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.     

Mechlorethamine-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells

Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.     

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.     

Activation of anchorage-independent growth of HT1080 human fibrosarcoma cells by dexamethasone

Anchorage independence is an important hallmark of the transformation that correlates with tumorigenicity. We have isolated a variant clone of HT1080 human fibrosarcoma cells (cl-2) that is specifically defective in anchorage-independent growth. Interestingly, 10(-7) M dexamethasone (DEX) substantially rescued the anchorage-independent growth of cl-2 cells in semisolid culture. DEX also promoted the anchorage-independent growth of parental HT1080 cells. However, the agent had no effect on the anchorage-dependent growth of cl-2 and parental cells in ordinary liquid culture. Cell cycle analysis demonstrated that the population of G0/G1 cells increased, whereas that of S and G2/M cells decreased in growth-arrested cl-2 cells in suspension culture. However, such an effect of anchorage loss on cell cycle progression was alleviated by adding 10(-7) M DEX. In cl-2 cells in semisolid culture, DEX suppressed the expression of P27Kip1, whereas it stimulated the expression of cyclin A and hyperphosphorylated retinoblastoma (Rb) proteins. On the other hand, DEX had no effect on cyclin D1 and P21Cap1 expression. These effects of DEX, except for the suppression of P27Kip1, were blocked by an antimicrofilament drug, cytochalasin D. Our results suggest that the stimulation of anchorage-independent growth by DEX involves at least two regulatory mechanisms, i.e., one that leads to the suppression of P27Kip1 protein without requiring cytoskeletal integrity, and another that requires cytoskeletal integrity, leading to stimulation of cyclin A and hyperphosphorylation of Rb protein.     

Activated N-ras controls the transformed phenotype of HT1080 human fibrosarcoma cells

To investigate whether the activated N-ras oncogene of HT1080 human fibrosarcoma cells contributes to the expression of the transformed phenotype, we have isolated flat revertants. In two independent revertant lines, an increase in chromosomal ploidy occurred without a concomitant increase in the number of copies of the N-ras transforming allele. Immunoprecipitation confirms that the level of the mutant N-ras p21 gene product in the revertants is correspondingly lower than in HT1080. Analysis of sporadic tumors derived from the revertant cells reveals an increased dosage of the transforming allele. The revertants also retransform after transfection of cloned activated ras oncogenes. These results imply direct participation of an N-ras oncogene in maintaining the transformed phenotype of a human tumor cell line.     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.     

Homocysteine inhibits the proliferation and invasive potential of HT-1080 human fibrosarcoma cells

The impairment of homocysteine metabolism has been related to several disorders and diseases. Recently, homocysteine has been shown to inhibit key steps of angiogenesis, including endothelial cell proliferation, invasion, and remodeling of the extracellular matrix. Since these are also key steps in tumor invasion and metastasis, it can be hypothesized that homocysteine can also interfere in these processes. Therefore, we studied the effects of homocysteine on tumor proliferation and invasion, as well as on urokinase, a key extracellular matrix-degrading protease, using a model human tumor cell line. This study demonstrates that, in fact, homocysteine inhibits HT-1080 proliferation and invasion, and is a potent inhibitor of tumor cell urokinase expression.     

Suppressive effects of electrochemically reduced water on matrix metalloproteinase-2 activities and in vitro invasion of human fibrosarcoma HT1080 cells

It has been demonstrated that hydrogen peroxide (H(2)O(2)) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H(2)O(2) in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H(2)O(2)-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH(2)-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H(2)O(2)-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.     

