SNAI1-mediated transcriptional regulation of epithelial-to-mesenchymal transition genes in breast cancer stem cells.

BACKGROUND: Triple-negative breast cancer (TNBC) tumors are composed of a heterogeneous population containing both cancer cells and cancer stem cells (CSCs). These CSCs are generated through an epithelial-to-mesenchymal transition (EMT), thus making it pertinent to identify the unique EMT-molecular targets that regulate this phenomenon. METHODS AND RESULTS: In the present study, we performed in silico analysis of microarray data from luminal, Her2+, and TNBC cell lines and identified 15 relatively unexplored EMT-related differentially expressed genes (DEGs) along with the markedly high expression of EMT-transcription factor (EMT-TF), SNAI1. Interestingly, stable overexpression of SNAI1 in MCF-7 induced the expression of DEGs along with increased migration, invasion, and in vitro tumorigenesis that was comparable to TNBCs. Next, stable SNAI1 overexpression led to increased expression of DEGs that was reverted with SNAI1 silencing in both breast cancer cells and CSCs sorted from various TNBC cell lines. Higher fold enrichment of SNAI1 on E-boxes in the promoter regions suggested a positive regulation of ALCAM, MMP2, MMP13, MMP14, VCAN, ANKRD1, KRT16, CTGF, TGFRIIß, PROCR negative regulation of CDH1, DSP and DSC3B by SNAI1 leading to EMT. Furthermore, SNAI1-mediated increased migration, invasion, and tumorigenesis in these sorted cells led to the activation of signaling mediators, ERK1/2, STAT3, Src, and FAK. Finally, the SNAI1-mediated activation of breast CSC phenotypes was perturbed by inhibition of downstream target, MMPs using Ilomastat. CONCLUSION: Thus, the molecular investigation for the gene regulatory framework in the present study identified MMPs, a downstream effector in the SNAI1-mediated EMT regulation.

Effect of GM6001 on the expression of syndecan-1 in rats with acute kidney injury and its protective effect on the kidneys.

Expression of syndecan-1 (SDC-1) in rats with acute kidney injury and the protective effect of GM6001 on the kidney were investigated. Fifty SD rats were selected and randomly divided into control group (CG) (n=15), treatment control group (TCG) (n=10), module group (MG) (n=15) and treatment group (TG) (n=10). In TG, the model of acute renal injury (AKI) in rats was established after pretreatment of intraperitoneal injection of GM6001 one day before modeling. In MG, the same amount of saline was injected intraperitoneally one day before modeling and the same treatment was done on the day of modeling. In CG, the same amount of saline was injected intraperitoneally one day before modeling but the model was not made. In TCG, rats were pretreated with intraperitoneal injection of GM6001 one day before modeling but the model was not made. The contents of blood urea nitrogen (BUN) in serum, serum creatinine (SCR), uric acid (UA) and blood ß2-microglobulin (ß2-MG) were detected by ELISA. The content of SDC-1 in renal tissues was detected by qRT-PCR and western blotting. Expression of SDC-1 in renal tissue of 24 rats after modeling was lower than that of MG (P<0.050). SDC-1 expression was the highest in TG (P<0.05). Compared with before modeling, the contents of BUN, SCR, UA and ß2-MG in MG and TG increased (P<0.05). After modeling, the contents of serum BUN, SCR, UA and ß2-MG in TG were significantly lower than those in MG (P<0.05). The levels of SDC-1 in renal tissue of rats with acute kidney injury increased. After GM6001 treatment, SDC-1 levels can be improved and has a certain protective effect on the kidneys.

Metalloproteinase inhibitor GM6001 delays regeneration in holothurians.

The effect of the GM6001 metalloproteinase inhibitor on the regeneration of ambulacral structures in Eupentacta fraudatrix has been investigated. Inhibition of proteinase activity exerts a marked effect on regeneration, being dependent on the time when GM6001 is injected. When administration of the inhibitor begins on day 3 post-injury, regeneration is completely abolished, and the animals die. This means that early activation of proteinases is crucial for triggering the regenerative process in holothurians. When GM6001 in first injected on day 7 post-injury, the regeneration rate decreases. However, this effect has proven to be reversible: when inhibition ceases, the regeneration resumes. The effect of the inhibitor is manifested as a retarded degradation of the extracellular matrix, the lack of cell dedifferentiation, and, probably, a slower cell migration. The gelatinase activity is detected in all the regenerating organs of E. fraudatrix. In the holothurian Cucumaria japonica, which is not capable of healing skin wounds and ambulacrum reparation, no gelatinase activity was observed at the site of damage. A suggestion is made that proteinases play an important role in regeneration in holothurians. The most probable morphogenesis regulators are matrix metalloproteinases with gelatinase activity.

