Siying Wang

Cyanidin-3-Glucoside inhibits ethanol-induced invasion of breast cancer cells overexpressing ErbB2

BackgroundEthanol is a tumor promoter. Both epidemiological and experimental studies suggest that ethanol may enhance the metastasis of breast cancer cells. We have previously demonstrated that ethanol increased the migration/invasion of breast cancer cells expressing high levels of ErbB2. Amplification of ErbB2 is found in 20-30% of breast cancer patients and is associated with poor prognosis. We sought to identify agents that can prevent or ameliorate ethanol-induced invasion of breast cancer cells. Cyanidin-3-glucoside (C3G), an anthocyanin present in many vegetables and fruits, is a potent natural antioxidant. Ethanol exposure causes the accumulation of intracellular reactive oxygen species (ROS). This study evaluated the effect of C3G on ethanol-induced breast cancer cell migration/invasion.ResultsC3G attenuated ethanol-induced migration/invasion of breast cancer cells expressing high levels of ErbB2 (BT474, MDA-MB231 and MCF7ErbB2) in a concentration dependent manner. C3G decreased ethanol-mediated cell adhesion to the extracellular matrix (ECM) as well as the amount of focal adhesions and the formation of lamellipodial protrusion. It inhibited ethanol-stimulated phosphorylation of ErbB2, cSrc, FAK and p130Cas, as well as interactions among these proteins. C3G abolished ethanol-mediated p130Cas/JNK interaction.ConclusionsC3G blocks ethanol-induced activation of the ErbB2/cSrc/FAK pathway which is necessary for cell migration/invasion. C3G may be beneficial in preventing/reducing ethanol-induced breast cancer metastasis.

Ethanol promotes mammary tumor growth and angiogenesis: the involvement of chemoattractant factor MCP-1.

Alcohol consumption is a risk factor for breast cancer in humans. Experimental studies indicate that alcohol exposure promotes malignant progression of mammary tumors. However, the underlying cellular and molecular mechanisms remain unclear. Alcohol induces a pro-inflammatory response by modulating the expression of cytokines and chemokines. Monocyte chemoattractant protein-1 (MCP-1), also known as chemokine (C–C motif) ligand 2, is a pro-inflammatory chemokine implicated in breast cancer development/malignancy. We investigated the role of MCP-1 in alcohol-promoted mammary tumor progression. Using a xenograft model, we demonstrated that alcohol increased tumor angiogenesis and promoted growth/metastasis of breast cancer cells in C57BL/6 mice. Alcohol up-regulated the expression of MCP-1 and its receptor CCR2 in breast cancer cells in vitro and in vivo. Using a three-dimensional tumor/endothelial cell co-culture system, we demonstrated MCP-1 regulated tumor/endothelial cell interaction and promoted tumor angiogenesis. More importantly, MCP-1 mediated alcohol-promoted angiogenesis; an antagonist of the MCP-1 receptor CCR2 significantly inhibited alcohol-stimulated tumor angiogenesis. The CCR2 antagonist abolished ethanol-stimulated growth of mammary tumors in mice. We further demonstrated that MCP-1 enhanced the migration, but not the proliferation of endothelial cells as well as breast cancer cells. These results suggest that MCP-1 plays an important role in ethanol-stimulated tumor angiogenesis and tumor progression.

