Lianhua Yin

Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells

PurposeAssociated with many molecules, metastasis includes cell adhesion to extracellular matrix, migration towards specific direction and invasion into local vessel of distant organs. The purpose of the present study was to evaluate the role of ecto-5′-nucleotidase (eN, ecto-5-NT, CD73) generated extracellular adenosine in biologically malignant behaviors of human breast cancer cell lines.Materials and methodsTwo human breast cancer cell lines, T-47D with lower expression of CD73 and MB-MDA-231 with higher expression of CD73, were used to investigate the functions of CD73. The effects of CD73 over-expression on invasion, migration and adhesion were observed in T-47D transfected with pcDNA-NT5E plasmid. The effects of specific CD73 inhibitor, α, ß-methylene ADP (APCP), were observed in MB-MDA-231 cells.ResultsThe results showed CD-73 overexpression increased invasion, migration and adhesion to ECM of the pcDNA-NT5E transfected T-47D cells compared to the saline and mock vector controls. The increased cell mobility of CD-73-overexpressed T-47D cells was blocked by APCP. Adenosine increased the mobility of wild type T-47D cells. APCP inhibited the mobility of the MB-MDA-231 cells.ConclusionTaken together, our results indicated that CD73 may facilitate the adhesion, migration and invasion of human breast cancer cells through its enzyme activity of generating adenosine. This study provided a possibly molecular mechanism of metastasis of breast carcinoma.

RNA interference of ecto-5′-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion

Metastasis is a leading cause of mortality and morbidity in breast cancer. Recently, dramatic overexpression of ecto-5′-nucleotidase (CD73), a glycosylphosphatidylinositol-anchored cell surface protein has been found in estrogen receptor-negative [ER (−)] breast cancer cell lines and in clinical samples. In this study, CD73 small interfering RNA (siRNA) plasmid was constructed and stably transfected into breast cancer cell MB-MDA-231 to determine the role of CD73 in breast cancer metastasis and the possible mechanism. Our study demonstrates that CD73 siRNA effectively inhibits CD73 gene expression at mRNA and protein level in MB-MDA-231 cells, leading to in vivo and in vitro growth suppression, prevention of adhesion to extracellular matrix (ECM), and inhibition of invasion and migration. These properties correlate with inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity as well as reduction of epidermal growth factor receptor (EGFR) expression. Demonstration of the role of CD73 in breast cancer may lead to new targeted therapies for breast cancer.

Overexpression of mitochondrial cholesterol delivery protein, StAR, decreases intracellular lipids and inflammatory factors secretion in macrophages

Hyperlipidemia is one of the most important risk factors for atherosclerosis. This can be amplified by a localized inflammatory response mediated by macrophages. Macrophages are capable of taking up excess cholesterol, and it is well known that delivery of cholesterol to the mitochondria by steroidogenic acute regulatory (StAR) protein is the rate-limiting step for cholesterol degradation in the liver. It has also been shown that overexpression of StAR in hepatocytes dramatically increases the amount of regulatory oxysterols in the nucleus, which play an important role in the maintenance of intracellular lipid homeostasis. The goal of the present study was to determine whether StAR plays a similar role in macrophages. We have found that overexpression of StAR in human THP-1 monocyte-derived macrophages decreases intracellular lipid levels, activates liver X receptor alpha (LXRalpha) and proliferation peroxysome activator receptor gamma (PPARgamma), and increases ABCG1 and CYP27A1 expression. Furthermore, it reduces the secretion of inflammatory factors, and prevents apoptosis. These results suggest that StAR delivers cholesterol to mitochondria where regulatory oxysterols are generated. Regulatory oxysterols can in turn activate nuclear receptors, which increase expression of cholesterol efflux transporters, and decrease secretion of inflammatory factors. These effects can prevent macrophage apoptosis. These results imply a potential role of StAR in the prevention of atherosclerosis.

RNAi-mediated CD73 suppression induces apoptosis and cell-cycle arrest in human breast cancer cells

Ecto‐5′‐nucleotidase (CD73), a cell surface protein that hydrolyzes extracellular AMP into adenosine and phosphate, is overexpressed in many solid tumors. In this study, we tested the hypothesis that increased CD73 may promote tumor progression by examining the effect of CD73 suppression via RNA interference and CD73 overexpression on tumor growth in vivo and in vitro. Using digitized whole‐body images, plate clone forming assay and TUNEL assay in frozen tissue sections, we found that the cell growth rate was significantly lower in vivo and in vitro after CD73 suppression and late apoptosis was much higher in xenograft tumors developed from the CD73‐siRNA transfected MB‐MDA‐231 clone (P1). By flow cytometry, the P1 cell cycle was arrested in the G0/G1 phase. Moreover, Bcl‐2 was downregulated, while Bax and caspase‐3 were upregulated with CD73 suppression. CD73 inhibitor α,β‐methylene adenosine‐5′‐disphosphate (APCP) functioned similarly with RNAi‐mediated CD73 suppression. In addition, in transfected MCF‐7 cells, we found that CD73 overexpression increased cell viability and promoted cell cycle progression, depending on its enzyme activity. More intriguingly, CD73 overexpression in MCF‐7 breast cancer cells produces a tumorigenic phenotype. We conclude that CD73 plays an important role in breast cancer growth by affecting cell cycle progression and apoptosis. (Cancer Sci 2010; 101: 2561–2569)

