Diallyl trisulfide inhibits migration,invasion and angiogenesis of human colon cancer HT‐29 cells and umbilical vein endothelial cells,and suppresses murine xenograft tumour growth

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis‐dependent diseases including cancer. We examined the cytotoxic, anti‐metastatic, anti‐cancer and anti‐angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases‐2, ‐7 and ‐9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary‐like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex‐vivo angiogenesis. We investigated the anti‐tumour effects of DATS against human colon cancer xenografts in BALB/c^nu/nu mice and its anti‐angiogenic activity in vivo. In this in‐vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.     

Type‐specific dysregulation of matrix metalloproteinases and their tissue inhibitors in end‐stage heart failure patients: relationship between MMP‐10 and LV remodelling

Although past studies observed the changes of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in end‐stage heart failure (HF) patients, a consistent and clear pattern of type‐specific MMPs and/or TIMPs has yet to be further defined. In this study, proteomic approach of human protein antibody arrays was used to compare MMP and TIMP expression levels of left ventricular (LV) myocardial samples from end‐stage HF patients due to dilated cardiomyopathy (DCM) with those from age‐ and sex‐ matched non‐failing patients. Western blot analysis, immunohistochemistry and ELISA were used for validation of our results. We observed that MMP‐10 and ‐7 abundance increased, accompanied by decreased TIMP‐4 in DCM failing hearts (n= 8) compared with non‐failing hearts (n= 8). The results were further validated in a cohort of 34 end‐stage HF patients derived from three forms of cardiomyopathies. Cardiac and plasma MMP‐10 levels were positively correlated with the LV end‐diastolic dimension in this HF cohort. In addition, we observed that insulin‐like growth factor‐2 promoted MMP‐10 production in neonatal rat cardiomyocytes. In conclusion, this study demonstrated a selective up‐regulation of MMP‐10 and ‐7 along with a discordant change of TIMP‐4, and a positive correlation between MMP‐10 levels and the degree of LV dilation in end‐stage HF patients. Our findings suggest that type‐specific dysregulation of MMPs and TIMPs is associated with LV remodelling in end‐stage HF patients, and MMP‐10 may act as a novel biomarker for LV remodelling.     

Differential effects of cyclic stretch on bFGF‐ and VEGF‐induced sprouting angiogenesis

How mechanical factors affect angiogenesis and how they and chemical angiogenic factors work in concert remain not yet well‐understood. This study investigated the interactive effects of cyclic uniaxial stretch and two potent proangiogenic molecules [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)] on angiogenesis using a stretchable three‐dimensional (3‐D) cell culture model. Endothelial cells seeded atop a 3‐D collagen gel underwent sprouting angiogenesis while being subjected to either 10 or 20% cyclic uniaxial stretch at a frequency of either 1/12 or 1 Hz, in conjunction with an elevated concentration of bFGF or VEGF. Without the presence of additional growth factors, 10 and 20% stretch at 1 Hz induced angiogenesis and the perpendicular alignment of new sprouts, and both inductive effects were abolished by cytochalasin D (an actin polymerization inhibitor). While “10% stretch at 1 Hz,” “20% stretch at 1 Hz,” bFGF, and VEGF were strong angiogenesis stimulants individually, only the combination of “20% stretch at 1 Hz” and bFGF had an additive effect on inducing new sprouts. Interestingly, the combination of “20% stretch at a lower frequency (1/12 Hz)” and bFGF decreased sprouting angiogenesis, even though the level of perpendicular alignment of new sprouts was the same for both stretch frequencies. Taken together, these results demonstrate that both stretch frequency and magnitude, along with interactions with various growth factors, are essential in mediating formation of endothelial sprouts and vascular patterning. Furthermore, work in this area is warranted to elucidate synergistic or competitive signaling mechanisms. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:879–888, 2014     

Glycation of fibronectin inhibits VEGF‐induced angiogenesis by uncoupling VEGF receptor‐2‐c‐Src crosstalk

