Human Fibrosarcoma HT1080 Cells Research Articles

The stem cell transcription factor ZFP57 induces IGF2 expression to promote anchorage-independent growth in cancer cells

Several common biological properties between cancer cells and embryonic stem (ES) cells suggest the possibility that some genes expressed in ES cells might have important roles in cancer cell growth. The transcription factor ZFP57 is expressed in self-renewing ES cells and its expression level decreases during ES cell differentiation. This study showed that ZFP57 is involved in the anchorage-independent growth of human fibrosarcoma HT1080 cells in soft agar. ZFP57 overexpression enhanced, whereas knockdown suppressed, HT1080 tumor formation in nude mice. Furthermore, ZFP57 regulates the expression of insulin-like growth factor 2 (IGF2), which has a critical role in ZFP57-induced anchorage-independent growth. ZFP57 also promotes anchorage-independent growth in ES cells and immortal fibroblasts. Finally, immunohistochemical analysis revealed that ZFP57 is overexpressed in human cancer clinical specimens. Taken together, these results suggest that the ES-specific transcription factor ZFP57 is a novel oncogene.

PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21Waf1/Cip1 and p27Kip1 descended in PPARγ1S84D stable HT1080 cell, whereas the expression of p18INK4C was not changed. Moreover, compared to the PPARγ1S84A, PPARγ1S84D up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs.

Morphological evaluation of apoptosis induced by salicylates in HT-1080 human fibrosarcoma cells

Salicylates are a group of phenolic compounds that are found naturally in plants. They exert anti-inflammatory and pro-apoptosis activities. Three salicylates (acetyl salicylic acid, ASA; acetyl salicylate calcium, ASCa and salicylate calcium, SACa) were tested in human fibrosarcoma cells for their apoptotic activities in HT-1080 cells using both biochemical and morphological approaches. Salicylates-treated HT-1080 cells exhibited typical apoptotic features, including membrane blebbing, shrinkage of the cell and fragmentation into apoptotic bodies. The effects of the DNA labelling agent, BrdU, resulted in more rounder smaller and a smooth membrane in HT-1080 cells. These salicylate compounds demonstrated anti-proliferation and reduced the metabolic activities of HT-1080 cells by time- and concentration-dependent manners.

Dietary gallic acid and anthocyanin cytotoxicity on human fibrosarcoma HT1080 cells. A study on the mode of action

Gallic acid and anthocyanins are abundant plant food bioactives present in many fruits and vegetables, being especially important in the composition of berries. Gallic acid has been shown to possess cytotoxic properties in several cancer cell lines and to inhibit carcinogenesis in animal models. However, its mechanism of action is not yet fully understood. The aim of this study was to elucidate whether the observed inhibitory activity of gallic acid against gelatinases corresponds to its cytotoxic activity in HT1080 cells and to determine if anthocyanins could exhibit a similar behavior. Gallic acid and delphinidin-3-glucoside have shown selective cytotoxicity towards HT1080 cells. Further analysis by a migration and invasion assay showed anti-invasive activities of gallic acid, delphinidin and pelargonidin-3-glucosides. Zymographic analysis demonstrated the inhibitory activity of gallic acid at the level of secreted and activated gelatinases. Moreover, gallic acid inhibited MMP-2 and MMP-9 proteolytic activity with very similar potency. NMR and molecular modelling experiments confirmed the interaction of gallic acid with MMP-2, and suggested that it takes place within the catalytic center. In this work we give some new experimental data supporting the role of these compounds in the inhibition of metalloproteases as the mechanism for their cytotoxic activity against fibrosarcoma.

A peptide functionalized poly(ethylene glycol) (PEG) hydrogel for investigating the influence of biochemical and biophysical matrix properties on tumor cell migration.