Activation of the NF-κB pathway as a mechanism of alcohol enhanced progression and metastasis of human hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC), the most common form of primary liver cancer, is the third leading cause of cancer-related death in human. Alcohol is a known risk factor for HCC. However it is still unclear whether and how alcohol enhances the progression and metastasis of existing HCC.Methods and resultsWe first retrospectively investigated 52 HCC patients (24 alcohol-drinkers and 28 non-drinkers), and found a positive correlation between alcohol consumption and advanced Tumor-Node-Metastasis (TNM) stages, higher vessel invasion and poorer prognosis. In vitro and in vivo experiments further indicated that alcohol promoted the progression and migration/invasion of HCC. Specifically, in a 3-D tumor/endothelial co-culture system, we found that alcohol enhanced the migration/invasion of HepG2 cells and increased tumor angiogenesis. Consistently, higher expression of VEGF, MCP-1 and NF-κB was observed in HCC tissues of alcohol-drinkers. Alcohol induced the accumulation of intracellular reactive oxygen species (ROS) and the activation of NF-κB signaling in HepG2 cells. Conversely, blockage of alcohol-mediated ROS accumulation and NF-κB signaling inhibited alcohol-induced expression of VEGF and MCP-1, the tumor growth, angiogenesis and metastasis.ConclusionThis study suggested that chronic moderate alcohol consumption may promote the progression and metastasis of HCC; the oncogenic effect may be at least partially mediated by the ROS accumulation and NF-ĸB-dependent VEGF and MCP-1 up-regulation.

MicroRNA-362-5p promotes tumor growth and metastasis by targeting CYLD in hepatocellular carcinoma

MicroRNAs are increasingly recognized as playing important roles in hepatocellular carcinoma (HCC) tumorigenesis. Here we identified an essential role for miR-362-5p in the regulation of HCC development. We found that miR-362-5p was significantly up-regulated in HCCs and associated with HCC progression. Inhibition of miR-362-5p in HCC cells dramatically decreased cell proliferation, clonogenicity, migration and invasion in vitro as well as tumor growth and metastasis in vivo. We subsequently identified that CYLD was a target gene of miR-362-5p. Furthermore, knockdown of CYLD expression partially counteracted the tumor suppressive effects of miR-362-5p inhibitors. Finally, we have shown that miR-362-5p acts through CYLD to activate the NF-κB signaling pathway, which contributes to HCC progression. Taken together, our findings indicate that miR-362-5p belongs to a new class of oncomiR that regulates HCC cell aggressiveness, thus providing new insight into the molecular mechanisms underlying HCC development. This study also suggests that miR-362-5p may serve as a novel therapeutic target for miRNA based HCC therapy.

GSK3β signaling is involved in ultraviolet B-induced activation of autophagy in epidermal cells.

Ultraviolet B (UVB) exposure causes damage to skin and represents the primary etiological agent for skin cancer formation. UVB induces DNA damage and apoptosis in epidermal cells. In this study, we demonstrated that UVB activated autophagy in JB6 epidermal cells, which was evident by the formation of LC3 puncta, the induction of LC3 lipidation, the increase in beclin 1 expression, and the decrease in the levels of p62. Autophagy appeared to be a protective response to UVB-induced damage because inhibition of autophagy exacerbated UVB-induced cell death, and stimulation of autophagy offered protection. Furthermore, we demonstrated that glycogen synthase kinase 3β (GSK3β) was involved in UVB-induced autophagy. UVB inhibited GSK3β activation by simultaneously enhancing phosphorylation at Ser9 and suppressing Tyr216 phosphorylation. GSK3β negatively regulated autophagy; overexpression of wild-type or S9A (constitutive-active) GSK3β mutant inhibited UVB-mediated autophagy, while overexpression of a dominant-negative K85R mutant enhanced UVB-mediated autophagy. Inhibition of GSK3β also offered protection against UVB-mediated damage. UVB activated AMP-activated protein kinase (AMPK), an important regulator of autophagy through the inhibition of GSK3β. Taken together, our results suggest that UVB-stimulated autophagy is a protective response for epidermal cells and is mediated by the GSK3β/AMPK pathway.

