separate experiments. Cells treated with Ad-mda7 showed less invasion ( P = 0.001) than cells treated with PBS or AdLuc. The inhibitory effect mediated by MDA-7 was similar to the inhibitory effect observed with LY294002 and MMP-II inhibitor. Bars denote standard error.
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FIG. 3. MDA-7/IL-24 down-regulates production of proteins associated with migration and invasion. Tumor cells (A549 and H1299) were treated with PBS, Ad-Luc, Ad-mda7, or LY294002. Cells were harvested at 48 h, total cell lysates were prepared, and proteins were separated by SDS – PAGE. Proteins were blotted using antibodies against MDA-7, p85 PI3K, pJNK, p38MAPK, p44/42MAPK, and pFAK. h-Actin was used as a loading control. The relative change in the protein expression was expressed as a ratio with the value for PBS-treated cells arbitrarily set to 1.
number of invading cells was significantly less after treatment with Ad-mda7 ( P = 0.001) than with PBS or Ad-Luc. We observed the inhibitory effect exerted by MDA-7/IL-24 as early as 24 h after Ad-mda7 treatment. Furthermore, MDA-7/IL-24-mediated inhibitory effect was similar to the inhibitory effect observed in cells treated with LY294002, a PI3K inhibitor, or with MMPII inhibitor. A cell viability assay showed that the inhibition was not a result of MDA-7/IL-24-mediated cell death (data not shown). These results show that MDA-7/IL-24 effectively inhibits cell invasion.
MDA-7 Down-regulates the Production of Proteins Associated with Cell Migration and Invasion We next examined the regulation of proteins that are associated with cell migration and invasion signaling pathways by Western blot analysis. We did not observe MDA-7/IL-24 production in PBS- or Ad-Luc-treated tumor cells, but high levels of MDA-7/IL-24 production were found in Ad-mda7-treated cells (Fig. 3). Overproduction of the MDA-7/IL-24 protein in both H1299 and A549 tumor cell lines resulted in decreased production of p85 PI3K and pFAK and increased production of pJNK,
FIG. 4. MDA-7/IL-24 inhibits MMP-2 and -9 production. Tumor cells were treated with PBS, Ad-Luc, or Ad-mda7. At 24 and 48 h after treatment, cell supernatant and cell lysate were collected and analyzed for MMP2 and -9 by zymography and Western blot analysis. (A) Inhibition of MMP-2 and -9 was observed in A549 cells treated with Admda7. Inhibition was shown by both zymography (top) and Western blot analyses (bottom). No change in MMP-2 or -9 was observed in cells treated with PBS or Ad-Luc. (B) Ad-mda7 inhibited MMP-2 but not MMP-9 production in H1299 cells. Inhibition of MMP-2 was observed on both zymography (top) and Western blot analyses (bottom).
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p38MAPK, and p44/42MAPK compared to PBS- or Ad-Luctreated cells (Fig. 3). In H1299 cells, treatment with AdLuc resulted in increased expression of p85 PI3K, pJNK, and pFAK compared to PBS-treated cells. We also observed the inhibition of p85 PI3K and pFAK in cells treated with the PI3K inhibitor LY294002, although the inhibition differed between the two cell lines. The decrease in pFAK expression in cell lines overproducing MDA-7 was greater than that observed with LY294004. Furthermore, LY294002 treatment resulted in increased production of p38MAPK and p44/42MAPK in H1299 cells. In A549 cells,
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LY294004 increased the production of pJNK and p44/ 42MAPK. These results indicate that MDA-7, like LY294002, effectively inhibits PI3K and pFAK while increasing the expression of other signaling proteins. Inhibition of Matrix Metalloproteinase Production in Tumor Cells by MDA-7/IL-24 We next examined tumor cells overproducing MDA-7 for MMP regulation by zymography and Western blot analysis. Zymography and Western blot analysis showed that production of the MMP-2 and -9 proteins was decreased
FIG. 5. MDA-7/IL-24 inhibits lung metastases. (A) A549 lung tumor cells were treated with PBS, Ad-Luc, and Ad-mda7 ex vivo. After 6 h, cells were harvested, washed, resuspended in PBS, and injected into female nude mice via the tail vein. Three weeks after the injection of the tumor cells, the animals were euthanized by CO2 inhalation, and the presence of lung tumors was determined by histopathologic examination (top; representative tumor indicated by circles) and the number of tumors determined by counting tumor nodules under a dissecting microscope (bottom). Significantly ( P = 0.01) fewer lung tumors were observed in animals injected with tumor cells treated with Ad-mda7 than in animals injected with tumor cells treated with PBS or Ad-Luc. Results are the means of two separate experiments. Bars denote standard error. (B) Mice bearing experimental A549 lung metastases were either untreated (control) or treated with DOTAP:Chol – CAT or DOTAP:Chol – mda7 complex. Animals were treated daily for six doses via tail vein injection. Three weeks after the last treatment animals were euthanized and the number of lung tumors was counted. Animals treated with DOTAP:Chol – mda7 complex demonstrated significant inhibition of lung metastasis compared to animals that were untreated or treated with DOTAP:Chol – CAT complex ( P = 0.001). Bars denote standard error.
