Decrease In Metalloproteinase Research Articles

MCL1 Inhibition Overcomes the Aggressiveness Features of Triple-Negative Breast Cancer MDA-MB-231 Cells.

Triple-Negative Breast Cancer (TNBC) is a particularly aggressive subtype among breast cancers (BCs), characterized by anoikis resistance, high invasiveness, and metastatic potential as well as Epithelial-Mesenchymal Transition (EMT) and stemness features. In the last few years, our research focused on the function of MCL1, an antiapoptotic protein frequently deregulated in TNBC. Here, we demonstrate that MCL1 inhibition by A-1210477, a specific BH3-mimetic, promotes anoikis/apoptosis in the MDA-MB-231 cell line, as shown via an increase in proapoptotic markers and caspase activation. Our evidence also shows A-1210477 effects on Focal Adhesions (FAs) impairing the integrin trim and survival signaling pathways, such as FAK, AKT, ERK, NF-κB, and GSK3β-inducing anoikis, thus suggesting a putative role of MCL1 in regulation of FA dynamics. Interestingly, in accordance with these results, we observed a reduction in migratory and invasiveness capabilities as confirmed by a decrease in metalloproteinases (MMPs) levels following A-1210477 treatment. Moreover, MCL1 inhibition promotes a reduction in EMT characteristics as demonstrated by the downregulation of Vimentin, MUC1, DNMT1, and a surprising re-expression of E-Cadherin, suggesting a possible mesenchymal-like phenotype reversion. In addition, we also observed the downregulation of stemness makers such as OCT3/4, SOX2, NANOG, as well as CD133, EpCAM, and CD49f. Our findings support the idea that MCL1 inhibition in MDA-MB-231 could be crucial to reduce anoikis resistance, aggressiveness, and metastatic potential and to minimize EMT and stemness features that distinguish TNBC.

MTOR inhibitor everolimus reduces invasiveness of melanoma cells

The mammalian target of rapamycin (mTOR) plays a key role in several cellular processes: proliferation, survival, invasion, and angiogenesis, and therefore, controls cell behavior both in health and in disease. Dysregulation of the mTOR signaling is involved in some of the cancer hallmarks, and thus the mTOR pathway is an important target for the development of a new anticancer therapy. The object of this study is recognition of the possible role of mTOR kinase inhibitors—everolimus single and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3 K), U0126 (ERK1/2), GDC-0879 (B-RAF), AS-703026 (MEK), MK-2206 (AKT), PLX-4032 (B-RRAF) in cell invasion in malignant melanoma. Treatment of melanoma cells with everolimus led to a significant decrease in the level of both phosphorylated: mTOR (Ser2448) and mTOR (Ser2481) as well as their downstream effectors. The use of protein kinase inhibitors produced a significant decrease in metalloproteinases (MMPs) activity, as well as diminished invasion, especially when used in combination. The best results in the inhibition of both MMPs and cell invasiveness were obtained for the combination of an mTOR inhibitor— everolimus with a B-RAF inhibitor—PLX-4032. Slightly less profound reduction of invasiveness was obtained for the combinations of an mTOR inhibitor—everolimus with ERK1/2 inhibitor—U126 or MEK inhibitor—AS-703026 and in the case of MMPs activity decrease for PI3 K inhibitor—LY294002 and AKT inhibitor—MK-2206. The simultaneous use of everolimus or another new generation rapalog with selected inhibitors of crucial signaling kinases seems to be a promising concept in cancer treatment.

Expression of Matrix Metalloproteinase-2/9 and TissueInhibitorof Metalloproteinase-1/2 as Predictive Factors in Oropharyngeal Squamous Cell Carcinoma.

Metalloproteinases and their tissue inhibitors play an important role in the metastases formation. A multistage process of carcinogenesis requires the involvement of numerous enzymes and compounds that facilitate the expansion of tumor cells. The formation of metastases depends on both metalloproteinases and tissue inhibitors activation leading to the activation of neoangiogenesis. The changes of the expression in stromal and tumor proteins could be prognostic factors in patients with oropharyngeal squamous cell carcinoma. This study was conducted on a total of 34 patients with squamous cell carcinoma of the oropharynx divided into 2 groups, including 20 patients with neck metastasis and 14 patients without lymph node metastasis. Immunohistochemistry was performed with a standard protocol. The results of the present analysis indicated a higher expression of metalloproteinases 2 in the stroma than in tumor with increasing tumor grade. The dynamics of changes in the expression of metalloproteinases showed the increase in metalloproteinases 2 and the decrease in metalloproteinases 9 depending on the tumor size. Dynamics of changes in the expression of tissue inhibitor 1 in the tumor stroma significantly increased with the tumor stage. In the assessment of nodal staging from N0 to N3, the expression of tissue inhibitor 1 and 2 were higher in the tumor tissues. The increase of metalloproteinases 2, tissue inhibitor 1 in the tumor, and metalloproteinases 9 in the stroma were characterized by a reduction in the odds ratio of patient's survival. The complex evaluation of the expression of metalloproteinases and tissue inhibitors may be used for the prognosis of the patient's survival.