Junctional adhesion molecule-C promotes metastatic potential of HT1080 human fibrosarcoma

The junctional adhesion molecule (JAM) family is a key molecule in a process called transendothelial migration or diapedesis. Here, we report implications of JAM-C in cancer metastasis. We first determined the mRNA expression of JAMs in 19 kinds of cancer cell lines. JAM-C was expressed in most of tumors having potent metastatic properties. Especially in murine K-1735 melanoma cell lines, the highly metastatic sublines (M2 and X21) strongly expressed JAM-C when compared with the poorly metastatic ones (C-10 and C23). Next, we investigated the role of JAM-C in cancer metastasis by using human JAM-C (hJAM-C) gene-transfected HT1080 fibrosarcoma cells. In comparison with mock-transfected HT1080 cells, these cells showed a significant increase in the adhesion to various extracellular substrates and the invasion across a Matrigel-coated membrane. The knockdown of hJAM-C using small interfering RNA resulted in the suppression of both the adhesion and the invasion of HT1080 cells, suggesting that endogenous hJAM-C might be involved in tumor metastasis. Finally, we studied the role of hJAM-C in an in vivo experimental metastatic model. The results showed that the overexpression of hJAM-C in HT1080 cells significantly decreased the life spans of the tumorbearing mice. In contrast, the knockdown of hJAM-C in HT1080 cells suppressed the weight gain of the lungs with metastatic colonies. We conclude that the expression of JAM-C promotes metastasis by enhancing both the adhesion of cancer cells to extracellular matrices and the subsequent invasion.     

Ral promotes anchorage-independent growth of a human fibrosarcoma, HT1080

Ral has been shown to act downstream of Ras oncoprotein. However, the role of Ral in Ras-induced cellular transformation has not been fully understood. To test the involvement of Ral in Ras-induced anchorage-independent growth, we ectopically expressed Ral mutants in HT1080 cells, whose ability to grow in the absence of anchorage depends on the oncogenic mutation of N-ras. Expression of an activated mutant of Ral resulted in enhanced growth of HT1080 cells in soft agar, whereas a dominant-negative mutant of Ral inhibited their anchorage-independent growth. Moreover, the activated Ral mutant decreased the amount of p27(Kip1) in the absence of adhesion, while the dominant-negative mutant increased it. These results suggest that Ral is involved in the Ras-dependent anchorage-independent growth of HT1080 cells by regulating p27(Kip1).     

Segregation of rat chromosomes in somatic cell hybrids between rat cells and HT 1080 human fibrosarcoma cells

Summary We produced somatic cell hybrids between HT 1080-6TG human fibrosarcoma cells and either rat white blood cells (WBC) or cells directly derived from rat spleen. Karyologic and isozyme analyses of hybrid cells indicated that they preferentially lose rat chromosomes. Hypoxanthine-aminopterine thymidine-selected hybrid clones expressing rat hypoxanthine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), and phosphoglycerate kinase (PGK) and containing the rat X chromosome were counterselected in a medium containing 30 g/ml of 6-thioguanine. Concordant loss of the rat X chromosome and of the expression of rat HPRT and G6PD was observed in the hybrid clones.     

Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.     

Similar radiation sensitivities of acutely and chronically hypoxic cells in HT 1080 fibrosarcoma xenografts

It has been suggested that chronically hypoxic tumor cells may be more radiosensitive than acutely hypoxic or even aerobic cells. In the present study we have used the fact that chronically, but not acutely, hypoxic cells that are transformed with a vector containing an enhanced green fluorescent protein (EGFP) driven by a hypoxia-responsive promoter become green (high EGFP) at low oxygen concentrations and can be viably sorted from transplanted tumors in vitro. We showed that the fluorescence of HT 1080 human fibrosarcoma cells stably transfected with this vector increases constantly with decreasing O2 concentrations (<2%, longer than 1 h, half maximum approximately 0.2% for longer than 8 h), and that cells subjected to repeated cycles of hypoxia/reoxygenation (simulating acutely hypoxic cells) showed only background fluorescence. To test the radiosensitivity of acutely and chronically hypoxic cells in tumors, we isolated high-EGFP ("chronically hypoxic") and low-EGFP cells (containing both acutely hypoxic and aerobic cells) from HT 1080 xenograft tumors by fluorescence-activated cell sorting (FACS), immediately after in situ treatment with 20 Gy (ambient or clamped), and plated the cells to determine clonogenic survival in vitro. We found that the survival of high-EGFP cells after irradiation was not affected by clamping, suggesting that all, or almost all, of these cells were fully (chronically) hypoxic. Also, the survival of the low-EGFP cells irradiated under clamped conditions (acutely hypoxic cells) was not significantly different from that of the high-EGFR cells (chronically hypoxic) cells irradiated under nonclamped (or clamped) conditions. We therefore conclude that, at least in this tumor model, the radiation sensitivity of chronically hypoxic cells is similar to that of the acutely hypoxic cells.     