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in A549 tumor cells treated with Ad-mda7, compared with cells that were treated with PBS or Ad-Luc (Fig. 4A). In H1299 cells treated with Ad-mda7, we observed a decrease in MMP-2 but not in MMP-9 compared with cells treated with PBS or Ad-Luc (Fig. 4B). Results of zymography analysis correlated with the results of the Western blot analysis (Figs. 4A and 4B). Thus Ad-mda7 can modulate MMP expression and activity in both p53 wild-type and p53-null NSCLC lines. MDA-7/IL-24 Inhibits Experimental Lung Metastases In an experimental lung metastasis model using A549 human lung cancer cells in nude mice, significantly ( P = 0.01) fewer lung tumor nodules formed in mice injected with tumor cells treated with Ad-mda7 than in mice injected with tumor cells treated with PBS or Ad-Luc (Fig. 5A). Inhibition of experimental metastasis correlated with histological tissue staining that demonstrated smaller number of tumors. To confirm further that inhibition of experimental metastasis was not due solely to cell death, we carried out additional in vivo experiments. Treatment of lung tumor-bearing animals with DOTAP:cholesterol (DOTAP:Chol) – mda7 resulted in significant inhibition ( P = 0.001) of experimental metastasis compared to mice that were untreated or treated with DOTAP:Chol – CAT complex (Fig. 5B). The ability to inhibit experimental metastasis is considered an indirect correlate of inhibition of migration and invasion of tumor cells. These results demonstrate that MDA-7 inhibits tumor cell migration and invasion in vivo and is in agreement with our observed in vitro results.
DISCUSSION In this study, we show for the first time that MDA-7 can inhibit the migration and invasion of human NSCLC cells in vitro and in vivo. In vitro, tumor cells treated with Ad-mda7 were significantly inhibited from growth factorinduced migration. Protein production analysis showed that the inhibition of cell migration was due to MDA-7. The ability of MDA-7 to inhibit tumor cell migration is not surprising and is consistent with our recent findings in which inhibition of normal endothelial cell migration by recombinant MDA-7 (rMDA-7) protein was demonstrated [15]. It could be argued that the observed inhibition of cell migration was due to the cytotoxicity of Admda7 as opposed to rMDA-7; however, that was ruled out by a cell proliferation assay. Note that at the doses and time frames employed in this study, more than 90% of the cells were viable. The ability of Ad-mda7 to inhibit cell migration of lung tumor cells supports the findings of Chen et al. [19], who recently reported the inhibition of rat smooth muscle cell (SMC) migration by Ad-mda7. Furthermore, in that study, the inhibition of cell migration was found to be independent of the antitumor
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activity of Ad-mda7. These results show that Ad-mda7 can inhibit tumor cell migration in vitro. Although the ability of Ad-mda7 to inhibit SMC migration has been shown previously [19], the mechanism by which MDA-7 inhibits cell migration was not studied. Cell migration is an important element in the physiology of a normal cell and of a cell in pathologic states. For example, the migration of inflammatory cells to a site of injury is a requisite for wound healing [20,21]. Similarly, the migration of tumor cells to distant organ sites plays a pivotal role in metastasis [22,23]. Cell migration has previously been shown to be regulated by numerous molecules, including PI3K, p38MAPK, p44/42MAPK, pJNK, and FAK [24 – 28]. Inhibition of PI3K and MAPK activity using specific inhibitors impaired epidermal growth factor (EGF)-stimulated cell migration of ovarian tumor cells [29]. In our study, we found that MDA-7 inhibited cell migration by downregulating the production of the p85, PI3K, and FAK proteins. Furthermore, the inhibitory effect exerted by MDA-7 was equivalent to that observed with the PI3K inhibitor LY294002. In support of these findings, Mhashilkar et al. [30] recently reported the inhibition of p85 PI3K by MDA-7 in human lung and breast cancer cells. Although LY294002 inhibited p85 PI3K and pFAK expression in A549 and H1299 cells, the inhibition