Glycation of extracellular matrix proteins has been demonstrated to contribute to the pathogenesis of vascular complications. However, no previous report has shown the role of glycated fibronectin (FN) in vascular endothelial growth factor (VEGF)‐induced angiogenesis. Thus, this study aimed to investigate the effects of glycated FN on VEGF signalling and to clarify the molecular mechanisms involved. FN was incubated with methylglyoxal (MGO) in vitro to synthesize glycated FN, and human umbilical vein endothelial cells (HUVECs) were seeded onto unmodified and MGO‐glycated FN. Then, VEGF‐induced angiogenesis and VEGF‐induced VEGF receptor‐2 (VEGFR‐2) signalling activation were measured. The results demonstrated that normal FN‐positive bands (260 kD) vanished and advanced glycation end products (AGEs) appeared in MGO‐glycated FN and glycated FN clearly changed to a higher molecular mass. The glycation of FN inhibited VEGF‐induced VEGF receptor‐2 (VEGFR‐2), Akt and ERK1/2 activation and VEGF‐induced cell migration, proliferation and tube formation. The glycation of FN also inhibited the recruitment of c‐Src to VEGFR‐2 by sequestering c‐Src through receptor for AGEs (RAGE) and the anti‐RAGE antibody restored VEGF‐induced VEGFR‐2, Akt and ERK1/2 phosphorylation, endothelial cell migration, proliferation and tube formation. Furthermore, the glycation of FN significantly inhibited VEGF‐induced neovascularization in the Matrigel plugs implanted into subcutaneous tissue of mice. Taken together, these data suggest that the glycation of FN may inhibit VEGF signalling and VEGF‐induced angiogenesis by uncoupling VEGFR‐2‐c‐Src interaction. This may provide a novel mechanism for the impaired angiogenesis in diabetic ischaemic diseases.     

IGFBP‐4 enhances VEGF‐induced angiogenesis in a mouse model of myocardial infarction

Vascular endothelial growth factor (VEGF) is a well‐known angiogenic factor, however its ability in promoting therapeutic angiogenesis following myocardial infarction (MI) is limited. Here, we aimed to investigate whether dual treatment with insulin‐like growth factor binding protein‐4 (IGFBP‐4), an agent that protects against early oxidative damage, can be effective in enhancing the therapeutic effect of VEGF following MI. Combined treatment with IGFBP‐4 enhanced VEGF‐induced angiogenesis and prevented cell damage via enhancing the expression of a key angiogenic factor angiopoietin‐1. Dual treatment with the two agents synergistically decreased cardiac fibrosis markers collagen‐I and collagen‐III following MI. Importantly, while the protective action of IGFBP‐4 occurs at an early stage of ischemic injury, the action of VEGF occurs at a later stage, at the onset angiogenesis. Our findings demonstrate that VEGF treatment alone is often not enough to protect against oxidative stress and promote post‐ischemic angiogenesis, whereas the combined treatment with IGFBP4 and VEGF can utilize the dual roles of these agents to effectively protect against ischemic and oxidative injury, and promote angiogenesis. These findings provide important insights into the roles of these agents in the clinical setting, and suggest new strategies in the treatment of ischemic heart disease.     

Matrix metalloproteases and TIMPs as prognostic biomarkers in breast cancer patients treated with radiotherapy: A pilot study

Breast cancer (BC) is the most common tumour in women and one of the most important causes of cancer death worldwide. Radiation therapy (RT) is widely used for BC treatment. Some proteins have been identified as prognostic factors for BC (Ki67, p53, E‐cadherin, HER2). In the last years, it has been shown that variations in the expression of MMPs and TIMPs may contribute to the development of BC. The aim of this pilot work was to study the effects of RT on different MMPs (‐1, ‐2, ‐3, ‐7, ‐8, ‐9, ‐10, ‐12 and ‐13) and TIMPs (‐1 to ‐4), as well as their relationship with other variables related to patient characteristics and tumour biology. A group of 20 BC patients treated with RT were recruited. MMP and TIMP serum levels were analysed by immunoassay before, during and after RT. Our pilot study showed a slight increase in the levels of most MMP and TIMP with RT. However, RT produced a significantly decrease in TIMP‐1 and TIMP‐3 levels. Significant correlations were found between MMP‐3 and TIMP‐4 levels, and some of the variables studied related to patient characteristics and tumour biology. Moreover, MMP‐9 and TIMP‐3 levels could be predictive of RT toxicity. For this reason, MMP‐3, MMP‐9, TIMP‐3 and TIMP‐4 could be used as potential prognostic and predictive biomarkers for BC patients treated with RT.     

TFPI‐2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour‐stromal cell interactions

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α₁ integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.     

