Qian Ning

Generation 4 polyamidoamine dendrimers is a novel candidate of nano-carrier for gene delivery agents in breast cancer treatment

Polyamidoamine dendrimer (PAMAM-D) is a new gene vector developed in recent years. In this study, we successfully prepared G4PAMAM and detected its unique structure by NMR, FITR and TEM. We revealed that G4PAMAM could bind to human erythrocytes and BSA through electrostatic interaction respectively, and caused haemolysis and reduced bioavailability. However, G4PAMAM-VEGF-ASODN (antisense oligodeoxynucleotides) complex could prevent G4PAMAM from binding to the erythrocytes and BSA and remained stable as a conjugate, therefore the toxicity of the complex was reduced. Meanwhile, we showed that G4PAMAM could be used as a gene vector to deliver AODNs into breast cancer MDA-MB-231 cells without significant cell toxicity, and it enhanced cellular uptake of ODNs. In vivo experiment of human breast tumor xenograft mice model, G4PAMAM also showed more efficiency of accumulating VEGF-ASODN to inhibit the tumor vascularization of breast tumor tissue than naked AODN. Furthermore, G4PAMAM could protect DNA in cytoplasm from digestion of restriction enzymes, which was important to become an effective tool in gene research and therapy.

Notch1 induces epithelial-mesenchymal transition and the cancer stem cell phenotype in breast cancer cells and STAT3 plays a key role.

Breast cancer is the most common malignancy in women. The Notch signaling pathway has been shown to be associated with the development and progression of many human cancers, including breast cancer, but the precise mechanism remains unknown. Here, the influence of Notch1 signaling in mammary epithelial cells was studied. We showed that Notch1 promotes proliferation in MCF7 and MCF10A cells. Transwell assay indicated that Notch1 overexpression promotes cell migration and the invasion of breast cancer cells. We showed that MCF7 and MCF10A cells overexpressing Notch1 acquired features of epithelial-mesenchymal transition (EMT) and displayed a cancer stem cell (CSC) phenotype. The expression levels of the epithelial markers E-cadherin and occludin were decreased, while the expression levels of the mesenchymal markers N-cadherin, vimentin and fibronectin were increased in cells overexpressing Notch1. We demonstrated that Notch1 induced phosphorylation of the signal transducer and activator of transcription 3 (STAT3) in breast cancer cells and increased the expression of p65 and interleukin (IL)-1β. Inhibition of STAT3 activity by JSI124 reduced the expression of p65 and IL-1. Treatment of MCF7-notch1 and MCF10A-notch1 cells with JSI124 also reduced the expression of N-cadherin, markers of epithelial mesenchymal transition and increased the expression of E-cadherin. Our results suggest that Notch1 promotes EMT and the CSC phenotype through induction of STAT3.

Vascular endothelial growth factor receptor-1 activation promotes migration and invasion of breast cancer cells through epithelial-mesenchymal transition.

Vascular endothelial growth factor receptor-1 (VEGFR-1 or Flt-1), a tyrosine kinase receptor, is highly expressed in breast cancer tissues, but near absent in normal breast tissue. While VEGFR-1 expression is associated with poor prognosis of women with breast cancer, it is not clear whether it is involved in the aggressiveness of breast cancer. Thus, the present study examined whether VEGFR-1 activation is associated with the invasiveness of breast cancer. We reported that VEGFR-1 was detected in 60.6% of invasive breast carcinoma tissue sections. In addition, VEGFR-1 expression positively correlated with lymph node-positive tumor status, low expression level of membranous E-cadherin, and high expression levels of N-cadherin and Snail. We found that PlGF-mediated VEGFR-1 activation promoted migration and invasion in MCF-7 (luminal) cells and led to morphologic and molecular changes of epithelial-mesenchymal transition (EMT). This was blocked by the down-regulation of VEGFR-1. Conversely, down-regulation of VEGFR-1 in MDA-MB-231 (post-EMT) cells resulted in morphologic and molecular changes similar to mesenchymal-epithelial transition (MET), and exogenous PlGF could not reverse these changes. Moreover, VEGFR-1 activation led to an increase in nuclear translocation of Snail. Finally, MDA-MB-231 cells expressing shRNA against VEGFR-1 significantly decreased the tumor growth and metastasis capacity in a xenograft model. Histological examination of VEGFR-1/shRNA-expressing tumor xenografts showed up-regulation of E-cadherin and down-regulation of N-cadherin and Snail. These findings suggest that VEGFR-1 may promote breast cancer progression and metastasis, and therapies that target VEGFR-1 may be beneficial in the treatment of breast cancer patients.