of these molecules varied between the two cell lines. The cause for the observed difference is not clear. One possibility is that the concentration of LY294002 required to inhibit the expression of the two molecules may vary among the cell types. Another possibility is that the expression of pFAK in H1299 cells is regulated by additional mechanisms heretofore unrecognized. We are currently examining these possibilities in our laboratory. Examination of the regulation of additional signaling molecules, such p38MAPK, p44/42MAPK, and JNK, that have previously been shown to participate in cell migration demonstrated increased production of these proteins upon MDA-7 expression. Increased expression of these molecules was also observed when cells were treated with LY294002. These observations are somewhat surprising and suggest that these signaling pathways are uncoupled from of the PI3K pathway. Thus, MDA-7, like LY294002, selectively inhibits the PI3K pathway without affecting other signaling pathways, resulting in reduced migration. In addition to migration, tumor cells need to invade to establish metastasis successfully at a distant site. Tumor cell invasion involves the degradation of the extracellular matrix, and this destruction has been attributed to the activity of proteolytic enzymes [31,32]. Among the proteases implicated in tumor cell dissemination are MMPs [33], which play an important role in tissue remodeling in normal cells. However, the production of MMPs has been observed in many invasive tumor cell lines and during tumor growth [34]. In fact,
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the MMP levels appear to be prognostically significant during tumor progression in the breast, lung, and colon [35 – 37]. Evidence that MMPs are involved in invasion and angiogenesis in lung cancer comes from the observation that MMP-2 and -9 have been found in several lung cancer cell lines and surgical specimens [38], that MMP-2 and -9 proteins are localized to the tumor neovasculature [39], and that the extent of MMP overproduction correlates with prognosis [40]. On the basis of these reports, we next investigated the effect of Ad-mda7 on tumor cell invasion. In this study, we found that MDA-7 inhibited tumor cell invasion in a manner similar to that of LY294002 and MMP-II inhibitor. Furthermore, MDA-7-mediated inhibition of invasion occurred via the down-regulation of MMP-2 and -9. However, the exact mechanism by which MDA-7 inhibits MMP-2 and -9 is not clear. Microarray analyses performed on H1299 cells indicated that MMP-2 and -9 were down-regulated by Ad-mda7 (data not shown). Thus MDA-7 production may activate a signaling cascade resulting in regulation of transcription of MMPs. However, further studies are warranted to explore these possibilities. Whatever the underlying mechanism, the ability of Ad-mda7 to inhibit tumor cell invasion has been clearly shown in this study. Finally, the inhibition of cell migration and invasion by MDA-7 was investigated in an experimental lung metastasis model. Intravenous injection of tumor cells transfected with Ad-mda7 ex vivo into nude mice demonstrated a significantly reduced number of tumor metastases than injection of tumor cells transfected with AdLuc. The inhibition of tumor metastasis was also observed when tumor-bearing animals were treated with a liposomal vector carrying the MDA-7 plasmid DNA. Although the results of the present study establish a role for MDA-7 in inhibiting tumor progression and metastasis, the inhibitory effect observed in vivo may still not be sufficient to inhibit metastasis completely. Thus, it is envisioned that a combination therapy of Ad-mda7 with a PI3K or MMP inhibitor may produce a synergistic inhibitory effect on tumor cell metastasis. These studies are currently in progress in our laboratory. In conclusion, we have shown for the first time that MDA-7 inhibits cell migration and invasion by human lung tumor cells both in vitro and in vivo, resulting in reduced experimental metastasis. Thus, MDA-7 genebased drugs may provide a novel therapeutic strategy that can inhibit tumor growth directly via induction of apoptosis and also prevent tumor invasion and ultimately reduce metastasis.
