Human Fibrosarcoma HT1080 Cells Research Articles

C-terminal domain of p16INK4a is adequate in inducing cell cycle arrest, growth inhibition and CDK4/6 interaction similar to the full length protein in HT-1080 fibrosarcoma cells

The tumor suppressor p16(INK4a) has earned widespread attention in cancer studies since its discovery as an inhibitor of cyclin-dependent kinases (CDKs) 4/6. Structurally, it consists of four complete ankyrin repeats, believed to be involved in CDK4 interaction. According to the previous disparities concerning the importance of domains and inactivating mutations in p16, we aimed to search for the domain possessing the functional properties of the full length protein. Upon our in silico screening analyses followed by experimental assessments, we have identified the novel minimum functional domain of p16 to be the C-terminal half including ankyrin repeats III, IV and the C-terminal flanking region accompanied by loops 2 and 3. Transfection of this truncated form into HT-1080 human fibrosarcoma cells, lacking endogenous p16, revealed that it is able to inhibit cell growth and proliferation equivalent to p16(INK4a). The functional analysis showed that this fragment like p16 can interact with CDK4/6, block the entry into S phase of the cell cycle and suppress growth as indicated by colony formation assay. Identification of p16 minimum functional domain can be of benefit to the future peptidomimetic drug design as well as gene transfer for cancer therapy.

Acteoside inhibits PMA‐induced matrix metalloproteinase‐9 expression via CaMK/ERK‐ and JNK/NF‐κB‐dependent signaling

Acteoside, an active phenylethanoid glycoside found in bitter tea and many medicinal plants, displays chemopreventive properties. The aim of our study was to determine the effect of acteoside on tumor invasion and migration; the possible mechanisms involved in this inhibition were investigated in human fibrosarcoma HT-1080 cells. We employed invasion, migration and gelatin zymography assays to characterize the effect of acteoside on HT-1080 cells. Transient transfection assays were performed to investigate gene promoter activities, and immunoblot analysis to study its molecular mechanisms of action. We found that acteoside suppresses phorbol-12-myristate-13-acetate (PMA)-enhanced matrix metalloproteinase-9 (MMP-9) expression at the protein, mRNA, and transcriptional levels through the suppression of NF-κB activation. In addition, acteoside repressed the PMA-induced phosphorylation of ERK1/2 (ERK, extracellular regulated kinase) and JNK1/2. Further, we found that acteoside decreased the PMA-induced influx of Ca(2+) and repressed PMA-induced calmodulin-dependent protein kinase (CaMK) phosphorylation. Furthermore, treatment with BAPTA/AM, W7, or capsazepine markedly decreased PMA-induced MMP-9 secretion and cell migration, as well as ERK and JNK/NF-κB activation. Acteoside inhibited PMA-induced invasion and migration of human fibrosarcoma cells via Ca(2+) -dependent CaMK/ERK and JNK/NF-κB-signaling pathways. Acteoside therefore has the potential to be a potent anticancer agent in therapeutic strategies for fibrosarcoma metastasis.

Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator

The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP-1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element.

Apoptosis of human fibrosarcoma HT-1080 cells by epigallocatechin-3-O-gallate via induction of p53 and caspases as well as suppression of Bcl-2 and phosphorylated nuclear factor-κB

Animal tumor bioassays and in vitro cell culture systems have demonstrated that epigallocatechin-3-O-gallate (EGCG), the predominant catechin in green tea, possesses anti-proliferative and pro-apoptotic effects on various cancer cells and tumors. In this study, we investigated the effects of EGCG on cell growth, cell cycle progression, and apoptosis in human fibrosarcoma HT-1080 cells. The involvement of p53, Bcl-2, Bax, caspases, and nuclear factor-κB (NF-κB) was examined as a mechanism for the anti-cancer activity of EGCG. Time-dependent intracellular trafficking of EGCG was also determined using fluorescein isothiocyanate (FITC)-conjugated EGCG (FITC-EGCG). Our data show that EGCG treatment caused dose-dependent cell growth inhibition, cell cycle arrest at the G(0)/G(1) phase, and DNA fragmentation suggesting the induction of apoptosis in HT-1080 cells. Immunoblot analysis revealed that the expression of p53, caspase-7 and -9 as well as the ratio of Bax/Bcl-2 protein increased significantly with higher EGCG concentrations and longer incubation times. Moreover, expression of phosphorylated NF-κB/p65 in HT-1080 cells was inhibited by EGCG treatment in a dose-dependent manner, while that of unphosphorylated NF-κB/p65 remained unaffected. Here we also reveal time-dependent internalization of FITC-EGCG into the cytosol of HT-1080 cells and its subsequent nuclear translocation. These results suggest that EGCG may interrupt exogenous signals directed towards genes involved in proliferation and cell cycle progression. Taken together, our data indicate that HT-1080 apoptosis may be mediated through the induction of p53 and caspases by the pro-oxidant activity of internalized EGCG, as well as suppression of Bcl-2 and phosphorylated NF-κB by the antioxidant activity of EGCG.

Engineered human tumor xenografts with functional human vascular networks

Animal models of human tumor angiogenesis would have multiple applications. However, they are extremely difficult to standardize. In this work, we tried to generate human tumor xenografts containing a human vascular bed in immunodeficient mice by subcutaneous co-implantation of matrigel-embedded human endothelial cells (EC), human mesenchymal stem cells (MSC) as a source of mural cells and HT1080 human fibrosarcoma cells. Unfortunately, in this context human EC were rapidly substituted by their murine counterparts, and by day 16 post-implantation human CD34 positive cells were hardly detectable in intratumoral vessels. In an attempt to inhibit host EC colonization of human xenografts and promote human EC grafting, we investigated the effect of radiation prior to implantation on the vascularization and growth of tumor xenografts. Nude mice underwent either localized (implantation area) or sublethal whole-body exposure to radiation. Localized radiation inhibited both human and murine neovascularization, and even the tumor growth rate was remarkably decreased when compared to control unirradiated mice and sublethally whole-body irradiated mice. Interestingly, numerous human vessels were detectable in sublethally irradiated mice at day 30, with murine EC only overpassing human EC when spontaneous hematopoietic reconstitution has taken place. This observation strongly suggests the implication of bone marrow-derived murine endothelial precursors in tumor neovascularization. In summary, we have established a model of human tumor neovascularization that is amenable to both the study of molecular aspects in the angiogenic process and the evaluation of potential new antiangiogenic drugs.