The effects of inflammatory cytokines on steroidogenic acute regulatory protein expression in macrophages

Abstract.Objective:To investigate the expression of steroidogenic acute regulatory protein (StAR) in macrophages and the effects of inflammatory cytokines on StAR expression.Methods:The macrophages isolated from ApoE knockout mice and C57BL/6J mice and RAW264.7 cells (a cell line from mouse macrophage. ATCC Number: TIB-71TM) were cultured in DMEM containing 10% fetal bovine serum. RAW264.7 cells were treated with different inflammatory cytokines (TNF-α, IFN-γ and TGF-β1) and 8-Br-cAMP, a cAMP analog. RT-PCR and Western blot analysis were applied to evaluate the effects of inflammatory cytokines on StAR expression.Results:RT-PCR and Western blot analysis demonstrated the expression of StAR in the macrophages isolated from ApoE knockout mice, C57BL/6J mice and RAW264.7 cells. Proinflammatory cytokines TNF-α and IFN-γ significantly decreased StAR mRNA and protein levels in RAW264.7 cells. The inhibition was dose- and time-dependent. In contrast, anti-inflammatory cytokine TGF-β1 increased StAR mRNA and protein levels. At 1:15 molecular ratio, TGF-β1 blocked the down-regulation of StAR expression mediated by TNF-α. cAMP also induced StAR expression in RAW264.7 cells. When the cells were co-treated with 8-Br-cAMP and TNF-α, 8-Br-cAMP failed to induce StAR expression.Conclusion:Our results provide interesting evidence that inflammatory cytokines regulate StAR expression in macrophages.

Potential prognostic biomarker CD73 regulates epidermal growth factor receptor expression in human breast cancer

CD73, an ecto‐enzyme overexpressed in breast‐cancer cells, catalyzes the dephosphorylation of adenosine monophosphates into adenosine. Anti‐CD73 slows breast cancer growth and its spread both in vivo and in vitro. In this study, we investigated the relation of CD73 to epidermal growth factor receptor (EGFR) expression using tissue array and breast cancer cell lines. We found that CD73 expression correlated positively to EGFR expression in vivo (n = 80, r = 0.425, P < 0.01) and in vitro. EGFR expression can be decreased by suppressing CD73 with an inhibitor or small shRNA, and this effect was reversed by adenosine and NECA (adenosine A2 receptor agonist), which suggested that adenosine is involved in EGFR expression regulated by CD73 (P < 0.01). We also showed that CD73 regulates EGFR phosphorylation by Src (P < 0.01). By transcription factor (TF) assay, CD73 was found to regulate some associated TFs activity such as PPARγ, which mediates EGFR expression, although whether PPARγ mediates the effect of CD73 on EGFR expression needs further study. The Kaplan–Meier recurrence‐free survival curves for CD73 were also plotted in www.kmplot.com. The curves show that CD73 expression separates the cases into significantly different prognostic groups among the estrogen receptor‐negative cancers (P < 0.01). Our results suggest that CD73 may be a potential prognostic biomarker associated with coexpression of EGFR in human breast cancer.

Tryptase promotes atherosclerotic plaque haemorrhage in ApoE-/- mice.

Tryptase, the most abundant mast cell (MC) granule protein, plays an important role in atherosclerosis plaque development. To test the hypothesis that tryptase participates directly in atherosclerosis plaque haemorrhage, the gene sequence and siRNA for tryptase were cloned into a lentivirus carrier and atherosclerosis plaque haemorrhage models in ApoE-/- mice were constructed. After a cuffing-cervical artery operation, the mice were randomly divided into 6 groups. Hematoxylin and eosin(HE) staining showed that the cervical artery plaque area was much larger in the tryptase overexpression group compared to the other groups, and there was greater artery stenosis. The artery stenosis from the cuff-side in all groups was more than 90%, except the siRNA group. Tryptase promotes plaque haemorrhage distinctively because 50% of the mice in the tryptase overexpression group had plaque haemorrhage, while only 10% in the siRNA group did. The immunohistochemistry of the cervical artery plaque showed that plasminogen activator inhibitor-1 (PAI-1) expression was the lowest while tissue plasminogen activator (tPA), CD31, CD34 and VEGF was the highest in the tryptase overexpression groups. This observation was completely contrary to what was observed in the siRNA group. Tryptase promoted bEnd.3 cell growth, migration and capillary-like tube formation, which suggests that tryptase can promote microvessel angiogenesis. PAI-1 expression was inhibited, while tPA expression was increased by tryptase in bEnd.3 cells. Our in vivo and in vitro studies suggest that trypase can promote atherosclerotic plaque haemorrhage by promoting angiogenesis and regulating the balance of PAI-1 and tPA. Thus, regulating tryptase expression in MCs may provide a potential target for atherosclerosis treatment.