Attenuation of NDEA‐induced hepatocarcinogenesis by naringenin in rats

Chemoprevention is one of the most promising and realistic approaches in the prevention of cancer. Several bioactive compounds present in fruits and vegetables have revealed their cancer curative potential on hepatocellular carcinoma. Naringenin is one such naturally occurring flavonoid widely found in citrus fruits. In this study, we examined the molecular mechanisms by which naringenin inhibited NDEA‐induced hepatocellular carcinoma in rats by analysing the expression patterns of proliferating cell nuclear antigen, Bcl‐2, NF‐κB, VEGF and MMP‐2/9. Enhanced cell proliferation and apoptotic evasion in NDEA‐induced hepatocarcinogenesis was associated with imbalance in pro‐apoptotic and anti‐apoptotic proteins together with upregulation of proliferating cell nuclear antigen (PCNA) and downregulation of caspase‐3. Administration of pretreatment and posttreatment of naringenin decreased the expression of PCNA and Bcl‐2 and increased the expression of Bax and caspase‐3, indicating antiproliferative and apoptotic effects, respectively. Administration of NDEA increased the tumour expression of NF‐κB, COX‐2, VEGF, MMP‐2 and MMP‐9 that was correlated with more aggressive lesions and tumour growth. Downregulation of NF‐κB, VEGF and MMPs by naringenin seen in the present study were correlated with the inhibition of liver tumour induced by NDEA. Our results suggest that naringenin could act as a legitimate agent by inhibiting cancer processes. Copyright © 2012 John Wiley & Sons, Ltd.     

Endothelin‐1 increases the expression of VEGF‐R1/Flt‐1 receptors in rat cultured astrocytes through ETB receptors

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin‐1 (ET‐1) were examined in rat cultured astrocytes. Expressions of VEGF‐R1 and ‐R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF‐R3 and Tie‐2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF‐R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF‐E, a selective VEGF‐R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF₁₆₅ were reduced by selective antagonists for VEGF‐R1 and ‐R2. Treatment with ET‐1 increased VEGF‐R1 mRNA and protein levels. The effects of ET‐1 on VEGF‐R1 mRNA were mimicked by Ala^1,3,11,15‐ET‐1, a selective agonist for ET_B receptors, and inhibited by BQ788, an ET_B antagonist. ET‐1 did not affect the mRNA levels of VEGF‐R2, ‐R3, and Tie‐2. Pre‐treatment with ET‐1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET‐1 induces up‐regulation of VEGF‐R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.

     

Phosphorylation of Dab2 is involved in inhibited VEGF‐VEGFR‐2 signaling induced by downregulation of syndecan‐1 in glomerular endothelial cell

Disabled‐2 (Dab2) and PAR‐3 (partitioning defective 3) are reported to play critical roles in maintaining retinal microvascular endothelial cells biology by regulating VEGF‐VEGFR‐2 signaling. The role of Dab2 and PAR‐3 in glomerular endothelial cell (GEnC) is unclear. In this study, we found that, no matter whether with vascular endothelial growth factor (VEGF) treatment or not, decreased expression of Dab2 could lead to cell apoptosis by preventing activation of VEGF‐VEGFR‐2 signaling in GEnC, accompanied by reduced membrane VEGFR‐2 expression. And silencing of PAR‐3 gene expression caused increased apoptosis of GEnC by inhibiting activation of VEGF‐VEGFR‐2 signaling and membrane VEGFR‐2 expression. In our previous research, we found that the silencing of syndecan‐1 gene expression inhibited VEGF‐VEGFR‐2 signaling by modulating internalization of VEGFR‐2. And our further research demonstrated that downregulation of syndecan‐1 lead to no significant change in the expression of Dab2 and PAR‐3 both at messenger RNA and protein levels in GEnC, while phosphorylation of Dab2 was significantly increased in GEnC transfected with Dab2 small interfering RNA (siRNA) compared with control siRNA. Atypical protein kinase C (aPKC) could induce phosphorylation of Dab2, thus negatively regulating VEGF‐VEGFR‐2 signaling. And we found that decreased expression of syndecan‐1 lead to activation of aPKC, and aPKC inhibitor treatment could block phosphorylation of Dab2 in GEnC. Besides, aPKC inhibitor treatment could activate VEGF‐VGEFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA in a dose‐dependent manner. In conclusion, we speculated that phosphorylation of Dab2 is involved in preventing activation of VEGF‐VEGFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA. This provides a new target for the therapy of GEnC injury and kidney disease.