Local inhibition of matrix metalloproteinases reduced M2 macrophage activity and impeded recovery in spinal cord transected rats after treatment with fibroblast growth factor-1 and nerve grafts.

Alternatively activated macrophages (M2 macrophages) promote central nervous system regeneration. Our previous study demonstrated that treatment with peripheral nerve grafts and fibroblast growth factor-1 recruited more M2 macrophages and improved partial functional recovery in spinal cord transected rats. The migration of macrophages is matrix metalloproteinase (MMP) dependent. We used a general inhibitor of MMPs to influence macrophage migration, and we examined the migration of macrophage populations and changes in spinal function. Rat spinal cords were completely transected at T8, and 5 mm of spinal cord was removed (group T). In group R, spinal cord-transected rats received treatment with fibroblast growth factor-1 and peripheral nerve grafts. In group RG, rats received the same treatment as group R with the addition of 200 µM GM6001 (an MMP inhibitor) to the fibrin mix. We found that MMP-9, but not MMP-2, was upregulated in the graft area of rats in group R. Local application of the MMP inhibitor resulted in a reduction in the ratio of arginase-1 (M2 macrophage subset)/inducible nitric oxide synthase-postive cells. When the MMP inhibitor was applied at 8 weeks postoperation, the partial functional recovery observed in group R was lost. This effect was accompanied by a decrease in brain-derived neurotrophic factor levels in the nerve graft. These results suggested that the arginase-1 positive population in spinal cord transected rats is a migratory cell population rather than the phenotypic conversion of early iNOS+ cells and that the migration of the arginase-1+ population could be regulated locally. Simultaneous application of MMP inhibitors or promotion of MMP activity for spinal cord injury needs to be considered if the coadministered treatment involves M2 recruitment.

Influence of MMP inhibitor GM6001 loading of fibre coated polypropylene meshes on wound healing: Implications for hernia repair.

Polypropylene meshes are standard for hernia repair. Matrix metalloproteinases play a central role in inflammation. To reduce the inflammatory response and improve remodelling with an associated reduction of hernia recurrence, we modified polypropylene meshes by nanofibre coating and saturation with the broad-spectrum matrix metalloproteinase inhibitor GM6001. The aim was to modulate the inflammatory reaction, increase collagen deposition and improve mesh biointegration. Polypropylene meshes were surface-modified with star-configured NCO-sP(EO -stat-PO) and covered with electrospun nanofibres (polypropylene-nano) and GM6001 (polypropylene-nano-GM). In a hernia model, defects were reconstructed with one of the meshes. Inflammation, neovascularization, bio-integration, proliferation and apoptosis were assessed histologically, collagen content and gelatinases biochemically. Mesh surface modification resulted in higher inflammatory response compared to polypropylene. Pro-inflammatory matrix metalloproteinase-9 paralleled findings while GM6001 reduced matrix metalloproteinase-9 significantly. Significantly increased matrix metalloproteinase-2 beneficial for remodelling was noted with polypropylene-nano-meshes. Increased vascular endothelial growth factor, neo-vascularization and collagen content were measured in polypropylene-nano-meshes compared to polypropylene. GM6001 significantly reduced myofibroblasts. This effect ended after d14 due to engineering limitations with release of maximal GM6001 loading. Nanofibre-coating of polypropylene-meshes confers better tissue vascularization to the cost of increased inflammation. This phenomenon can be only partially compensated by GM6001. Future research will enable higher GM6001 uptake in nano-coated meshes and may alter mesh biointegration in a more pronounced way.

Extracellular matrix remodeling and matrix metalloproteinase inhibition in visceral adipose during weight cycling in mice.

Extracellular matrix (ECM) remodeling is necessary for a health adipose tissue (AT) expansion and also has a role during weight loss. We investigate the ECM alteration during weight cycling (WC) in mice and the role of matrix metalloproteinases (MMPs) was assessed using GM6001, an MMP inhibitor, during weight loss (WL). Obesity was induced in mice by a high-fat diet. Obese mice were subject to caloric restriction for WL followed by reintroduction to high-fat diet for weight regain (WR), resulting in a WC protocol. In addition, mice were treated with GM6001 during WL period and the effects were observed after WR. Activity and expression of MMPs was intense during WL. MMP inhibition during WL results in inflammation and collagen content reduction. MMP inhibition during WL period interferes with the period of subsequent expansion of AT resulting in improvements in local inflammation and systemic metabolic alterations induced by obesity. Our results suggest that MMPs inhibition could be an interesting target to improve adipose tissue inflammation during WL and to support weight cyclers.