MATERIALS AND METHODS Cell culture. Human NSCLC cell line A549 (adenocarcinoma) was purchased from the American Type Culture Collection (Rockville, MD, USA). Human large-cell lung carcinoma cell line H1299 was a gift from Drs. A.
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Gazdar and J. D. Minna (The University of Texas Southwestern Medical Center, Dallas, TX, USA). Tumor cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS; GIBCO BRL, Grand Island, NY, USA), antibiotics (GIBCO), and L-glutamine. Before the experiments were started, the absence of mycoplasma from the cells was verified. Cells were used in the log phase of growth. Recombinant adenoviral vector. Construction of the replication-deficient human type 5 adenoviral vector carrying the MDA-7 gene (Ad-mda7) has been previously described [6,7]. Viruses were propagated in human embryonic kidney 293 cells and purified by chromatography. Cell migration assay. Tumor cells (H1299 and A549) were seeded at a density of 5 105 cells/well in six-well tissue culture plates. The next day, cells were infected with Ad-mda7 or Ad-Luc at a multiplicity of infection (m.o.i.) of 2500 viral particles (vp)/cell. At 6 h after infection, the cells were trypsinized, washed in PBS, and resuspended in serumfree RPMI 1640 medium. A cell migration assay was performed in a 24well Transwell unit (Millipore, Cambridge, MA, USA), as described previously [15]. Briefly, polycarbonate filters with 8-Am pores were used. The lower chambers of the Transwell units were filled with serum-free medium, and the upper chambers were seeded with 1 104 cells from each treatment group in triplicate wells. After 24- and 48-h incubations, the cells that had passed through the filter into the lower wells were counted, and the number was expressed as a percentage of the sum of the cells in the upper and lower wells. The experiments were performed four times, and the results were recorded as the mean of these experiments. In a parallel set of experiments, tumor cells subjected to various treatments as described above were subjected to cell viability assays at 24 and 48 h, as described previously [6,7]. These experiments were performed to exclude the possibility that the inhibition of cell migration by MDA-7/IL-24 was a result of cytotoxicity. Cell invasion assay. Tumor cells (H1299 and A549) were seeded at 5 105 cells/well in 6-well tissue culture plates. The next day, cells were infected with Ad-mda7 or Ad-Luc at an m.o.i. of 2500 vp/cell or treated with 10 AM LY294002 (Cell Signaling, Beverly, MA, USA) or 1 Ag/ml MMP-II inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After transfection, cultures were replenished with complete medium. At 6 h after treatment, cells were trypsinized, washed in PBS, and resuspended in serum-free RPMI 1640 medium. A cell invasion assay was performed in a 24-well Transwell unit coated with Matrigel (Becton – Dickinson, Franklin Lakes, NJ, USA), as described previously [16]. Briefly, the lower chambers of the Matrigel-coated Transwell units were filled with serumfree medium, and the upper chambers were seeded with 1 104 cells from each treatment in triplicate wells. After 24- and 48-h incubations, the cells that had passed through the Matrigel-coated filter membrane into the lower well were counted as a measure of invasion. The invading cells were counted for each treatment and expressed as a percentage of the sum of the cells in the upper and lower wells. Experiments were performed at least three times, and the results were recorded as the mean of these experiments. Gelatin zymography analysis. To determine the effect of Ad-mda7 treatment on MMP production, a gelatin zymography assay was performed, as described previously [17]. Briefly, tumor cells (H1299 and A549) grown in low-serum (1% FBS) medium were seeded at 5 105 cells/well in six-well tissue culture plates and infected with Ad-mda7 or Ad-Luc at an m.o.i. of 2500 vp/cell. Cells treated with PBS served as a negative control in these experiments. At 6 h after infection, the culture medium was removed and replaced with fresh medium containing 1% FBS. At 24 and 48 h after infection, cell culture supernatants were collected, clarified by centrifugation, and subjected to electrophoresis in sodium dodecyl sulfate (SDS) – polyacrylamide gels copolymerized with gelatin (Sigma Chemicals, St. Louis, MO, USA). The gels were then washed and incubated with reaction buffer (50 mM Tris – HCl (pH 7.4), 0.02% NaN3, and 10 mM CaCl2) with constant shaking for 16 h at 37jC, stained, and destained. The protein concentration in the culture supernatant was measured to confirm that equal amounts were used for the assays. Relative