To address the challenges associated with defined control over matrix properties in 3D cell culture systems, we employed a peptide functionalized poly(ethylene glycol) (PEG) hydrogel matrix in which mechanical modulus and adhesive properties were tuned. An HT-1080 human fibrosarcoma cell line was chosen as a model for probing matrix influences on tumor cell migration using the PEG hydrogel platform. HT-1080 speed varied with a complex dependence on both matrix modulus and Cys-Arg-Gly-Asp-Ser (CRGDS) adhesion ligand concentration, with regimes in which motility increased, decreased, or was minimally altered being observed. We further investigated cell motility by forming matrix interfaces that mimic aspects of tissue boundaries that might be encountered during invasion by taking advantage of the spatial control of the thiol-ene photochemistry to form patterned regions of low and high cross-linking densities. HT-1080s in 100 Pa regions of patterned PEG hydrogels tended to reverse direction or aggregate at the interface when they encountered a 360 Pa boundary. In contrast, HT-1080s were apparently unimpeded when migrating from the stiff to the soft regions of PEG peptide hydrogels, which may indicate that cells are capable of "reverse durotaxis" within at least some matrix regimes. Taken together, our results identified matrix regimes in which HT-1080 motility was both positively and negatively influenced by cell adhesion or matrix modulus.

Analysis of the androgen receptor/filamin a complex in stromal cells.

The androgen receptor (AR), a ligand-regulated nuclear transcription factor, mediates differentiation and proliferation of target tissues. Its action is frequently associated with human proliferative diseases, mainly the prostate cancer. We have recently analyzed in mouse embryo NIH3T3 fibroblasts and human fibrosarcoma HT1080 cells the molecular basis and the biological role of AR interaction with the full-length filamin A (FLNa), an actin-cross-linking protein. Here, we describe a procedure revealing the AR/FLNa complex in stromal cells. Upon physiological (10 nM) androgen stimulation of quiescent NIH3T3 cells, FLNa co-immunoprecipitates with AR and co-localizes with the receptor at intermediate actin filaments. The AR/FLNa complex specifically regulates AR extranuclear functions leading to Rac1 activation and consequent cell motility. This complex adds a new and unexpected piece to the growing evidence of the role of signalling effectors, scaffolds, and cytoskeletal proteins in the rapid androgen action and in progression of hormone-dependent cancers.

A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype.

Caveolae are specialized cell membrane invaginations known to regulate several cancer cell functions and oncogenic signaling pathways. Among other caveolar proteins, they are characterized by the presence of proteins of the cavin family. In this study, we assessed the impact of cavin-1, cavin-2, and cavin-3 on cell migration in a human HT-1080 fibrosarcoma model. We found that all cavin-1, -2 and -3 transcripts were expressed and that treatment with phorbol 12-myristate 13-acetate (PMA), which is known to prime cell migration and proliferation, specifically upregulated cavin-3 gene and protein expression. PMA also triggered matrix metalloproteinase (MMP)-9 secretion, but reduced the global cell migration index. Overexpression of recombinant forms of the three cavins demonstrated that only cavin-3 was able to reduce basal cell migration, and this anti-migratory effect was potentiated by PMA. Interestingly, cavin-3 overexpression inhibited PMA-induced MMP-9, while cavin-3 gene silencing led to an increase in MMP-9 gene expression and secretion. Furthermore, recombinant cavin-3 significantly prevented PMA-mediated dephosphorylation of AKT, a crucial regulator in MMP-9 transcription. In conclusion, our results demonstrate that cellular cavin-3 expression may repress MMP-9 transcriptional regulation in part through AKT. We suggest that the balance in cavin-3-to-MMP-9 expression regulates the extent of extracellular matrix degradation, confirming the tumor-suppressive role of cavin-3 in controlling the invasive potential of human fibrosarcoma cells.

A Quantitative Comparison of Human HT-1080 Fibrosarcoma Cells and Primary Human Dermal Fibroblasts Identifies a 3D Migration Mechanism with Properties Unique to the Transformed Phenotype

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels (“synthetic extracellular matrix” or “synthetic ECM”). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

