Hydrophilic extract from Posidonia oceanica inhibits activity and expression of gelatinases and prevents HT1080 human fibrosarcoma cell line invasion

Posidonia oceanica (L.) Delile is an endemic Mediterranean sea-grass distributed in the infralittoral zones, where it forms meadows playing a recognized ecological role in the coastal marine habitat. Although its use as a traditional herbal remedy is poorly documented, recent literature reports interesting pharmacological activities as antidiabetic, antioxidant and vasoprotective. Differently from previous literature, this study presents a hydrophilic extraction method that recovers metabolites that may be tested in biological buffers. We showed for the first time in the highly invasive HT1080 human fibrosarcoma cell line that our hydrophilic extract from P. oceanica was able to strongly decrease gene and protein expression of gelatinases MMP-2 and MMP-9 and to directly inhibit in a dose-dependent manner gelatinolytic activity in vitro. Moreover, we have revealed that our extract strongly inhibited HT1080 cell migration and invasion. Biochemical analysis of the hydrophilic extract showed that catechins were the major constituents with minor contribution of gallic acid, ferulic acid and chlorogenic plus a fraction of uncharacterized phenols. However, if each individual compound was tested independently, none by itself was able to induce a direct inhibition of gelatinases as strong as that observed in total extract, opening up new routes to the identification of novel compounds. These results indicate that our hydrophilic extract from P. oceanica might be a source of new pharmacological natural products for treatment or prevention of several diseases related to an altered MMP-2 and MMP-9 expression.     

Lysophosphatidic acid receptor-5 negatively regulates cell motile and invasive activities of human sarcoma cell lines

LPA signaling via LPA receptors [LPA receptor-1 (LPA₁)–LPA₆] mediates the several cellular responses in cancer cells, including cell motility and invasion. In the present study, to investigate a role of LPA₅ in the cell motile and invasive activities of sarcoma cells, LPAR5 knockdown (HOSL5 and HT1080L5) cells were generated from human osteosarcoma HOS and fibrosarcoma HT1080 cells, respectively. In cell motility assays with cell culture inserts, HOSL5 and HT1080L5 cells indicated the high cell motile activities, compared with control cells. The cell invasive activities of HOSL5 and HT1080L5 cells were significantly higher than those of control cells. Moreover, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by gelatin zymography. MMP-2 was significantly activated in HOSL5 cells, but not MMP-9. The elevated activities of MMP-2 and MMP-9 were found in HT1080L5 cells, in comparison with control cells. These results suggest that LPA signaling via LPA₅ negatively regulates the cell motile and invasive activities of human sarcoma cells.     

Functional mimetic peptide discovery isolated by phage display interacts selectively to fibronectin domain and inhibits gelatinase

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cell invasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies.     

Resveratrol with antioxidant activity inhibits matrix metalloproteinase via modulation of SIRT1 in human fibrosarcoma cells

AimsResveratrol, a silent information regulator 1 (SIRT1) activator, has been reported to act as an antioxidant contained in red wine and prevent the development of cardiovascular diseases. Histone deacetylase such as SIRT1 is involved in the regulation of lifespan extension. In this study, the effect of resveratrol on matrix metalloproteinases (MMPs) that play an important role in metastasis was examined in human fibrosarcoma cell line, HT1080.Main methodsThe effect of resveratrol on MMPs' activity was evaluated using gelatin zymography. Western blot analysis and RT-PCR assay were used to determine the effect of resveratrol on the expression level of MMP-9, MAPK and SIRT1 proteins and genes, respectively.Key findingsIt was observed that resveratrol exhibited not only antioxidant effects on lipid peroxidation and DNA oxidation but also inhibitory effects on the expression of MMP-2 and 9 in HT1080 cells stimulated with either phorbol myristate acetate or phenazine methosulfate. Furthermore, it was found that treatment with resveratrol decreased the level of MMP-9 expression via down-regulation of p-ERK, c-fos and p65. In addition, the level of SIRT1 gene expression was also enhanced by treatment of resveratrol alone but the level of MMP-9 gene expression was decreased.SignificanceThese results suggest that the activation of SIRT1 in the presence of resveratrol especially inhibits the expression of MMP-9 in HT1080 cells, providing evidence that resveratrol can be a potential candidate for chemoprevention of cancer.     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity

Matrix metalloproteinase (MMP) inhibitors have been identified as potential therapeutic candidates for metastasis, arthritis, chronic inflammation and wrinkle formation. For the first time here we report a detailed study on the inhibitory effects of phlorotannins in brown algae, Ecklonia cava (EC) on MMP activities in cultured human cell lines. A novel gelatin digestion assay could visualize complete inhibition of bacterial collagenase-1 activity at 20 microg/ml of EC extract during preliminary screening studies. Sensitive fluorometric assay revealed that EC extract can specifically inhibit both MMP-2 and MMP-9 activities significantly (P < 0.001) at 10 microg/ml. In addition, artificially induced activities of MMP-2 and MMP-9 in human dermal fibroblasts and HT1080 cells were inhibited by EC extract in a more or less similar manner to the positive control doxycycline. Even though the expression levels of MMPs differ from one cell type to the other, gelatin zymography clearly revealed that both MMP expression and activity in cells can be inhibited by EC extract. More interestingly, EC extract did not exert any cytotoxic effect even at 100 microg/ml anticipating its potential use as a safe MMP inhibitor.     

Impaired DNA repair and genomic stability in hereditary tyrosinemia type 1

The autosomal recessive disorder, hereditary tyrosinemia type 1 (HT1), is caused by a defective fumarylacetoacetate hydrolase enzyme. Consequently intermediate metabolites such as fumarylacetoacetate, succinylacetone and p-hydroxyphenylpyruvic acid accumulate. Characteristic to HT1 is the development of hepatocellular carcinoma, irrespective of dietary intervention or pharmacological treatment. Carcinogenesis may occur through a chromosomal instability mutator phenotype or a microsatellite instability phenotype, and deficient DNA repair may be a contributing factor thereof. The purpose of this study was to investigate the expression of DNA repair proteins, and the possible occurrence of microsatellite instability in HT1. Gene expression analyses show low expression of hOGG1 and ERCC1 in HT1 patient lymphocytes. Results from microsatellite instability analyses show allelic imbalance on chromosome 7 of the fah^−/− mouse genome, and instability of the D2S123, D5S346 and (possibly) D17S250 microsatellite markers, in HT1 patient lymphocytes.     

Wild chrysanthemum extract prevents UVB radiation-induced acute cell death and photoaging

Wild chrysanthemum (Chrysanthemum indicum L.) is traditionally used in folk medicine as an anti-inflammatory agent. It is also used in the southwest plateau region of China to prevent ultraviolet-induced skin damage. However, the role and mechanism by which wild chrysanthemum prevents UV-induced skin damage and photoaging have never been investigated in vitro. In the present study, we found that aqueous extracts from wild chrysanthemum strongly reduced high-dose UVB-induced acute cell death of human immortalized keratinocytic HaCat cells. Wild chrysanthemum extract was also demonstrated to reduce low-dose UVB-induced expression of the photoaging-related matrix metalloproteinases MMP-2 and MMP-9. The ROS level elevated by UVB irradiation was strongly attenuated by wild chrysanthemum extract. Further study revealed that wild chrysanthemum extract reduced UVB-triggered ERK1/2 and p38 MAPK phosphorylation and their protective role, which is partially dependent on inhibiting p38 activation. These results suggest that wild chrysanthemum extract can protect the skin from UVB-induced acute skin damage and photoaging by reducing the intracellular reactive oxygen species (ROS) level and inhibiting p38 MAPK phosphorylation. The present study confirmed the protective role of wild chrysanthemum against UV-induced skin disorders in vitro and indicated the possible mechanism. Further study to identify the active components in wild chrysanthemum extract would be useful for developing new drugs for preventing and treating skin diseases, including skin cancer and photoaging, induced by UV irradiation.     

Identifying specific matrix metalloproteinase-2-inhibiting peptides through phage display-based subtractive screening

Gelatinases A and B, which are members of the matrix metalloproteinase (MMP) family, play essential roles in cancer development and metastasis, as they can break down basal membranes. Therefore, the determination and inhibition of gelatinases is essential for cancer treatment. Peptides that can specifically block each gelatinase may, therefore, be useful for cancer treatment. In this study, subtractive panning was carried out using a 12-mer peptide library to identify peptides that block gelatinase A activity (MMP-2), which is a key pharmacological target. Using this method, 17 unique peptide sequences were determined. MMP-2 inhibition by these peptides was evaluated through zymogram analyses, which revealed that four peptides inhibited MMP-2 activity by at least 65%. These four peptides were synthesized and used for in vitro wound healing using human umbilical vein endothelial cells, and two peptides, AOMP12 and AOMP29, were found to inhibit wound healing by 40%. These peptides are, thus, potential candidates for MMP-2 inhibition for cancer treatment. Furthermore, our findings suggest that our substractive biopanning screening method is a suitable strategy for identifying peptides that selectively inhibit MMP-2.     

