Shanzhi Gu

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.

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.

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.

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.

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro

ObjectiveTo study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells.MethodsWe examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells.ResultsThe compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly.ConclusionWith a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.

An antimicrobial peptide containing NGR motif has potent antitumor activity against CD13+ and CD13− tumor cells

Antimicrobial peptides (AMPs) with Asn-Gly-Arg (NGR) motif have potent cytotoxicity, preferably against tumor cells due to their binding to CD13 on tumor cells. However, the importance of αvβ3 expression for antitumor activity of AMPs containing NGR has not been clarified. This study was aimed at designing a new AMP containing NGR and testing their biological activity against different types of tumor cells with varying CD13 and αvβ3 expression. We first synthesized the new AMP containing NGR motif (CK21), which effectively entered into CD13+ HT-1080, but less into CD13− αvβ3+ MDA-MB-435 and further less into stable αvβ3-silencing MDA-MB-435 cells. Furthermore, CK21 displayed dose-dependent antiproliferation against these tumor cells and induced cell cycling arrest at G2/M phases and apoptosis of these tumor cells. In addition, CK21 inhibited the invasion of these tumor cells in vitro and inhibited the growth of implanted tumor cells in vivo. Particularly, the antitumor effect of CK21 in CD13+ HT-1080 was stronger than that of CD13− αvβ3+ MDA-MB-435 and much stronger than that of stable αvβ3-silencing MDA-MB-435. Our data indicated that the new AMPs containing NGR had potent antitumor activity against CD13+ or αvβ3+ tumor cells, preferably against CD13+ tumor cells, possibly through binding to CD13 or αvβ3 on tumor cells.

Estrogen promotes progression of hormone-dependent breast cancer through CCL2-CCR2 axis by upregulation of Twist via PI3K/AKT/NF-κB signaling

The chemokine (C-C motif) ligand 2 (CCL2) with its cognate receptor chemokine (C-C motif) receptor 2 (CCR2) plays important roles in tumor invasion and metastasis. However, the mechanisms and mediators for autocrine CCL2 and CCL2-CCR2 axis remain elusive in breast cancer. Here we examined the levels of CCL2 in 4 breast cancer cell lines along with 57 human breast cancer specimens and found them significantly increased with presence of 17β-estradiol (E2) in estrogen receptor (ER)-positive breast cancer cells, while anti-estrogen treatment weakened this enhancement. CCL2 expression positively correlated with Twist staining and aggressiveness of breast cancer. Estrogen exposure facilitated the proliferation, invasion and metastasis of hormone-dependent breast cancer and promoted angiogenesis via the increased secretion of CCL2 in vitro and in vivo, which could be suppressed by disruption of CCL2-CCR2 axis with CCR2 antagonist RS102895. Knockdown of Twist in MCF-7 cells significantly inhibited E2-induced CCL2 production, indicating an essential role of Twist in CCL2 regulation under estrogenic condition. Our data show the hormonal regulation on CCL2-CCR2 axis is associated with enhanced Twist expression via activation of ERα and PI3K/AKT/NF-κB signaling. Thus, CCL2-CCR2 axis may represent as a novel therapeutic target eagerly needed for hormone-dependent breast cancer.

Thymoquinone inhibits the metastasis of RCC cells by inducing autophagy via AMPK/mTOR signaling pathway

Thymoquinone (TQ, 2‐methyl‐5‐isopropyl‐1,4‐benzoquinone), a bioactive constituent extracted from the seeds of Nigella sativa, has been proved to exert anti‐tumor efficiency in various cancers. Autophagy is a self‐digestion phenomenon, and its role in tumor formation and progression remains controversial. In the present study, we investigated the effects of TQ on renal cell cancer (RCC) cell lines (786‐O and ACHN) using wound healing assay, transwell assay and western blot analysis. We found that TQ effectively inhibited the metastatic capacity of RCC cells in vitro, which was also verified in a xenograft model. Meanwhile, we observed LC3 puncta and detected the expression of LC3 in TQ‐treated RCC cells, and then found that autophagy was induced by TQ in 786‐O and ACHN cell lines. In addition, TQ inhibited the migration and invasion as well as the EMT in RCC cells in an autophagy‐dependent manner. To further explore the underlying mechanism, we detected the AMPK/mTOR signaling pathway. The results indicated that TQ inhibited the metastasis of RCC cells by inducing autophagy via AMPK/mTOR signaling pathway. In conclusion, our findings provide a novel therapeutic strategy that aims at TQ‐induced autophagy in RCC treatment.

Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo

Distinguishing the surface markers of cancer stem cells (CSCs) is a useful method for early diagnosis and treatment of tumors, as CSCs may participate in tumorigenesis and metastasis by migrating into the circulatory system. However, the potential targets of CSCs are expressed at low levels in the natural state and are always changing. Thus, dynamic screening has been reported to be an effective measure for exploring CSC markers. In recent years, diverse single‐chain variable fragments (scFvs) have been widely used in immunotherapy. In this study, we determined that the scFvs, screened using RD, had a high affinity to microspheres and could inhibit their progression. We also observed that the selected scFvs underwent evolution in vitro, and antitumor‐associated proteins were successfully expressed. Combined with chemotherapy, the scFvs had a synergistic effect on the inhibition of the microspheres’ progression in vitro and in vivo, which could be ascribed to their high affinity for stem‐like cells and the inhibition of the microspheres’ collective behaviors. In addition, proteins inhibiting CD44+/CD24+ and MAPK were involved. Our data indicated that dynamic screening of the scFvs in a natural state was of great significance in the inhibition of the microspheres in vitro and in vivo.