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.2013 Feb 13;15(1):R12.
doi: 10.1186/bcr3385.

Anti-matrix metalloproteinase-9 DNAzyme decreases tumor growth in the MMTV-PyMT mouse model of breast cancer

Anti-matrix metalloproteinase-9 DNAzyme decreases tumor growth in the MMTV-PyMT mouse model of breast cancer

Miranda A Hallett et al. Breast Cancer Res..

Abstract

Introduction: Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still overcome by cancer metastasis. Tumor cell proliferation, invasion and metastasis are mediated, at least in part, through degradation of basement membrane by neutral matrix metalloproteinases (MMP) produced by tumor and stromal cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression.

Methods: The application of anti-MMP-9 DNAzyme (AM9D) for the treatment of primary and metastatic breast cancer was evaluated in vitro and in vivo using MDA-MB-231 cells and the MMTV-PyMT transgenic breast cancer mouse model. Spontaneously developed mammary tumors in MMTV-PyMT transgenic mice were treated intratumorally with naked AM9D, once a week for 4 weeks. The stability of DNAzyme was determined in vitro and in vivo using fluorescently labeled DNAzyme.

Results: AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%. Weekly intratumoral treatment of spontaneously developed mammary tumors in MMTV-PyMT transgenic mice was sufficient to significantly reduce the rate of tumor growth and final tumor load in a dose dependent and statistically significant manner (P < 0.05). This decrease in tumor growth was correlated with decreased MMP-9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascularized and contained more apoptotic cells compared to control and untreated tumors.

Conclusions: These results show that targeting and down regulation of MMP-9 by AM9D could prove useful as a therapy against breast carcinoma tumor growth and invasion.