DCE-MRI biomarkers for monitoring an anti-angiogenic triple combination therapy in experimental hypopharynx carcinoma xenografts with immunohistochemical validation.

BACKGROUND: Novel anti-angiogenic treatments are increasingly complementing established cancer therapy strategies in head and neck tumors. Contrast-enhanced magnetic resonance imaging (MRI) can be applied for early and non-invasive therapy monitoring by non-invasive quantitative assessment of tumor microcirculation as in vivo imaging biomarkers of therapy response. PURPOSE: To monitor the anti-angiogenic effects of a novel combination therapy on experimental head and neck squamous cell carcinomas (HNSCC) with dynamic contrast-enhanced (DCE)-MRI. MATERIAL AND METHODS: Athymic rats (n = 18) with subcutaneous HNSCC xenografts were investigated by DCE-MRI before and after 7 days of a daily triple therapy regimen combining the COX-II-inhibitor celecoxib, the matrix-metalloproteinase-inhibitor GM6001, and the uPA-inhibitor upamostat. Quantitative measurements of tumor blood flow (tBF), tumor blood volume (tBV), and permeability-surface area product (PS) were calculated and validated by immunohistochemistry. RESULTS: Mean tBF and tBV in triple-therapy animals decreased significantly from day 0 to day 7 (tBF, 41.0 ± 14.2 to 20.4 ± 5.7 mL/100 mL/min; P < 0.01; tBV, 17.7 ± 3.9 to 7.5 ± 3.3%; P < 0.01). No significant effects on PS were observed in either group (P > 0.05). Immunohistochemical analysis showed a significantly lower tumor vascularity in the therapy group than in the control group (CD31), significantly fewer Ki-67+ proliferating tumor cells and significantly more Capase-3+ apoptotic tumor cells (P < 0.05). Significant (P < 0.05) correlations were observed between tBF/tBV and CD31 (tBF, r = 0.84; tBV, r = 0.70), tBV and Ki-67 (r = 0.62), as well as tBF and caspase-3 (r = -0.64). CONCLUSION: DCE-MRI may be a suitable tool for the non-invasive monitoring of the anti-vascular effects of this innovative triple therapy regimen with potential for clinical translation.

The phosphoinositide-dependent protein kinase 1 inhibitor, UCN-01, induces fragmentation: possible role of metalloproteinases.

Phosphoinositide-dependent protein kinase 1 (PDK1) is a key enzyme, master regulator of cellular proliferation and metabolism; it is considered a key target for pharmacological intervention. Using membranes obtained from DDT1 MF-2 cells, phospho-PDK1 was identified by Western blotting, as two major protein bands of Mr 58-68 kDa. Cell incubation with the PDK1 inhibitor, UCN-01, induced a time- and concentration-dependent decrease in the amount of phospho-PDK1 with a concomitant appearance of a ≈42 kDa phosphorylated fragment. Knocking down PDK1 diminished the amount of phospho-PDK1 detected in membranes, accompanied by similarly decreased fragment generation. UCN-01-induced fragment generation was also observed in membranes from cells stably expressing a myc-tagged PDK1 construct. Other PDK1 inhibitors were also tested: OSU-03012 induced a clear decrease in phospho-PDK1 and increased the presence of the phosphorylated fragment in membrane preparations; in contrast, GSK2334470 and staurosporine induced only marginal increases in the amount of PDK1 fragment. Galardin and batimastat, two metalloproteinase inhibitors, markedly attenuated inhibitor-induced PDK1 fragment generation. Metalloproteinases 2, 3, and 9 co-immunoprecipitated with myc-PDK1 under baseline conditions and this interaction was stimulated by UCN-01; batimastat also markedly diminished this effect of the PDK1 inhibitor. Our results indicate that a series of protein kinase inhibitors, namely UCN-01 and OSU-03012 and to a lesser extent GSK2334470 and staurosporine induce PDK1 fragmentation and suggest that metalloproteinases could participate in this effect.

Tissue-engineered 3D tumor angiogenesis models: potential technologies for anti-cancer drug discovery.

Angiogenesis is indispensable for solid tumor expansion, and thus it has become a major target of cancer research and anti-cancer therapies. Deciphering the arcane actions of various cell populations during tumor angiogenesis requires sophisticated research models, which could capture the dynamics and complexity of the process. There is a continuous need for improvement of existing research models, which engages interdisciplinary approaches of tissue engineering with life sciences. Tireless efforts to develop a new model to study tumor angiogenesis result in innovative solutions, which bring us one step closer to decipher the dubious nature of cancer. This review aims to overview the recent developments, current limitations and future challenges in three-dimensional tissue-engineered models for the study of tumor angiogenesis and for the purpose of elucidating novel targets aimed at anti-cancer drug discovery.