Antitumor Activity of Antimicrobial Peptides Containing CisoDGRC in CD13 Negative Breast Cancer Cells

Backgroud isoAsp-Gly-Arg (isoDGR) is a derivative of the Asn-Gly-Arg (NGR) motif, which is used as a targeted delivery tool to aminopeptidase N (CD13) positive cells. Recent studies have shown that cyclic isoDGR (CisoDGRC) has a more efficient affinity with αvβ3, a type of integrin that overexpresses in tumor cells. Antimicrobial peptides (AMPs) are an efficient antitumor peptide that specifically kills tumor cells. In the present study, we designed antimicrobial peptides containing the CisoDGRC motif (CDAK) and assessed its antitumor activity for CD13−/αvβ3 + breast cancer cells (MCF-7 and MDA-MB-231) in vitro and in vivo. Methods In vitro: We assessed the cytotoxicity of CDAK for MCF-7 and MDA-MB-231 breast cancer cells, the human umbilical vein endothelial cell (HUVEC), and human foreskin fibroblasts (HFF). We performed an apoptosis assay using Annexin-V/PI, DNA ladder, mitochondrial membrane potential, and Caspase-3 and Bcl-2. The effect on cell cycles and affinity with cell were tested using flow cytometry and fluorescent microscopy and the effect on invasion was analyzed using an invasion assay. CDAK was injected intravenously into tumor-bearing athymic nude mice in vivo experiment. Results CDAK showed cytotoxic activity in MCF-7 and MDA-MB-231 cells, whereas HUVEC and HFF were less sensitive to the peptides. CDAK induced apoptosis, reduced mitochondrial membrane potential, promoted Caspase-3, and inhibited Bcl-2 expression in the two breast cancer cell lines. In addition, CDAK inhibited proliferation of cancer cell through S phase arrest, and own selective affinity with MCF-7 and MDA-MB-231cells, inhibited the invasion of MDA-MB-231 cells. In vivo, CDAK significant inhibited the progression of the tumor and the generation of neovascularization. Conclusion Antimicrobial peptides containing the CisoDGRC (CDAK) motif could efficiently exhibit the antitumor activity for CD13−/αvβ3 + breast cancer cells.

Antitumor Effect of a Generation 4 Polyamidoamine Dendrimer/Cyclooxygenase-2 Antisense Oligodeoxynucleotide Complex on Breast Cancer In Vitro and In Vivo

Cyclooxygenase (COX)-2 plays critical roles in tumorigenesis, tumor cell growth, and angiogenesis, and inhibiting the expression of COX-2 by gene therapy has showed promising prospects. Vectors are crucial for gene therapy. Polyamidoamine (PAMAM) dendrimers are one type of nano-vectors. In this study, we synthesized a generation 4 polyamidoamine (G4PAMAM) dendrimer/COX-2 antisense oligodeoxynucleotide complex (G4PAMAM/COX-2ASODN), determined the transfection rate of G4PAMAM/COX-2ASODN on cultured breast cancer cells, assessed the cell viability, cell cycle dynamics, and cell invasiveness after transfection, and investigated the effects of G4PAMAM/COX-2ASODN on the expression of COX-2 mRNA and protein and microvessel density (MVD) levels in the tumor tissues of a breast cancer nude mouse model. The results showed that G4PAMAM/COX-2ASODN had a high transfection rate, decreased the cell viability, induced apoptosis and G0/G1 cell cycle arrest, and suppressed cell invasiveness. After treatment with G4PAMAM/COX-2ASODN, the copy number of COX-2 mRNA and protein expression in the tumor tissue were decreased markedly, MVD in the tumor tissue was also decreased, and tumor growth was restrained (p<0. 05). We conclude that COX-2ASODN can be delivered into the cultured and transplanted breast cancer cells efficiently by G4PAMAM, can reduce the expression of COX-2 mRNA and protein, and can lower the MVD of tumor tissues. The G4PAMAM/COX-2ASODN complex has antitumor properties in vitro and in vivo.

Lentivirus vector-mediated gene transduction of CNGRC peptide in rat adipose stem cells.

The aim of the present study was to investigate the feasibility of lentiviral-mediated Cys-Asn-Gly-Arg-Cys (CNGRC) peptide gene transduction in adipose stem cells. Adipose stem cells were prepared using enzymatic digestion and repeated adherence methods and identified in culture by immunofluorescence staining of surface markers. The pluripotency of the cultured adipose stem cells was confirmed by their induced differentiation into bone and fat cells. Following polymerase chain reaction amplification, the gene sequence for the CNGRC peptide was cloned into a lentiviral vector, which was then co-transfected into 293T cells with packaging plasmids Helper 1.0 and Helper 2.0. The lentiviruses carrying the CNGRC peptide gene were then harvested and used to transfect adipose stem cells. Following transduction, expression of CNGRC in adipose stem cells was detected using western blot analysis. Adipose stem cells in culture were successfully induced to differentiate into adipocytes and osteoblasts and the lentiviral vector containing CNGRC-3Flag-EGFP was successfully constructed. Following transduction, western blot analysis and immunofluorescence staining demonstrated expression of the CNGRC protein in adipose stem cells. This suggested that adipose stem cell lines expressing the CNGRC peptide were successfully established.