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

Hallett MA, Teng B, Hasegawa H, Schwab LP, Seagroves TN, Pourmotabbed T - Breast Cancer Res. (2013)

Bottom Line: DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression.AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%.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.

View Article: PubMed Central - HTML - PubMed

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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Effect of AM9D treatment on metalloproteinase (MMP) expression in MDA-MB-231 cells. (A) Expression levels of MMP9, MMP1, MMP13, MMP14, MMP19, and MMP21, and BACT (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 and MMP9, MMP1, MMP13, MMP14, MMP19 and MMP21, and BACT (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).
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Figure 1: Effect of AM9D treatment on metalloproteinase (MMP) expression in MDA-MB-231 cells. (A) Expression levels of MMP9, MMP1, MMP13, MMP14, MMP19, and MMP21, and BACT (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 and MMP9, MMP1, MMP13, MMP14, MMP19 and MMP21, and BACT (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).

Mentions: The specificity of AM9D toward MMP9 mRNA was demonstrated in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells express MMP1, MMP9, MMP13, MMP14, MMP19, and MMP21 (Figure 1A, lane 3). As shown in Figure 1A and 1B, contrary to control DNAzyme (lane 2), AM9D treatment (lane 1) significantly decreased the activity (Figure 1B) and the level of MMP9 mRNA (Figure 1A) in MDA-MB-231 cells without having an effect on MMP1, MMP13, MMP14, MMP19 or MMP21 mRNA levels. Although MMP-2 and -3 have also been reported to contribute to breast tumorigenesis [30], we did not detect MMP2 or MMP3 mRNA expression in cultured MDA-MB-231 cells. These data demonstrate that the AM9D therapy is specific as it only affects the production of MMP-9 in cells, and that reduction of MMP9 mRNA leads to reduction in enzymatic activity, as expected.


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

Hallett MA, Teng B, Hasegawa H, Schwab LP, Seagroves TN, Pourmotabbed T - Breast Cancer Res. (2013)

Effect of AM9D treatment on metalloproteinase (MMP) expression in MDA-MB-231 cells. (A) Expression levels of MMP9, MMP1, MMP13, MMP14, MMP19, and MMP21, and BACT (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 and MMP9, MMP1, MMP13, MMP14, MMP19 and MMP21, and BACT (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).
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Figure 1: Effect of AM9D treatment on metalloproteinase (MMP) expression in MDA-MB-231 cells. (A) Expression levels of MMP9, MMP1, MMP13, MMP14, MMP19, and MMP21, and BACT (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 and MMP9, MMP1, MMP13, MMP14, MMP19 and MMP21, and BACT (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).
Mentions: The specificity of AM9D toward MMP9 mRNA was demonstrated in MDA-MB-231 human breast cancer cells. MDA-MB-231 cells express MMP1, MMP9, MMP13, MMP14, MMP19, and MMP21 (Figure 1A, lane 3). As shown in Figure 1A and 1B, contrary to control DNAzyme (lane 2), AM9D treatment (lane 1) significantly decreased the activity (Figure 1B) and the level of MMP9 mRNA (Figure 1A) in MDA-MB-231 cells without having an effect on MMP1, MMP13, MMP14, MMP19 or MMP21 mRNA levels. Although MMP-2 and -3 have also been reported to contribute to breast tumorigenesis [30], we did not detect MMP2 or MMP3 mRNA expression in cultured MDA-MB-231 cells. These data demonstrate that the AM9D therapy is specific as it only affects the production of MMP-9 in cells, and that reduction of MMP9 mRNA leads to reduction in enzymatic activity, as expected.

Bottom Line: DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression.AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%.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.

View Article: PubMed Central - HTML - PubMed

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.

Show MeSH
Related in: MedlinePlus