Diabetes mellitus as a protective factor of abdominal aortic aneurysm: Possible mechanisms

Abdominal aortic aneurysm (AAA) shares several risk factors with atherosclerosis. Among these, diabetes mellitus (DM) could have a negative effect on the formation, growth and expansion of AAA. Several systematic reviews and meta-analyses reported up to 2016 have shown concordant results regarding the possible protective effect on AAA formation.However, the pathophysiological mechanisms of this supposed protective effect are still unknown. It appears that both hyperglycaemia and hyperinsulinemia, which are closely associated with DM, cause an increase in advanced glycation end-products, a decrease in fibrinolysis, and alterations in smooth muscle cells, leading to a decreased risk of aneurysm growth and expansion. This protective role is mainly mediated by a decrease in metalloproteinases, mainly type 2 and 9. In addition, drugs used to treat type 2 DM, as well as those for hypertension and dyslipidaemia could also play an important role in this protective effect. Metformin, thiazolidinediones, DPP4 inhibitors, statins, and angiotensin-converting enzyme inhibitors have been evaluated in this field.In conclusion, studies reported so far seem to confirm the protective effect of DM in both the formation and expansion of AAA, although future long-term studies are needed to confirm the pathophysiological mechanisms involved, as well as the role of concomitant medication.

La diabetes mellitus como factor protector del aneurisma de aorta abdominal: posibles mecanismos

Abdominal aortic aneurysm (AAA) shares several risk factors with atherosclerosis. Among these, diabetes mellitus (DM) could have a negative effect on the formation, growth and expansion of AAA. Several systematic reviews and meta-analyses reported up to 2016 have shown concordant results regarding the possible protective effect on AAA formation.However, the pathophysiological mechanisms of this supposed protective effect are still unknown. It appears that both hyperglycaemia and hyperinsulinemia, which are closely associated with DM, cause an increase in advanced glycation end-products, a decrease in fibrinolysis, and alterations in smooth muscle cells, leading to a decreased risk of aneurysm growth and expansion. This protective role is mainly mediated by a decrease in metalloproteinases, mainly type 2 and 9. In addition, drugs used to treat type 2 DM, as well as those for hypertension and dyslipidaemia could also play an important role in this protective effect. Metformin, thiazolidinediones, DPP4 inhibitors, statins, and angiotensin-converting enzyme inhibitors have been evaluated in this field.In conclusion, studies reported so far seem to confirm the protective effect of DM in both the formation and expansion of AAA, although future long-term studies are needed to confirm the pathophysiological mechanisms involved, as well as the role of concomitant medication.

Nafamostat mesilate negatively regulates the metastasis of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) lacking of oestrogen receptor, progesterone receptor, and epidermal growth factor receptor type 2 is a highly malignant disease which results in a poor prognosis and rare treatment options. Despite the use of conventional chemotherapy for TNBC tumours, resistance and short duration responses limit the treatment efficacy. Therefore, a need exists to develop a new chemotherapy for TNBC. The aim of this study was to examine the anti-cancer effects of nafamostat mesilate (NM), a previously known serine protease inhibitor and highly safe drug on breast cancer cells. Here, we showed that NM significantly inhibits proliferation, migration, and invasion in MDA-MB231 cells, induces G2/M phase cell-cycle arrest, and inhibits the expression of cyclin-dependent kinase 1 (CDK1). Exposure of MDA-MB231 cells to NM also resulted in decreased transcription factor activities accompanied by the regulated phosphorylation of signalling molecules and a decrease in metalloproteinases, the principal modulators of the extracellular environment during cancer progression. Especially, inhibition of TGFβ-stimulated Smad2 phosphorylation and subsequent metastasis-related gene expression, and downregulation of ERK activity may be pivotal mechanisms underlying inhibitory effects of NM on NM inhibits lung metastasis of breast cancer cells and growth of colonized tumours in mice. Taken together, our data revealed that NM inhibits cell growth and metastasis of TNBC cells and indicated that NM is a multi-targeted drug that could be an adjunct therapy for TNBC treatment.

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.