Inhibitory effects of p-dodecylaminophenol on the invasiveness of human fibrosarcoma cell line HT1080

Cancer is a major cause of death, and the development of new anticancer drugs is urgently needed. Invasion and metastasis are the primary causes of death due to cancer rather than growth of the primary tumor. In the current study, we examined the anti-invasive effects of p-dodecylaminophenol (1), which was developed based on N-(4-hydroxyphenyl)retinamide (2), a synthetic amide of all-trans-retinoic acid (3). In HT1080 cells 1 inhibited growth, induced apoptosis and arrested the cell cycle in S phase in a dose-dependent manner. In addition, 1 significantly suppressed cell invasion, and the activity and mRNA expression of matrix metalloproteinase-9 (MMP-9). Furthermore, the expression of the reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is a negative regulator of MMP-9, was increased by treatment with 1. These results suggest that 1 could be an effective anti-cancer agent that suppresses cell growth through apoptosis induction and cell cycle arrest, which also inhibits cell invasion by decreasing MMP-9 expression due to an increase in RECK. Compound 1 might be useful clinically as a new and potent anticancer agent that could overcome adverse side effects of the retinoids.     

Calcium salicylate-mediated apoptosis in human HT-1080 fibrosarcoma cells

Salicylates are novel biologically active compounds that exhibit multiple therapeutic activities. The anti-cancer effectiveness of calcium salicylate has been investigated on human HT-1080 fibrosarcoma cell lines at relatively low concentrations (predominantly 0.4 mM) compared to those previously reported. Although low calcium salicylate concentrations did not retard tumour growth progression significantly, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and time-lapse assays, its cytotoxic characteristics were proven to be prominent by various morphological and immunocytological techniques. The results here demonstrate evidence for approximately 25% apoptosis after treatment with calcium salicylate, which up-regulatd the expression of p53, p21 and Bax, and down-regulated Bcl-2 in HT-1080 cells.     

Inhibitory effect of obovatal on the migration and invasion of HT1080 cells via the inhibition of MMP-2

Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, compounds with the ability to inhibit MMP activity have a potential in the treatment of tumor. From the examination of 2000 plant extracts, obovatal isolated from the extract of the leaves of Magnolia obovata THUNB was a potent inhibitor of MMP-2 enzyme in vitro. In human fibrosarcoma cells (HT1080) activated with MMP-2, obovatal inhibited MMP-2 enzyme activity and expression. In addition, the compound blocked migration and invasion of the cells. This study demonstrates that obovatal exerts its anticancer effects through blocking migration and invasion by inhibition of MMP-2 expression and activity, and also will be a good lead molecule for the development of anti-tumor drug.     

Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells

We present evidence for differential roles of Rho-kinase and myosin light chain kinase (MLCK) in regulating shape, adhesion, migration, and chemotaxis of human fibrosarcoma HT1080 cells on laminin-coated surfaces. Pharmacological inhibition of Rho-kinase by Y-27632 or inhibition of MLCK by W-7 or ML-7 resulted in significant attenuation of constitutive myosin light chain phosphorylation. Rho-kinase inhibition resulted in sickle-shaped cells featuring long, thin F-actin-rich protrusions. These cells adhered more strongly to laminin and migrated faster. Inhibition of MLCK in contrast resulted in spherical cells and marked impairment of adhesion and migration. Inhibition of myosin II activation with blebbistatin resulted in a morphology similar to that induced by Y-27632 and enhanced migration and adhesion. Cells treated first with blebbistatin and then with ML-7 also rounded up, suggesting that effects of MLCK inhibition on HT1080 cell shape and motility are independent of inhibition of myosin activity.