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.     

Assessing effects of fishing prohibition on Posidonia oceanica seagrass meadows in the Marine Natural Reserve of Cerbère-Banyuls

The potential of alleviated fishing pressure measures established at the Marine Natural Reserve of Cerbère-Banyuls to affect phenological characters of P. oceanica, the main canopy-forming seagrass, was studied. Our results show differences in some leaf parameters between meadows under fishing prohibition measures compared to those without. In addition shallow P. oceanica meadows inside MPAs had lower non-structural carbohydrate content, yet are able to maintain themselves and, moreover, with an increased density. On the other hand, sexual reproduction was reduced inside protected areas.     

Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor

The 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) acting as a receptor for prions and viruses is overexpressed in various cancer cell lines, and their metastatic potential correlates with LRP/LR levels. We analyzed the tumorigenic fibrosarcoma cell line HT1080 regarding 37-kDa/67-kDa LRP/LR levels and its invasive potential. Compared to the less invasive embryonic fibroblast cell line NIH3T3, the tumorigenic HT1080 cells display approximately 1.6-fold higher cell-surface levels of LRP/LR. We show that anti-LRP/LR tools interfere with the invasive potential of HT1080 cells. Anti-LRP/LR single-chain variable fragment antibody (scFv) iS18 generated by chain shuffling from parental scFv S18 and its full-length version immunoglobulin G1-iS18 reduced the invasive potential of HT1080 cells significantly by 37% and 38%, respectively. HT1080 cells transfected with lentiviral plasmids expressing small interfering RNAs directed against LRP mRNA showed reduced LRP levels by approximately 44%, concomitant with a significant decrease in the invasive potential by approximately 37%. The polysulfated glycans HM2602 and pentosan polysulfate (SP-54), both capable of blocking LRP/LR, reduced the invasive potential by 20% and 35%, respectively. Adhesion of HT1080 cells to laminin-1 was significantly impeded by scFv iS18 and immunoglobulin G1-iS18 by 60% and 68%, respectively, and by SP-54 and HM2602 by 80%, suggesting that the reduced invasive capacity achieved by these tools is due to the perturbation of the LRP/LR-laminin interaction on the cell surface. Our in vitro data suggest that reagents directed against LRP/LR or LRP mRNA such as antibodies, polysulfated glycans, or small interfering RNAs, previously shown to encompass an anti-prion activity by blocking or downregulating the prion receptor LRP/LR, might also be potential cancer therapeutics blocking metastasis by interfering with the LRP/LR-laminin interaction in neoplastic tissues.     

Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

Background

Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).

Experimental

Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR.

Conclusion

This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.     

Cytotoxic and pro-apoptotic effects of Abrus precatorius L. on human metastatic breast cancer cell line, MDA-MB-231

Abrus precatorius is highly regarded as a universal panacea in the herbal medicine with diverse pharmacological activity spectra. This experimental study on the mechanism of the anticancer activity of A. precatorius leaf extracts, may offer new evidence for A. precatorius in the treatment of breast cancer in clinical practice. Cell death was determined by using MTT assay. Further analyses were carried out by doing DNA laddering, PARP cleavage, FACS, semi-quantitative RT-PCR and detection of cellular reactive oxygen species (ROS) by DCFDA assay. A. precatorius showed very striking inhibition on MDA-MB-231 cells. MTT assay showed more than 75 % inhibition of the cells and treated cells indicated visible laddering pattern with thick compact band. PARP cleavage produced 89 kDa cleavage product which was associated with apoptosis. Flow cytometer exhibited a sub-G0/G1 peak as an indicative of apoptosis. mRNA expression level of apoptosis-related genes p21 and p53 was markedly increased in cells treated with the extract as compared to control. The up-regulation of p21 and p53 may be the molecular mechanisms by which A. precatorius extract which induces apoptosis. An increase in the concentration of A. precatorius extract does not generate ROS, instead it reduces ROS formation in MDA-MB-231 cells, as evident from the shift in fluorescence below untreated control. This is the first report showing that A. precatorius leaf extract exhibits a growth inhibitory effect by induction of apoptosis in MDA-MB-231 cells. Our results contribute towards validation of the A. precatorius extract as a potentially effective chemopreventive or therapeutic agent against breast cancer.