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activities of MMP-2 and -9 were quantified using ImageQuant software (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Immunoblotting. Immunoblotting using various antibodies was performed as described previously [6]. Briefly, cells were harvested by trypsinization and resuspended in lysis buffer (62.5 mM Tris – HCl, 2% SDS, 10% glycerol, and 4 M urea). Protein samples (50 Ag each) were diluted into a 20-Al solution of lysis buffer and 5% 2-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA, USA) and heated in a water bath at 95jC for 5 min. Then, protein extracts were separated by 10% SDS – polyacrylamide gel electrophoresis (SDS-PAGE) in a vertical-slab gel electrophoresis cell (Bio-Rad). Next, the separated proteins were transferred from the gel to a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech) and then blocked in a blocking solution (5% dry milk and 0.3% Tween 20 in PBS) for 1 h. Then, the membranes were incubated with the primary antibodies for MMP-2, MMP-9, and p85 PI3K (Santa Cruz Biotechnology), phosphorylated FAK (Pharmingen, San Diego, CA, USA), MDA-7 (Introgen Therapeutics, Inc., Houston, TX, USA), and h-actin (Sigma Chemicals). The membranes were then incubated with horseradish peroxidaselabeled secondary antibodies (Amersham). Finally, the proteins were visualized on enhanced chemiluminescence film (Hyperfilm; Amersham) using Amersham’s Enhanced Chemiluminescence Western Blotting Detection System. The relative changes in the protein expression levels after various treatments were quantified using ImageQuant software (Amersham Pharmacia Biotech) and expressed as a ratio with 1 being the value for PBS-treated cells. Experimental lung metastasis model. To determine whether MDA-7/IL24 production can inhibit metastasis, animal experiments were performed using an experimental lung metastasis model [18]. Briefly, A549 tumor cells (5 106) seeded in 150-mm tissue culture plates were treated with PBS, Ad-Luc, or Ad-mda7 (2500 vp/cell). At 6 h after infection, cells were harvested, washed, and resuspended to a final volume of 1 ml in sterile PBS (1 106 cells/100 Al). Cells were injected into female nude mice intravenously via the tail vein. There were seven animals in each treatment group. Three weeks after the injection of the cells, the animals were euthanized by CO2 inhalation. Lungs from five mice from the three groups were injected intratracheally with India ink and fixed in Fekete’s solution, as described previously [18]. The effect of MDA-7/IL-24 on tumor metastasis was determined by counting the number of metastatic tumors in each lung under a dissecting microscope. Lungs from the remaining two mice in each group were subjected to hematoxylin and eosin staining and examined for tumors. Experiments were performed two times, and the results were recorded as the mean of the two experiments. To eliminate the possibility that inhibition of tumor growth by Admda7 was due to cell killing, additional in vivo experiments were performed using an expression plasmid carrying the MDA-7 or chloramphenicol acetyl transferase (CAT) gene and a cationic DOTAP:Chol liposome vector. Experimental lung metastasis was established in nude mice by injecting 1 106 A549 cells via the tail vein. Ten days later, animals were divided into three groups and treated as follows: group 1 was not treated, group 2 was treated with DOTAP:Chol – CAT complex, and group 3 was treated with DOTAP:Chol – mda7 complex. Preparation of complex was carried out as previously described [18]. Animals were treated daily for a total of six doses (50 Ag/dose) via the tail vein. Animals were euthanized and examined for lung metastasis as described above [18]. Ability to inhibit lung metastasis by DOTAP:Chol – mda7 complex was considered a measure of inhibition of tumor cell migration and invasion. Statistical analysis. The statistical significance of the experimental results was calculated using ANOVA and the Mann – Whitney rank-sum test. The differences among groups were interpreted as statistically significant if the P value was less than 0.05.