항산화 활성을 가진 그래핀이 HT1080 세포에서 기질금속단백질분해효소에 미치는 영향

그래핀(graphene)은 원자 하나의 두께를 가지는 흑연(graphite)의 단층으로서 탄소구조체들 중 하나이다. 그래핀은 최근 의학분야에서 광열요법을 이용한 암 발생의 예방효과와 DNA의 산화에 대한 보효효과를 가진다고 밝혀진 바 있다. 본 연구에서는 사람 섬유아육종세포(HT1080)에서 산화 스트레스와 MMPs에 대한 그래핀의 효과가 조사되었다. 항산화 효과에 대한 연구에서 그래핀은 DNA 산화에 대한 억제효과를 특이하게 나타내었으나 다른 산화 스트레스는 억제하지 않았다. 뿐만 아니라 그래핀은 세포 내 과산화수소를 생성시키는 phenazinemethosulfate(PMS)에 의하여 자극된 MMP-2 및 MMP-9의 발현과 활성을 감소시켰다. 특히 superoxide dismutase(SOD-2)와 같은 항산화 효소의 발현이 HT1080세포에서 감소하였는데, 이것이 시사하는 바는 SOD 발현수준의 감소가 그래핀의 항산화 효과로부터 기인 되었다는 것을 나타낸다. 이상의 결과로 그래핀의 존재에서 산화스트레스의 억제효과가 HT1080 세포에서 MMP-9의 활성과 발현을 감소시킬 수 있다는 것을 암시하고 있다. 이러한 연구 결과를 바탕으로 그래핀은 암 전이와 관련 있는 MMP-2 및 MMP-9의 활성과 발현의 억제를 통하여 암 억제에 도움을 줄 수 있어, 산업화를 위한 하나의 우수한 생의학 응용소재로 이용될 수 있으리라 기대된다. Graphene is an allotrope of carbon that is composed of one-atom-thick planar sheets. It is known to have a preventive effect on cancer in photothermal therapy and a protective effect in DNA oxidation. The effect of graphene on oxidative stress and matrix metalloproteinases (MMPs) was investigated in human fibrosarcoma HT1080 cells. The results showed that graphene specifically exerted an inhibitory effect on DNA oxidation, but it did not inhibit other oxidative stress. In addition, graphene decreased the expression and the activation of MMP-2 and MMP-9 stimulated by phenazine methosulfate-m, which induces the production of intracellular hydrogen peroxide. In particular, the expression of antioxidant enzymes, such as superoxide dismutase (SOD-2), was decreased in the HT1080 cells, indicating that the decrease in the expression level of SOD was due to the antioxidant effect of graphene. These results suggest that the inhibitory effect of oxidative stress in the presence of graphene could inhibit the expression of MMPs in HT1080 cells. Based on the above results, graphene may have chemoprevention properties through inhibition of MMP-2 and MMP-9 related to metastasis.

FAM129B is a novel regulator of Wnt/β-catenin signal transduction in melanoma cells

The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/β-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/β-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of β-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/β-catenin signaling. Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a β-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/β-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/β-catenin signaling to apoptosis in melanoma.

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

BackgroundHormones 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.ConclusionThis 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.

The DNA hypomethylating agent, 5‐aza‐2′‐deoxycytidine, enhances tumor cell invasion through a transcription‐dependent modulation of MMP‐1 expression in human fibrosarcoma cells

In diseases such as cancer, cells need to degrade the extracellular matrix (ECM) and therefore require high protease levels. Thus, aberrant tissue degradation is associated to matrix metalloproteinases (MMPs) overexpression resulting from different mechanisms including epigenetic events. One of the most characterized epigenetic mechanisms is DNA methylation causing changes in chromatin conformation, thereby decreasing the accessibility to the transcriptional machinery and resulting in a robust gene silencing. Modulation of DNA methylation by DNA hypomethylating agents such as 5-aza-2'-deoxycytidine (5-azadC) is widely used in epigenetic anticancer treatments. Here, we focus on the effects of this drug on the expression level of MMP-1, -2, and -9 in human HT1080 fibrosarcoma cells. We demonstrate that 5-azadC increases MMP expression at both mRNA and protein levels, and promotes invasion potential of HT1080 cells. Using broad-spectrum and specific MMP inhibitors, we establish that MMP-1, but not MMP-2 and -9, plays a key role in 5-azadC-enhanced cell invasion. We show that 5-azadC induces MMP-1 expression through a transcriptional mechanism without affecting MMP-1 promoter methylation status. Finally, we demonstrate that 5-azadC treatment increases the nuclear levels of Sp1 and Sp3 transcription factors, and modulates their recruitment to the MMP-1 promoter, resulting in chromatin remodeling associated to 5-azadC-induced MMP-1 expression. All together, our data indicate that the hypomethylating agent 5-azadC modulates, mainly via Sp1 recruitment, MMP-1 expression resulting in an increased invasive potential of HT1080 cells.