Relationship of gelatinases-tight junction proteins and blood-brain barrier permeability in the early stage of cerebral ischemia and reperfusion.

Gelatinases matrix metalloproteinase-2 and matrix metalloproteinase-9 have been shown to mediate claudin-5 and occludin degradation, and play an important regulatory role in blood-brain barrier permeability. This study established a rat model of 1.5-hour middle cerebral artery occlusion with reperfusion. Protein expression levels of claudin-5 and occludin gradually decreased in the early stage of reperfusion, which corresponded to the increase of the gelatinolytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. In addition, rats that received treatment with matrix metalloproteinase inhibitor N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpenthanoyl]-L-tryptophan methylamide (GM6001) showed a significant reduction in Evans blue leakage and an inhibition of claudin-5 and occludin protein degradation in striatal tissue. These data indicate that matrix metalloproteinase-2 and matrix metalloproteinase-9-mediated claudin-5 and occludin degradation is an important reason for blood-brain barrier leakage in the early stage of reperfusion. The leakage of the blood-brain barrier was present due to gelatinases-mediated degradation of claudin-5 and occludin proteins. We hypothesized that the timely closure of the structural component of the blood-brain barrier (tight junction proteins) is of importance.

Vaccinium myrtillus (Bilberry) Extracts Reduce Angiogenesis In Vitro and In Vivo.

Vaccinium myrtillus (Bilberry) extracts (VME) were tested for effects on angiogenesis in vitro and in vivo. VME (0.3-30 µg ml(-1)) and GM6001 (0.1-100 µM; a matrix metalloproteinase inhibitor) concentration-dependently inhibited both tube formation and migration of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor-A (VEGF-A). In addition, VME inhibited VEGF-A-induced proliferation of HUVECs. VME inhibited VEGF-A-induced phosphorylations of extracellular signal-regulated kinase 1/2 (ERK 1/2) and serine/threonine protein kinase family protein kinase B (Akt), but not that of phospholipase Cγ (PLCγ). In an in vivo assay, intravitreal administration of VME inhibited the formation of neovascular tufts during oxygen-induced retinopathy in mice. Thus, VME inhibited angiogenesis both in vitro and in vivo, presumably by inhibiting the phosphorylations of ERK 1/2 and Akt. These findings indicate that VME may be effective against retinal diseases involving angiogenesis, providing it can reach the retina after its administration. Further investigations will be needed to clarify the major angiogenesis-modulating constituent(s) of VME.

Research on the intervention of TIMPs-GM6001 in the traumatic proliferative vitreoretinopathy / 眼科

Objective To investigate the expression of MMP-2, MMP-9 and TIMP-1. TIMP-2 during the course of traumatie pro- liferative vitreoretinopathy (tPVR) in rat retina treated with GM6001 and without GM6001 and evaluate the interventiunal effect uf GM6001 on tPVR. Design Experimental study. Participants 108 SD rats. Methods Rats were divided randomly into three groups: the Ns control group, the tPVR group, the tPVR treated with GM6001 group. The expression of MMP-2. MMP-9 and TIMP-1. TIMP-2 in retina was analyzed by Western Blot on day 1, 3. 7, 14, 21 and 28. Main Outcome Measures The expression of MMP-2, MMP-9, TIMP-2, TIMP-1 in the SD rats' retina of every group. Results The results of Western Blot showed that the expression of MMP-2 in the SD rats' retina of the tPVR group was stronger than the one in other groups at day 3, 7, 14, 21 and 28: the tPVR treated with GM6001 group was weaker than the one in the tPVR group at day 14, 21 and 28d. The expression of MMP-9 in the tPVR group was stronger than the one in other groups at all time; the cireumstance of tPVR treated with GM6001 group was weaker than the one in the tPVR group at day 1, 3, 7, 14 and 21. The rate of MMP-2/TIMP-2 of the SD rats" retina in the tPVR group increased at day 14, 21 and 28, and it was lower in the tPVR treated with GM6001 group compared with the tPVR group. The rate of MMP-9/TIMP-1 in the tPVR group increased at all time, and it degraded in the tPVR treated with GM6001 group as compared with the tPVR group. Conclusions The dishalance of MMP-2 and TIMP-2, MMP-9 and TIMP-1 involves with the course of tPVR. GM6001 playes an important role in in- terfering the course of tPVR by regulating the balance of these cell faetors.