ACKNOWLEDGMENTS The authors thank David Galloway for editorial assistance. This study was partially supported by a Texas Higher Education Coordinating Board ATP/ARP
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grant (003657-0078-2001), by an M. D. Anderson Cancer Center Institutional Research Grant, by M. D. Anderson Cancer Center Support Grant CA16672, by the W. M. Keck Foundation Fund for Human Cancer Gene Prevention and Therapy, by NCI Grant R43 CA86587, and by a sponsored research agreement with Introgen Therapeutics, Inc. RECEIVED FOR PUBLICATION AUGUST 14, 2003; ACCEPTED JANUARY 27, 2004.
REFERENCES 1. Jiang, H., et al. (1995). Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11: 2477 – 2486. 2. Jiang, H., et al. (1996). The melanoma differentiation associated gene mda-7 suppresses cancer cell growth. Proc. Natl. Acad. Sci. USA 93: 9160 – 9165. 3. Ellerhorst, J. A., et al. (2001). Loss of MDA-7 expression with progression of melanoma. J. Clin. Oncol. 15: 1069 – 1074. 4. Su, Z. Z., et al. (1998). The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc. Natl. Acad. Sci. USA 95: 14400 – 14405. 5. Madireddi, M. T., et al. (2000). Mda-7, a novel melanoma differentiation associated gene with promise for cancer gene therapy. In Cancer Gene Therapy: Past Achievements and Future Challenges (N. Habib, Ed.), pp. 239-261. Kluwer Academic/Plenum, New York. 6. Saeki, T., et al. (2000). Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro. Gene Ther. 7: 2051 – 2057. 7. Mhashilkar, A., et al. (2001). Melanoma-differentiation associated gene-7 (Mda-7): A novel anti-tumor gene for cancer gene therapy. Mol. Med. 7: 271 – 282. 8. Saeki, T., et al. (2002). Inhibition of lung cancer growth following adenovirus mediated mda7 gene expression in vivo. Oncogene 21: 4558 – 4566. 9. Lebedeva, I. V., et al. (2002). The cancer growth suppressing gene mda-7 induces apoptosis selectively in human melanoma cells. Oncogene 21: 708 – 718. 10. Sarkar, D., et al. (2002). mda-7/IL-24 mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38MAPK. Proc. Natl. Acad. Sci. USA 99: 10054 – 10059. 11. Began, G., et al. (2003). Gene therapeutic approach for treatment of human ovarian cancer using mda-7/IL-24. Proc. Am. Assoc. Cancer Res. 44: 2382. 12. Saito, Y., et al. (2003). Selective induction of apoptosis and cell cycle arrest in prostate cancer cells by adenovirus-mediated mda-7 gene. Proc. Am. Assoc. Cancer Res. 44: 247. 13. Yacoub, A., et al. (2003). Mda-7 (IL-24) inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling. Cancer Biol. Ther. 2: 347 – 353. 14. Cao, X. X., et al. (2002). Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Mol. Med. 8: 869 – 876. 15. Ramesh, R., et al. (2003). The melanoma differentiation associated-7 gene (mda-7)/ interleukin-24 (IL-24) is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res. 63: 5105 – 5113. 16. Stewart, A., et al. (2002). PI-3 kinase blockade by Ad-PTEN inhibits invasion and induces apoptosis in RPG and metastatic melanoma cells. Mol. Med. 8: 451 – 461. 17. Zhang, Y., et al. (2002). A role for focal adhesion kinase in hyaluronan-dependent MMP-2 secretion in a human small-cell lung carcinoma cell line, QG90. Biochem. Biophys. Res. Commun. 290: 1123 – 1127. 18. Ramesh, R., et al. (2001). Successful treatment of primary and disseminated human lung cancer by systemic delivery of tumor suppressor genes using an improved liposome vector. Mol. Ther. 3: 337 – 350. 19. Chen, J., Chada, S., Mhashilkar, A., and Miano, J. M. (2003). Tumor suppressor MDA-7/ IL-24 selectively inhibits vascular smooth muscle cell growth and migration. Mol. Ther. 8: 207 – 219. 20. Deregibus, M. C., Buttiglieri, S., Russo, S., Bussolati, B., and Camussi, G. (2003). CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis. J. Biol. Chem. 278: 18008 – 18014. 21. Fan, J., and Malik, A. B. (2003). Toll-like receptor-4 (TLR4) signaling augments chemokine-induced neutrophil migration by modulating cell surface expression of chemokine receptors. Nat. Med. 9: 315 – 321. 22. Berens, M. E., et al. (1994). The role of extracellular matrix in human astrocytoma migration and proliferation studied in a microliter scale assay. Clin. Exp. Metastasis 12: 405 – 415. 23. Giese, A., et al. (1999). Thromboxane synthase regulates the migratory phenotype of human glioma cells. Neuro-Oncology 1: 3 – 13. 24. Wang, F., Nohara, K., Olivera, A., Thompson, E. W., and Speigel, S. (1999). Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphinosine 1-phosphate. Exp. Cell Res. 247: 17 – 28. 25. Neudauer, C. L., and McCarthy, J. B. (2003). Insulin-like growth factor I-stimulated
517
ARTICLE
26.