The Effect of Hydroxybenzoate Lithium Complexes in Inducing Apoptosis in HT-1080 Human Fibrosarcoma Cells

There has been a growing interest in the beneficial effects of simple phenolic acids and their ability to exhibit various biological activities. The aim of this study was to assess in vitro biological activities of 2-, 3-, and 4-hydroxybenzoate lithium (HBLi) complexes on HT-1080 human fibrosarcoma cells by methods of using a metabolic activity assay, immunochemical and morphological techniques. Results showed that HBLi complexes exert their cytotoxic activities in a concentration- and chemical structure-dependent manner in the following order: 4-HBLi > 3-HBLi > 2-HBLi. Flow cytometry displayed evidence of apoptosis induced by 3-HBLi (21.8%) and 4-HBLi (33.2%). These results were verified by SEM, which revealed the formation of apoptotic bodies. In addition, these 3-HBLi and 4-HBLi caused an increase in HT-1080 cell cycle arrest in G0/G1 phase when compared to the controls (25% and 30.6%, resp.) when cells were treated with 6 mM for 24 hours. Immunochemical studies related to the molecular mechanism of apoptosis indicated that HBLi complexes downregulated the expression of Bcl-2 and upregulated Bax, p53, and caspases-3 in a concentration-dependent manner. HBLi complexes lowered Bcl-2/Bax ratios and induced the expression of p53 and caspase-3. These results suggest that HBLi complexes may exert their apoptotic effects through mitochondrial-mediated, caspase-dependent, apoptotic mechanisms.

Recombinant TIMP-1-GPI inhibits growth of fibrosarcoma and enhances tumor sensitivity to doxorubicin

Fibrosarcomas show a high incidence of recurrence and general resistance to apoptosis. Limiting tumor regrowth and increasing their sensitivity to chemotherapy and apoptosis represent key issues in developing more effective treatments of these tumors. Tissue inhibitor of metalloproteinase 1 (TIMP-1) broadly blocks matrix metalloproteinase (MMP) activity and can moderate tumor growth and metastasis. We previously described generation of a recombinant fusion protein linking TIMP-1 to glycosylphophatidylinositol (GPI) anchor (TIMP-1-GPI) that efficiently directs the inhibitor to cell surfaces. In the present report, we examined the effect of TIMP-1-GPI treatment on fibrosarcoma biology. Exogenously applied TIMP-1-GPI efficiently incorporated into surface membranes of human HT1080 fibrosarcoma cells. It inhibited their proliferation, migration, suppressed cancer cell clone formation, and enhanced apoptosis. Doxorubicin, the standard chemotherapeutic drug for fibrosarcoma, was tested alone or in combination with TIMP-1-GPI. In parallel, the influence of treatment on HT1080 side population cells (exhibiting tumor stem cell-like characteristics) was investigated using Hoechst 33342 staining. The sequential combination of TIMP-1-GPI and doxorubicin showed more than additive effects on apoptosis, while TIMP-1-GPI treatment alone effectively decreased “stem-cell like” side population cells of HT1080. TIMP-1-GPI treatment was validated using HT1080 fibrosarcoma murine xenografts. Growing tumors treated with repeated local injections of TIMP-1-GPI showed dramatically inhibited fibrosarcoma growth and reduced angiogenesis. Intraoperative peritumoral application of GPI-anchored TIMP-1 as an adjuvant to surgery may help maintain tumor control by targeting microscopic residual fibrosarcoma cells and increasing their sensitivity to chemotherapyElectronic supplementary materialThe online version of this article (doi:10.1007/s11523-013-0294-5) contains supplementary material, which is available to authorized users.

Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca2+ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca2+-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca2+. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.

