Xinhan Zhao

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 signaling regulates the epithelial–mesenchymal transition and invasion of breast cancer in a Slug-dependent manner

BackgroundThe epithelial–mesenchymal transition (EMT) is crucial for the invasion and metastasis of breast cancer. However, how Notch signaling regulates the EMT process and invasion in breast cancer remains largely unknown.MethodsThe impact of Notch1 silencing by specific shRNAs on the EMT and invasion of human breast cancer MCF-7 and MDA-MB-231 cells as well as xenografts was tested by western blot, real-time polymerase chain reaction (RT-PCR), immunofluorescence, transwell, and immunohistochemistry assays. The effect of Slug silencing or upregulation on the EMT and invasion of breast cancer cells was analyzed, and the effect of Notch1 signaling on Slug expression was determined by the luciferase reporter assay.ResultsThe Notch1 intracellular domain (N1ICD) and Jagged1 were expressed in breast cancer cells. Notch1 silencing reversed the spontaneous EMT process and inhibited the migration and invasion of breast cancer cells and the growth of xenograft breast cancers. The expression of N1ICD was upregulated significantly by Jagged1-mediated Notch signaling activation. Moreover, Jagged1-mediated Notch signaling promoted the EMT process, migration, and invasion of breast cancer cells, which were abrogated by Notch silencing. Furthermore, the N1ICD positively regulated the Slug expression by inducing Slug promoter activation. Importantly, the knockdown of Slug weakened the invasion ability of breast cancer cells and reversed the Jagged1-induced EMT process with significantly decreased expression of vimentin and increased expression of E-cadherin. In addition, Slug overexpression restored the Notch1 knockdown-suppressed EMT process.ConclusionsOur novel data indicate that Notch signaling positively regulates the EMT, invasion, and growth of breast cancer cells by inducing Slug expression. The Notch1–Slug signaling axis may represent a potential therapeutic target for breast cancer therapy.

VEGF/NRP-1axis promotes progression of breast cancer via enhancement of epithelial-mesenchymal transition and activation of NF-κB and β-catenin

Autocrine vascular endothelial growth factor (VEGF) can regulate the survival and progression of cancers through its various receptors. But the mechanisms and mediators for these functions are largely uncovered, especially in breast cancer. We examined the potential roles and mechanisms of VEGF/neuropilin-1 (NRP-1) axis in regulating the tumorigenesis and metastasis of breast cancer and found the expression of VEGF and NRP-1 correlated with aggressiveness of breast cancer. Knockdown of VEGF or NRP-1 inhibited the proliferation, migration and invasion, but enhanced the apoptosis of MDA-MB-231 cells. In contrast, induction of NRP-1 over-expression promoted the proliferation, migration and invasion of MCF-7 cells. VEGF or NRP-1 silencing attenuated the epithelial-mesenchymal transition (EMT) process and the activation of NF-κBp65, but enhanced GSK-3β expression in MDA-MB-231 cells while NRP-1 over-expression reversed the effects in MCF-7 cells. Treatment with hVEGF165 did not change the inhibition in NRP-1 silencing MDA-MB-231 cells, but enhanced the aggressiveness of NRP-1 over-expressing MCF-7 cells. In addition, VEGF-silencing inhibited the growth and metastasis of implanted MDA-MB-231 tumors in vivo. Our novel data suggest that the positive regulation of the VEGF/NRP-1 axis on the tumorigenesis and metastasis of breast cancer may be associated with enhancing the EMT process and the NF-κB and β-catenin signaling. Hence, the VEGF/NRP-1 axis may be a valuable target for design of therapies for intervention of breast cancer.

Epigallocatechin gallate, the main ingredient of green tea induces apoptosis in breast cancer cells.

Green tea has been suggested for prevention of cancers. In this study, the effect of the main constituent of green tea, epigallocatechin gallate (EGCG), on apoptosis of breast cancer cells was examined. EGCG induced apoptosis in T-47D cells through caspase cascade and the cells were detained at the G1 phase. The rate of apoptosis and activity of caspase-3 induced by EGCG was time and dose dependent. These findings suggest that EGCG might be useful in treatment and/or prevention of breast cancer by inducing apoptosis.