27.
28.
29.
30.
31. 32.
melanoma cell migration requires phosphoinositide 3-kinase but not extracellular-regulated kinase activation. Exp. Cell Res. 286: 128 – 137. Goncharova, E. A., et al. (2002). PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 283: L354 – L363. Kadri, Z., et al. (2000). Erythropoietin induction of tissue inhibitors of metalloproteinase-1 expression and secretion is mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. Cell Growth Differ. 11: 573 – 580. Lakka, S. S., et al. (2000). Regulation of MMP-9 (type IV collagenase) production and invasiveness in gliomas by the extracellular signal-regulated kinase and jun aminoterminal kinase signaling cascades. Clin. Exp. Metastasis 18: 245 – 252. Ellerbroek, S. M., et al. (2001). Phosphatidylinositol 3-kinase activity in epidermal growth factor-stimulated matrix metalloproteinase-9 production and cell surface association. Cancer Res. 61: 1855 – 1861. Mhashilkar, A., et al. (2003). MDA-7 regulates tumor suppressor gene activation and oncogene inactivation via h-catenin and PI3K pathways in breast and lung tumor cells. Mol. Ther. 8: 220 – 229. Goldfarb, J. R., and Liotta, L. A. (1986). Proteolytic enzymes in cancer invasion and metastasis. Semin. Thromb. Hemostasis 12: 294 – 307. Liotta, L. A. (1986). Tumor invasion and metastases—role of the extracellular matrix: Rhoads Memorial Award Lecture. Cancer Res. 46: 1 – 7.
518
doi:10.1016/j.ymthe.2004.01.019
33. Matrisian, L. M. (1992). The matrix degrading metalloproteinases. Bioessays 14: 455 – 463. 34. Shapiro, S. D. (1998). Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr. Opin. Cell Biol. 10: 602 – 608. 35. Pritchard, S. C., et al. (2001). Expression of matrix metalloproteinases 1, 2, 9 and their tissue inhibitors in stage II non-small cell lung cancer: implications for MMP inhibition therapy. Oncol. Rep. 8: 421 – 424. 36. Nakopoulou, L., et al. (2003). MMP-2 protein in invasive breast cancer and the impact of MMP-2/TIMP-2 phenotype on overall survival. Breast Cancer Res. Treat. 77: 145 – 155. 37. Oberg, A., Hoyhtya, M., Tavelin, B., Stenling, R., and Lindmark, G. (2000). Limited value of preoperative serum analyses of matrix metalloproteinases (MMP-2, MMP-9) and tissue inhibitors of matrix metalloproteinases (TIMP-1, TIMP-2) in colorectal cancer. Anticancer Res. 20: 1085 – 1091. 38. Ohbayashi, H. (2002). Matrix metalloproteinases in lung diseases. Curr. Protein Pept. Sci. 3: 409 – 421. 39. Sienel, W., et al. (2003). Prognostic impact of matrix metalloproteinase-9 in operable non-small cell lung cancer. Int. J. Cancer 103: 647 – 651. 40. Herbst, R., et al. (2000). Differential expression of E-cadherin and type IV collagenase genes predicts outcome in patients with stage I non-small cell lung carcinoma. Clin. Cancer Res. 6: 790 – 797.
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