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Figures

Figure 1
Figure 1
Effect of AM9D treatment on metalloproteinase (MMP) expression in MDA-MB-231 cells. (A) Expression levels ofMMP9,MMP1,MMP13,MMP14,MMP19, andMMP21, andBACT(ß-actin) mRNA in MDA-MB-231-transfected cells. MDA-MB-231 cells were transfected with Oregon Green 488-labeled DNAzymes, control DNAzyme or mock transfection reagents as described in Materials and methods. Positively transfected cells were identified by flow cytometry. Total RNA was isolated andMMP9,MMP1,MMP13,MMP14,MMP19andMMP21, andBACT(ß-actin) mRNA were amplified by reverse-transcription (RT)-PCR and the PCR products were subjected to agarose gel and visualized by ethidium bromide staining. Lane 1, AM9D; lane 2, control DNAzyme; lane 3, cells treated with DOTAP (N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate) transfection reagent only. (B) Gelatin zymography of culture media from transfected MDA-MB-231 cells. The cultured media from MDA-MB-231 cells transfected with AM9D (lane 1), control DNAzyme (lane 2), or treated with DOTAP alone (lane 3) were separated on 8% SDS polyacrylamide gel containing 1 mg/ml gelatin. (C) Histogram showing the percentage of carcinoma cells invading the ECMatrix™ matrigel matrix after treatment with AM9D compared to cells treated with control DNAzyme. Cells were transfected with Oregon Green 488 labeled-DNAzymes, sorted and cultured in a matrigel matrix invasion chamber as described in Materials and methods. *P<0.05 compared with control (one-way analysis of variance).
Figure 2
Figure 2
Immunohistochemical staining for metalloproteinase (MMP)-9 and α-smooth muscle actin (α-SMA) in mammary tumor sections. Tumors were resected from mice and double stained with antibodies to α-SMA to detect stromal cells(A)and MMP-9(B). When channels were merged(C), these data show that MMP-9 was present in both stromal and tumor cells. Magnification 200×; scale bar is equivalent to 100 µm.
Figure 3
Figure 3
Stability of DNAzyme in mammary tumors,in vitro, andin vivo. (A) Stability of DNAzyme in mammary tumors. Mammary tumors were injected (as described in Materials and methods) with fluorescently-labeled AM9D and resected at either (a) 7 days, (b)10 days, or (c) 14 days post-injection; (d) DNAzyme injected into the 2R tumor of a mouse was found to be distributed to an adjacent, non-injected mammary tumor, 3R, which emerged after intratumoral injections were first initiated. Scale bar is equivalent to 100 µm. (B)Urea-polyacrylamide gel electrophoresis of cleavedMMP9 RNA by AM9D. AM9D was incubated in PBS at 37ºC for 14 days; an equal amount was removed at days 1, 3, 5, 7, 10, and 14 (1D to 14D, respectively) and incubated withMMP9RNA substrate at 37ºC for 2 hours. The products were then visualized on a 4% urea-polyacrylamide gel. Lane 1, RNA substrate alone; lane 2, AM9D without prior incubation at 37°C (0) cleaved RNA substrate into two fragments. AM9D incubated at 37°C for 1, 3, 5, 7, 10, and 14 days, lanes 3 to 8 respectively, did not lose its catalytic activity toward RNA substrate. (C) Stability of AM9Din vivo. The MDA-MB-231 cells were transfected with Oregon Green fluorescently labeled AM9D for 72 hours, and fixed and analyzed for the uptake and stability of AM9D molecule in the cells by fluorescent microscopy (400× magnification). (a) The nucleus is stained with 4',6-diamidino-2-phenylindole (DAPI) and (b) AM9D is shown in green.(c)The overlap of AM9D with DAPI staining indicates that AM9D is present in both the cell cytosol and nuclei, as shown by the arrow.
Figure 4
Figure 4
Effect of AM9D on the rate of tumor growth, final mean tumor volume, and metalloproteinase (MMP)-9 expression. (A) AM9D-treated tumors (■) grew at a slower rate than either untreated tumors (no injections) (▼), or tumors treated with control DNAzyme (▲); at the study endpoint, age 12 weeks,P<0.05. (B) Weekly intratumoral treatment of transgenic mice with 10 µg (n= 9 tumors) or 25 µg AM9D (n= 21 tumors) per tumor reduced mean tumor burden by 39.5% or 50.1%, respectively, when compared to tumors treated with control DNAzyme (n= 24 tumors) (P<0.01 ANOVA). (C) Mammary tumors treated with either 25 µg AM9D or control DNAzyme were stained with an MMP-9 antibody. (a) Mammary tumors treated with 25 µg control DNAzyme for 4 weeks showed increased MMP-9 staining (arrows) compared to (b) mammary tumors treated with AM9D for 4 weeks. Images are shown at 200× magnification; scale bar is equivalent to 100 µm. (D)Mmp9mRNA expression levels in control DNAzyme- or AM9D-treated tumors. Total RNA was isolated and pooled from optical cutting temperature (OCT) compound-embedded tumor sections scraped from glass slides of individual control DNAzyme- or AM9D-treated tumors.Mmp9andBact(ß-actin) mRNA were amplified by RT-PCR and the PCR products were subjected to agarose gel and visualized by ethidium bromide staining. Lane 1, AM9D; lane 2, control DNAzyme.
Figure 5
Figure 5
Angiogenesis and apoptosis immunohistochemical staining of tumors. (A) Staining for CD31 (red) to detect the vasculature in (a) untreated mammary tumors and mammary tumors harvested from mice treated with (c) 25 µg of AM9D or (b) control DNAzyme. Untreated tumors (a) and tumors treated with control DNAzyme (b) contained more blood vessels than tumors treated with AM9D (c). Images are shown at 200× magnification. Scale bar is equivalent to 100 µm. (B) Immunohistochemical staining for caspase-3 to indicate apoptosis in mammary tumors harvested from mice treated for 4 weeks with (a) control DNAzyme or (b) 25 µg AM9D. Very few caspase-3-positive cells (green) were detected in tumors treated with control DNAzyme (a) compared to AM9D-treated (b) tumors, where large regions stained positive for caspase-3 (green). Scale bar is equivalent to 100 µm. (C) Histogram showing the mean CD31-immunostained endothelial area and mean apoptotic area in mammary tumors following treatment with AM9D as compared to untreated tumors and control DNAzyme-treated tumors. *P<0.05 compared with control (one-way analysis of variance and Student'st-test).
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