Overexpression of SHP2 tyrosine phosphatase promotes the tumorigenesis of breast carcinoma

Expression of Src homology phosphotyrosine phosphatase 2 (SHP2) has been observed in human breast cancer. SHP2 is known to promote cell migration and invasiveness. However, the pathophysiologic role of SHP2 and its relevance to tumorigenesis are still largely unknown. In the present study, we aimed to evaluate the effect of SHP2 on the malignant phenotype of human breast cancer. An SHP2-overexpressing human breast cancer cell line was established by stable transfection of the SHP2 vector. The expression of SHP2 protein was detected using western blotting. The effects of SHP2 overexpression on cell proliferation were examined by an MTS assay. Invasion and migration abilities of the SHP2-overexpressing cells were determined using a Matrigel-based Boyden chamber invasion assay and a monolayer wound-healing assay. Increased SHP2 expression was detected following SHP2-vector transfection in the MDA-MB-231 cells. Overexpression of SHP2 was associated with increased cell proliferation and clone formation, and decreased chemotherapeutic sensitivity. Furthermore, transfection of SHP2 into breast cancer cells significantly promoted tumor growth in a mouse xenograft model. The mechanism of the promotion of tumorigenesis by SHP2 appears to involve its ability to increase the activity of ERK/AKT-mediated signaling pathways. In conclusion, our data suggest an important role of SHP2 in the molecular etiology of tumor growth, and implicate the potential application of SHP2 in cancer therapy.

Alcohol promotes mammary tumor growth through activation of VEGF‑dependent tumor angiogenesis

Alcohol consumption has been recognized as a risk factor for breast cancer. Experimental studies demonstrate that alcohol exposure promotes the progression of existing mammary tumors. However, the mechanisms underlying this effect remain unclear. In the present study, the role of vascular endothelial growth factor (VEGF) in alcohol promotion of breast cancer development was investigated using a mouse xenograft model of mammary tumors and a three-dimensional (3D) tumor/endothelial cell co-culture system. For the mouse xenograft model, mouse E0771 breast cancer cells were implanted into the mammary fat pad of C57BL6 mice. These mice were exposed to alcohol in their drinking water. For the 3D co-culture system, E0771 cells and MDA-MB231 breast cancer cells were co-cultured with SVEC4-10EE2 and human umbilical vein endothelial cells, respectively. The results demonstrated that alcohol increased tumor angiogenesis and accelerated tumor growth. Furthermore, it appeared that alcohol induced VEGF expression in breast cancer cells in vitro and in vivo. Blocking VEGF signaling by SU5416 inhibited tumor angiogenesis in the 3D tumor/endothelial cell co-culture system. Furthermore, injection of SU5416 into mice inhibited alcohol-promoted mammary tumor growth in vivo. These results indicate that alcohol may promote mammary tumor growth by stimulating VEGF-dependent angiogenesis.

MiR-362-5p promotes the malignancy of chronic myelocytic leukaemia via down-regulation of GADD45α

BackgroundMicroRNAs (miR, miRNAs) play pivotal roles in numerous physiological and pathophysiological contexts. We investigated whether miR-362-5p act as an oncogene in chronic myeloid leukaemia (CML) and aimed to understand its potential underlying mechanisms.MethodsWe compared the miR-362-5p expression levels between CML and non-CML cell lines, and between fresh blood samples from CML patients and normal healthy controls using quantitative real-time PCR (qPCR). Cell counting kit-8 (CCK-8) and Annexin V-FITC/PI analyses were used to measure the effects of miR-362-5p on proliferation and apoptosis, and Transwell assays were used to evaluate migration and invasion. A xenograft model was used to examine in vivo tumourigenicity. The potential target of miR-362-5p was confirmed by a luciferase reporter assay, qPCR and western blotting. Involvement of the JNK1/2 and P38 pathways was investigated by western blotting.ResultsmiR-362-5p was up-regulated in CML cell lines and fresh blood samples from CML patients, and was associated with Growth arrest and DNA damage-inducible (GADD)45α down-regulation. Inhibition of miR-362-5p simultaneously repressed tumour growth and up-regulated GADD45α expression in a xenograft model. Consistently, the knockdown of GADD45α expression partially neutralized the effects of miR-362-5p inhibition. Furthermore study suggested that GADD45α mediated downstream the effects of miR-362-5p, which might indirectly regulates the activation of the JNK1/2 and P38 signalling pathways.ConclusionmiR-362-5p acts as an oncomiR that down-regulates GADD45α, which consequently activates the JNK1/2 and P38 signalling. This finding provides novel insights into CML leukaemogenesis and may help identify new diagnostic and therapeutic targets.