Abstract 4669: Targeting hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

Abstract Melanoma represents the deadliest of all skin cancers being responsible for more than 75% of skin cancer-related deaths. Although the results with BRAF inhibitors have been very promising, practically all of the patients treated thus far have developed resistance to these drugs. The Hedgehog (Hh) signaling pathway has been implicated in a variety of malignancies, including melanoma. However, there are no reports on the activation of this pathway in the setting of vemurafenib-resistance. Herein, we first identified that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, showed increased levels of Hh pathway component genes glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared to naïve cells. We also observed these findings in melanoma samples from patients. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with non-canonical Hh signaling, involving TGFβ/SMAD signaling. Knockdown of GLI1 and GLI2 restored the vemurafenib-resistant cells to sensitivity, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a 3D skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphtalmia transcription factor (MITF) upregulation. Our findings also demonstrated that GLI1/GLI2 modulation could be a useful strategy to prevent drug resistance. Alternating pre-treatment with vemurafenib and Gant61 significantly reduced IC50 of subsequent vemurafenib treatment in naïve melanoma cells, demonstrating a promising approach for the control of vemurafenib-resistance in patients with unresectable melanoma. The modulation of vemurafenib chemosensitivity was triggered by GLI1/GLI2 repression during the acquisition of resistance, which did not occur in the treatment protocols with only vemurafenib continuously or in alternate days. The alternating treatment regimen with vemurafenib and Gant61 was able to suppress GLI expression, delaying or decreasing vemurafenib resistance. Taken together, our data demonstrated an unprecedented mechanism of vemurafenib resistance by GLI1/GLI2 upregulation, shedding light on the development of Hh pathway inhibitors as a promising strategy for melanoma treatment. Citation Format: Fernanda Faiao-Flores, Debora Kristina Magalhaes Alves-Fernandes, Paula Comune Pennacchi, Silvana Sandri, Anna Luiza Silva Almeida Vicente, Cristovam Scapulatempo-Neto, Vinicius de Lima Vazquez, Rui Manuel Reis, Keiran S. Smalley, Silvya Stuchi Maria-Engler. Targeting hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4669.

Suppressive Effect of Dietary Fucoidan on Proinflammatory Immune Response and MMP-1 Expression in UVB-Irradiated Mouse Skin.

It is well known that ultraviolet B irradiation leads to dermal inflammation. In this study, we found that Mekabu fucoidan suppressed edema, decreased the thickness of the prickle cell layer, and decreased matrix metalloproteinase 1 in the skin of mice irradiated with ultraviolet B. Moreover, we found that the mean level of interferon gamma of Mekabu fucoidan-treated, ultraviolet B-irradiated mice (approximately 2.2 ng/mL) was not significantly different from that in normal mice (approximately 2.5 ng/mL). In contrast, a significant decrease in the mean level of interferon gamma (approximately 1.3 ng/mL) in ultraviolet B-irradiated control mice was observed compared with that in Mekabu fucoidan-treated, ultraviolet B-irradiated mice. The mean thickness of the prickle cell layer in the skin of Mekabu fucoidan-treated, ultraviolet B-irradiated mice was less than that in the ultraviolet B-irradiated control mice. Metalloproteinase 1 activity was significantly higher in the skin of ultraviolet B-irradiated mice than in the skin of untreated, nonirradiated normal mice. Metalloproteinase 1 in the skin of ultraviolet B-irradiated, Mekabu fucoidan- or L(+)-ascorbic acid (vitamin C)-treated mice was significantly lower than that in the ultraviolet B-irradiated control mice. Mitigation of the morphological changes in Mekabu fucoidan-treated mice was correlated with a decrease in metalloproteinase 1 levels. These data indicate that Mekabu fucoidan is an effective suppressor of inflammation in an ultraviolet B-irradiated mouse model.

MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9

AbstractMegakaryoblastic leukemia 1 (MAL) is a transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding one twenty-two (OTT). Herein, we show that MAL expression increases during the late differentiation steps of neonate and adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin. MAL knockdown in megakaryocyte progenitors reduced the percentage of cells forming filopodia, lamellipodia, and stress fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic megakaryocytes with disorganized demarcation membranes and α granules heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression after MAL inhibition. Luciferase assays in HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro. Megakaryocyte migration in response to stromal cell–derived factor 1, through Matrigel was considerably decreased after MAL knockdown, implicating MMP9 in migration. Finally, the use of a shRNA to decrease MYL9 expression showed that MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved in platelet formation and megakaryocyte migration by regulating MYL9 and MMP9.

Development of the heart in ANP−/− mice during early postnatal period

Disruption of the proANP gene results in marked cardiac hypertrophy, chronic and salt‐sensitive hypertension. It is unclear when cardiac hypertrophy in ANP−/− mice appears during development. The present study was designed to examine cardiac hypertrophy in ANP−/− mice during early postnatal development and correlate it with expression of the renin‐angiotensin (RAS) and the extracellular matrix (ECM) systems. Hearts were excised from 1–5 week old neonatal and 12 week adult male ANP+/+ and ANP−/− mice. Total mRNA was extracted from whole neonatal hearts and adult left ventricles, and evaluated using Northern blot analysis and quantitative RT‐PCR. Cardiac hypertrophy as indicated by an increase in heart/body weight ratio was evident in ANP−/− mice at all age groups. The most significant molecular changes were an increase in the expression of cardiac BNP mRNA level and a decrease in metalloproteinase 2 (MMP‐2) mRNA of ANP−/− as compared to the ANP+/+ mice. These data support the idea that the renin‐angiotensin system plays a minor role in the development of cardiac hypertrophy in the proANP gene disrupted mice and suggest the essential role of ANP and ECM in the development and maintenance of cardiac hypertrophy in the ANP−/− mice. Supported by the Heart and Stroke Foundation of Ontario.