Inhibition of invasion, angiogenesis, tumor growth, and metastasis by adenovirus-mediated transfer of antisense uPAR and MMP-9 in non-small cell lung cancer cells

Abstract

Lung cancer is currently the leading cause of cancer deaths in the United States. Conventional therapeutic treatments, including surgery, chemotherapy, and radiation therapy, have achieved only limited success. The overexpression of proteases, such as urokinase-type plasminogen activator (uPA), its receptor (uPAR), and matrix metalloproteinases (MMP), is correlated with the progression of lung cancer. In the present study, we used a replication-deficient adenovirus capable of expressing antisense uPAR and antisense MMP-9 transcripts to simultaneously down-regulate uPAR and MMP-9 in H1299 cells. Ad-uPAR-MMP-9 infection of H1299 cells resulted in a dose- and time-dependent decrease of uPAR protein levels and MMP-9 activity as determined by Western blotting and gelatin zymography, respectively. Corresponding immunohistochemical analysis also showed that Ad-uPAR-MMP-9 infection inhibited uPAR and MMP-9 expression. As shown by Boyden chamber assay, Ad-uPAR-MMP-9 infection significantly decreased the invasive capacity of H1299 cells compared with mock and Ad-CMV (empty vector)-infected cells in vitro. Furthermore, Ad-uPAR-MMP-9 infection inhibited capillary-like structure formation in H1299 cells cocultured with endothelial cells in a dose-dependent manner compared with mock- and Ad-CMV-infected cells. Ad-uPAR-MMP-9 injection caused the regression of s.c. induced tumors after s.c. injection with H1299 lung cancer cells and inhibited lung metastasis in the metastatic model with A549 cells. These data suggest that Ad-uPAR-MMP-9 shows its antitumor activity against both established and early phases of lung cancer metastases by causing the destruction of the tumor vasculature. In summary, adenovirus-mediated inhibition of uPA-uPAR interaction and MMP-9 on the cell surface may be a promising anti-invasion and antimetastatic strategy for cancer gene therapy.

Figure 1. Ad-uPAR-MMP-9 decreased uPAR and MMP-9 expression in lung cancer cells

(A) Western blot analysis of uPAR protein expression in cell lysates from H1299 infected with mock, Ad-EV (100 MOI), or the indicated doses of Ad-uPAR and Ad-uPAR-MMP-9. (C) Conditioned medium was collected from the H1299 cells, 60 μg of protein from these samples were mixed with Laemmli sample buffer and run on 10% SDS-PAGE gels containing 0.1% gelatin (gelatin zymography). Densitometric quantitation of uPAR and MMP-9 was performed and the data represent average values from 3 separate experiments. uPAR (B) & MMP-9 (D) expression in H1299 cells infected with 50 MOI of Ad-uPAR-MMP-9 at the indicated time points.

Figure 2. Ad-uPAR-MMP-9 inhibits lung cancer cell invasiveness through Matrigel

H1299 cells were trypsinized and counted 3 days after infection with mock, Ad-EV (empty vector), or the indicated doses of Ad-uPAR and Ad-uPAR-MMP-9. Invasion assays were carried out in a 12-well transwell units (1 × 10⁶ cells/treatment condition in triplicate). After a 24 h incubation period, the cells that had passed through the filter into the lower wells were fixed and photographed (A). The percentage of invasion was quantitated as described in materials and methods (B). Values are mean ± S.D. from 5 different experiments.

Figure 3. Ad-uPAR-MMP-9 inhibits cell migration from lung cancer cell spheroids

H1299 spheroids were infected with mock, Ad-EV, or the indicated doses of Ad-uPAR and Ad-uPAR-MMP-9. After 3 days, single spheroids were placed in the center of wells in 8-well chamber slides and cultured at 37 °C for 48 h. At the end of the migration assay, spheroids were fixed, stained and photographed.

Figure 4. Ad-uPAR-MMP-9 inhibits lung tumor cell-induced angiogenesis

H1299 cells were infected with mock, Ad-EV, or the indicated doses of Ad-uPAR and Ad-uPAR-MMP-9. Four days after infection, conditioned media from these cells was used to culture human endothelial cells for 72 h. The cells were then washed, stained and photographed (A). Image pro analysis of the same showing the length (B) and the branch points (C).

Figure 5. Inhibiton of subcutaneous tumor growth

(A) Nude mice were injected with H1299 cells (5 × 10⁶ /100 μl). When the tumor reached 5–6 mm in size, mice were injected tumorally with Ad-EV (5 × 10⁹) or Ad-uPAR-MMP-9 (5 × 10⁸ or 5 × 10⁹) 3 times. Tumor size was measured using calipers as described in methods. Inhibition of subcutaneous tumor growth and lung metastasis. A549 cells were injected subcutaneously into nude mice. After 2 weeks, the mice received intravenous injections of PBS or 5 × 10⁸ PFU of either Ad-EV or Ad-uPAR-MMP-9 three times at 5-day intervals. Representative subcutaneous tumors are shown for each treatment condition (B). H & E staining of lung sections from the same mice (C).