Role of MCP‐1 in alcohol‐induced aggressiveness of colorectal cancer cells

Epidemiological studies demonstrate that alcohol consumption is associated with an increased risk of colorectal cancer (CRC). In addition to promoting carcinogenesis, alcohol may also accelerate the progression of existing CRC. We hypothesized that alcohol may enhance the aggressiveness of CRC. In this study, we investigated the effect of alcohol on the migration/invasion and metastasis of CRC. Alcohol increased the migration/invasion of colorectal cancer cells (DLD1, HCT116, HT29, and SW480) in a concentration‐dependent manner. Among these colon cancer cell lines, HCT116 cells were most responsive while HT29 cells were the least responsive to ethanol‐stimulated cell migration/invasion. These in vitro results were supported by animal studies which demonstrated that ethanol enhanced the metastasis of colorectal cancer cells to the liver and lung. Monocyte chemoattractant protein‐1 (MCP‐1) is a chemokine that plays an important role in regulating tumor microenvironment and metastasis. Alcohol increased the expression of MCP‐1 and its receptor CCR2 at both protein and mRNA levels. The pattern of alcohol‐induced alterations in MCP‐1 expression was consistent with its effect on migration/invasion; HCT116 cells displayed the highest up‐regulation of MCP‐1/CCR2 in response to alcohol exposure. An antagonist of CCR2 blocked alcohol‐stimulated migration. Alcohol caused an initial cytosolic accumulation of β‐catenin and its subsequent nuclear translocation by inhibiting GSK3β activity. Alcohol stimulated the activity of MCP‐1 gene promoter in a β‐catenin‐dependent manner. Furthermore, knock‐down of MCP‐1/CCR2 or β‐catenin was sufficient to inhibit alcohol‐induced cell migration/invasion. Together, these results suggested that alcohol may promote the metastasis of CRC through modulating GSK3β/β‐catenin/MCP‐1 pathway.

Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38γ

Both epidemiological and experimental studies suggest that ethanol may enhance aggressiveness of breast cancer. We have previously demonstrated that short term exposure to ethanol (12–48 hours) increased migration/invasion in breast cancer cells overexpressing ErbB2, but not in breast cancer cells with low expression of ErbB2, such as MCF7, BT20 and T47D breast cancer cells. In this study, we showed that chronic ethanol exposure transformed breast cancer cells that were not responsive to short term ethanol treatment to a more aggressive phenotype. Chronic ethanol exposure (10 days - 2 months) at 100 (22 mM) or 200 mg/dl (44 mM) caused the scattering of MCF7, BT20 and T47D cell colonies in a 3-dimension culture system. Chronic ethanol exposure also increased colony formation in an anchorage-independent condition and stimulated cell invasion/migration. Chronic ethanol exposure increased cancer stem-like cell (CSC) population by more than 20 folds. Breast cancer cells exposed to ethanol in vitro displayed a much higher growth rate and metastasis in mice. Ethanol selectively activated p38γ MAPK and RhoC but not p38α/β in a concentration-dependent manner. SP-MCF7 cells, a derivative of MCF7 cells which compose mainly CSC expressed high levels of phosphorylated p38γ MAPK. Knocking-down p38γ MAPK blocked ethanol-induced RhoC activation, cell scattering, invasion/migration and ethanol-increased CSC population. Furthermore, knocking-down p38γ MAPK mitigated ethanol-induced tumor growth and metastasis in mice. These results suggest that chronic ethanol exposure can enhance the aggressiveness of breast cancer by activating p38γ MAPK/RhoC pathway.