Quercetin Suppresses Intracellular ROS Formation, MMP Activation, and Cell Motility in Human Fibrosarcoma Cells

Cell metastasis is a major cause of death from cancer and can arise from excessive levels of oxidative stress. The objective of this study was to investigate whether the natural flavonoid quercetin can inhibit matrix metalloproteinase (MMP)-2 and -9 activities through the attenuation of reactive oxygen species (ROS) formation, an event expected to lead to the inhibition of cell motility. To induce sustained ROS formation, cells were treated with phenazine methosulfate (PMS; 1 μM). Noncytotoxic concentrations of quercetin inhibited PMS-induced increases in cell motility in HT1080 human fibrosarcoma (HT1080) cells. While nearly 100% of cells were observed to migrate after 24 h of PMS treatment, quercetin significantly (P < 0.01) suppressed this effect. We also found that quercetin, up to 10 μg/mL, attenuated PMS-induced MMP-2 activation. We then investigated whether the decreased levels of MMP-2 activation could be attributable to lower levels of ROS formation by quercetin. We found that quercetin treatments significantly attenuated PMS-induced ROS formation (P < 0.01) and resulted in decreased cell motility associated with a reduction in MMP-2 and -9 activitiy in HT1080 cells, even in the absence of PMS treatment. Collectively, these results suggest that quercetin inhibits cell motility via the inhibition of MMP activation in HT1080 cells in the presence and absence of PMS. This is likely to be a result of the suppression of intracellular ROS formation by quercetin.

FAM129B is a novel regulator of Wnt/β-catenin signal transduction in melanoma cells

The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/β-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/β-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of β-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/β-catenin signaling. Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a β-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/β-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/β-catenin signaling to apoptosis in melanoma.

CD133+ subpopulation of the HT1080 human fibrosarcoma cell line exhibits cancer stem-like characteristics

The cancer stem cell (CSC) theory holds that a minority population within tumors possesses stem cell properties of self-renewal and multilineage differentiation capacity and provides the initiating cells from which tumors are derived and sustained. However, verifying the existence of these CSCs has been a significant challenge. The CD133 antigen is a pentaspan membrane glycoprotein proposed to be a CSC marker for cancer-initiating subpopulations in the brain, colon and various other tissues. Here, CD133+ cells were obtained and characterized from the HT1080 cell line to determine the utility of this marker for isolating CSCs from human fibrosarcoma cells. In this study, CD133+ cells were separated from HT1080 cells using magnetic beads and characterized for their proliferation rate and resistance to chemotherapeutic drugs, cisplatin and doxorubicin, by MTS assay. Relative expression of tumor-associated genes Sox2, Oct3/4, Nanog, c-Myc, Bmi-1 and ABCG2 was measured by real-time polymerase chain reaction (PCR). Clonal sphere formation and the ability of CD133+ cells to initiate tumors in BALB/c nude mice was also evaluated. We found that CD133+ cells showed a high proliferation rate, increased resistance to chemotherapy drugs and overexpression of tumor-associated genes compared with these features in CD133- cells. Additionally, CD133+ cells were able to form spherical clusters in serum-free medium with high clonogenic efficiency, indicating a significantly greater tumor-initiating potential when compared with CD133- cells. These findings indicate that CD133+ cells identified within the HT1080 human fibrosarcoma cell line possess many CSC properties and may facilitate the development of improved therapies for fibrosarcoma.

Physalin A Induces Apoptotic Cell Death and Protective Autophagy in HT1080 Human Fibrosarcoma Cells

Physalin A (1) is a withanolide isolated from Physalis alkekengi var. franchetii. In this study, the selective growth inhibitory effects on tumor cells induced by 1 were screened, and the mechanism was investigated on 1-induced growth inhibition, including apoptosis and autophagy, in human fibrosarcoma HT1080 cells. Apoptosis induced by 1 in HT1080 cells was associated with up-regulation of caspase-3 and caspase-8 expression. However, there were no significant changes in caspase-9, Bid, Bax, and Bcl-2 expression, indicating that 1-induced apoptosis in HT1080 cells occurs mainly through activation of the death receptor-associated extrinsic apoptotic pathways. Autophagy induced by 1 was found to antagonize apoptosis in HT1080 cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor 3-methyladenine (3MA). Loss of beclin 1 (as an autophagic regulator) function led to similar results to 3MA. However, 1 did not show inhibitory effects on normal human cells (human peripheral blood mononuclear cells). Taken together, these results suggest that 1 may be a promising agent for the treatment of cancer.

Fibroblast Growth Factor Inducible (Fn14)-specific Antibodies Concomitantly Display Signaling Pathway-specific Agonistic and Antagonistic Activity

Fn14 is a therapeutic target in various diseases. Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.