Overexpression of steroidogenic acute regulatory protein in rat aortic endothelial cells attenuates palmitic acid-induced inflammation and reduction in nitric oxide bioavailability

BackgroundEndothelial dysfunction is a well documented evidence for the onset of atherosclerosis and other cardiovascular diseases. Lipids disorder is among the main risk factors for endothelial dysfunction in these diseases. Steroidogenic acute regulatory protein (StAR), one of the cholesterol transporters, plays an important role in the maintenance of intracellular lipid homeostasis. However, the effect of StAR on endothelial dysfunction is not well understood. Palmitic acid (PA) has been shown to decrease eNOS activity and induce inflammation, both are the causes of endothelial dysfunction, in an endothelial cell culture model.MethodsStAR gene was introduced into primary rat aortic endothelial cells by adenovirus infection. Real-time PCR and Western blotting were performed to determine the relative genes and proteins expression level to elucidate the underlying mechanism. The free fatty acid and cholesterol quantification kits were used to detect total cellular free fatty acid and cholesterol. The levels of inflammatory factors and nitric oxide were determined by ELISA and classic Griess reagent methods respectively.ResultsWe successfully overexpressed StAR in primary rat aortic endothelial cells. Following StAR overexpression, mRNA levels of IL-1β, TNFα, IL6 and VCAM-1 and protein levels of IL-1β, , TNFα and IL-6 in culture supernatant were significantly decreased, which duing to blocke NFκB nuclear translocation and activation. Moreover, StAR overexpression attenuated the PA-induced reduction of nitric oxide bioavailability by protecting the bioactivity of pAkt/peNOS/NO pathway. Furthermore, the key genes involved in lipid metabolism were greatly reduced following StAR overexpression. In order to investigate the underlying mechanism, cerulenin and lovastatin, the inhibitor of fatty acid and cholesterol synthase, were added prior to PA treatment. The results showed that both cerulenin and lovastatin had a similar effect as StAR overexpression. On the other hand, the role of StAR was inhibited when siRNA was introduced to reduce StAR expression.ConclusionsOur results showed that StAR attenuated lipid synthesis and uptake as well as PA-induced inflammation and reduction in NO bioavailability in aortic endothelial cells. StAR can ameliorate endothelial dysfunction induced by PA via reducing the intracellular lipid levels.

Mitochondrial Cholesterol Transporter, StAR, Inhibits Human THP-1 Monocyte-Derived Macrophage Apoptosis

Steroidogenic acute regulatory protein (StAR) plays an important role in the maintenance of intracellular lipid homeostasis. Macrophages are the key cellular player in the pathophysiology of atherosclerosis. Imbalance of macrophage lipid homeostasis causes cellular apoptosis, which is the key process in the initiation of atherosclerosis. The present study has investigated the effects of StAR in the apoptotic process of human THP-1 derived macrophages induced by serum withdrawal or Ox-LDL. Overexpression of StAR significantly decreased the number of apoptotic macrophages by decreasing the expression of pro-apoptotic genes Caspase-3 and Bax mRNA and protein levels, as well as through increasing expression of anti-apoptotic gene Bcl-2 mRNA and protein levels in the absence and presence of Ox-LDL. The results indicate that StAR plays an important role in macrophage and foam cell apoptotic processing, which may provide a potential method for preventing atherosclerosis.

Overexpression of the steroidogenic acute regulatory protein increases the expression of ATP-binding cassette transporters in microvascular endothelial cells (bEnd.3)

ObjectiveTo determine the effect of steroidogenic acute regulatory protein (StAR) overexpression on the levels of adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) in an endothelial cell line (bEnd.3).MethodsThe StAR gene was induced in bEnd.3 cells with adenovirus infection. The infection efficiency was detected by fluorescence activated cell sorter (FACS) and fluorescence microscopy. The expressions of StAR gene and protein levels were detected by real-time polymerase chain reaction (PCR) and Western blot. The gene and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blot after StAR overexpression.ResultsThe result shows that StAR was successfully overexpressed in bEnd.3 cells by adenovirus infection. The mRNA and protein expressions of ABCA1 and ABCG1 were greatly increased by StAR overexpression in bEnd.3 cells.ConclusionOverexpression of StAR increases ABCA1 and ABCG1 expressions in endothelial cells.