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.

Region-specific expression of brain-derived neurotrophic factor splice variants in morphine conditioned place preference in mice.

It is well established that brain-derived neurotrophic factor (BDNF) plays a pivotal role in brain plasticity-related processes, such as learning, memory and drug addiction. However, changes in expression of BDNF splice variants after acquisition, extinction and reinstatement of cue-elicited morphine seeking behavior have not yet been investigated. Real-time PCR was used to assess BDNF splice variants (I, II, IV and VI) in various brain regions during acquisition, extinction and reinstatement of morphine-conditioned place preference (CPP) in mice. Repeated morphine injections (10mg/kg, i.p.) increased expression of BDNF splice variants II, IV and VI in the hippocampus, caudate putamen (CPu) and nucleus accumbens (NAcc). Levels of BDNF splice variants decreased after extinction training and continued to decrease during reinstatement induced by a morphine priming injection (10mg/kg, i.p.). However, after reinstatement induced by exposure to 6 min of forced swimming (FS), expression of BDNF splice variants II, IV and VI was increased in the hippocampus, CPu, NAcc and prefrontal cortex (PFC). After reinstatement induced by 40 min of restraint, expression of BDNF splice variants was increased in PFC. These results show that exposure to either morphine or acute stress can induce reinstatement of drug-seeking, but expression of BDNF splice variants is differentially affected by chronic morphine and acute stress. Furthermore, BDNF splice variants II, IV and VI may play a role in learning and memory for morphine addiction in the hippocampus, CPu and NAcc.

Chemoprevention of breast cancer: current status and future prospects.

Chemoprevention plays an important role in the prevention of cancer. Due to being an estrogen-dependent cancer, breast carcinomas are ideal candidate for chemoprevention. The two main approaches to chemoprevention of breast cancer are to attain a balance in estrogen or to eliminate all estrogens by selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs). A series of clinical trials have proven tamoxifene quite useful as a preventive strategy for women at high risk of breast cancer. The third-generation, AIs, hold a great promise in chemoprevention of breast cancer. In this review, the mechanisms, side effects and main clinical trials of SERMs and AIs are discussed. The use of other drugs including CoQ10, retinoids, cyclooxygenase (COX)-2 inhibitors, vitamin E, soy isoflavones, tea polyphenols and statins in chemoprevention is introduced. Since most studies on these drugs were conducted on animal models or cell culture, further studies are needed to determine the efficacy of these drugs in chemoprevention of human cancers.

Molecular mechanism of epigallocatechin-3-gallate in human esophageal squamous cell carcinoma in vitro and in vivo

Epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has been shown to inhibit proliferation in various types of tumors. However, few studies concerning the role and mechanism of EGCG in esophageal squamous cell carcinoma are available. Therefore, the antitumor mechanism of EGCG needs to be investigated. The present study aimed to examine the antitumor effect of EGCG on the human esophageal squamous cell carcinoma cell lines, Eca-109 and Te-1, in vitro and in vivo. Cell viability was assessed using the MTT assay and tumor formation and growth in murine xenograft models with or without EGCG treatment. Cell cycle analysis and levels of reactive oxygen species (ROS) were detected using flow cytometry. Apoptosis was measured by Annexin/propidium iodide staining. Caspase-3 cleavage and vascular endothelial growth factor (VEGF) expression were detected using western blot analysis and immunohistochemistry in tumor cell lines and tumor xenografts, respectively. The results showed that EGCG inhibited proliferation in the Eca-109 and Te-1 cells in a time- and dose-dependent manner. Tumor cells were arrested in the G1 phase and apoptosis was accompanied by ROS production and caspase-3 cleavage. In a mouse model, EGCG significantly inhibited the growth of Eca-109 tumors by increasing the expression of cleaved-caspase-3 and decreasing VEGF protein levels. Taken together, the results suggest that EGCG inhibits proliferation and induces apoptosis through ROS production, caspase-3 activation, and a decrease in VEGF expression in vitro and in vivo. Furthermore, EGCG may have future clinical applications for novel approaches to treat esophageal squamous cell carcinoma.