건조 마 추출물의 항산화 및 인체 암세포 증식 억제 효과

We investigated the inhibitory effects of solvent extracts from dried yam on H₂O₂-induced oxidative stress and growth of cancer cell lines (HT1080 human fibrosarcoma and HT-29 human colon cancer cells). Yam (Dioscoreacea) has been recognized as a healthy food due to its various biological activities, such as anti-obesity, anti-constipation, anti-proliferation, and anti-mutagenic activities, as well as its ability to decrease blood glucose and cholesterol levels. In order to determine the protective effect on H₂O₂-induced oxidative stress, DCFH-DA (dichlorodihydrofluorescin diacetate) assay was conducted. Acetone with methylene chloride (A+M) extract of dried yam appeared to reduce the levels of intracellular reactive oxygen species (ROS) with dose responses. Among the fractions, 85% aq. methanol fraction showed the highest protective effect on production of lipid peroxides. Inhibitory effects of A+M and methanol (MeOH) extracts on the growth of HT1080 and HT-29 cancer cells increased in a dose dependent manner. The treatments of n-hexane, 85% aq. methanol and n-butanol fractions (? 0.5 ㎎/ml concentrations) significantly inhibited the growth of both cancer cells (p＜0.05). From these results, 85% aq. methanol fraction showed inhibitory effects on cellular oxidation and growth of human cancer cells, suggesting that this fraction may contain active compounds of dried yam.

Effect of the degree of oxidation on the physicochemical and biological properties of Grifola frondosa polysaccharides

Polysaccharide extracted from Grifola frondosa was subjected to 2,2,6,6-tetramethyl-1-piperidine oxoammonium ion (TEMPO)-mediated oxidation of which effects on the structural and biological properties of the polysaccharide were investigated as a function of degree of oxidation. Successive oxidation of the polysaccharide was confirmed by 13C NMR spectroscopy and the molecular weight change of the oxidized polysaccharide was observed, decreasing from 10.6 × 10 5 Da to 7.5 × 10 5 Da by 100% oxidation. The oxidation also caused an increase in the water solubility of the polysaccharide while its viscosity was significantly reduced. In addition, when human fibrosarcoma HT1080 cells were treated with 100% oxidized polysaccharide, their in vitro growth was effectively inhibited. However, the oxidation reduced the activity of superoxide dismutase up to 82% at a concentration of 2.5 mg/mL. Thus, the chemical modification by TEMPO-mediated oxidation was shown to play a significant role in the biological properties of the polysaccharides derived from Grifola frondosa.

Cell adhesive peptide screening of the mouse laminin α1 chain G domain

Cell adhesive peptides have been widely applied for therapeutic drugs, drug delivery systems, and biomaterials. Previously, we identified various cell adhesive sequences in the G domains of four laminin α chains (α2–α5) by the systematic soluble peptide screening. We also identified five cell-binding sequences in the laminin α1 chain G domain using synthetic peptide-polystyrene beads. Here, we re-screened cell adhesive peptides in the laminin α1 chain G domain by the systematic soluble peptides screening. The 110 soluble peptides were evaluated for their cell adhesive activities using human fibrosarcoma HT1080 cells and human dermal fibroblasts. Fourteen peptides were newly identified as a cell adhesive. Additionally, four peptides (AG22: SSFHFDGSGYAM, AG42: TFDLLRNSYGVRK, AG76: HQNQMDYATLQLQ, AG86: LGGLPSHYRARNI) promoted integrin-mediated cell adhesion. Further, neurite outgrowth activity with rat pheochromocytoma PC12 cells was evaluated and two peptides (AG20: SIGLWNYIEREGK, AG26: SPNGLLFYLASNG) were newly identified for neurite outgrowth activity. These results suggested that the systematic soluble peptides screening approach is an accurate and powerful strategy for finding biologically active sequences. The active sequences newly identified here could be involved in the biological functions of this domain. The active peptides are useful for evaluating molecular mechanisms of laminin–receptor interactions and for developing cell adhesive biomaterials.

Mitogen-activated protein/extracellular signal-regulated kinase kinase 1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells.

Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified alpha tubulin and beta tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability.

UV light killing efficacy of fluorescent protein‐expressing cancer cells in vitro and in vivo

We investigated the cell-killing efficacy of UV light on cancer cells expressing GFP in the nucleus and RFP in the cytoplasm (dual-color cells). After exposure to various doses of UVA, UVB, or UVC, apoptotic and viable cells were quantitated under fluorescence microscopy using dual-color 143B human osteosarcoma cells, HT-1080 human fibrosarcoma cells, Lewis lung carcinoma (LLC), and XPA-1 human pancreatic cancer cells in vitro. UV-induced cancer cell death was wave-length and dose dependent, as well as cell-line dependent. After UVA exposure, most cells were viable even when the UV dose was increased up to 200 J/m(2). With UVB irradiation, cell death was observed with irradiation at 50 J/m(2). For UVC, as little as 25 J/m(2) UVC irradiation killed approximately 70% of the 143B dual-color cells. This dose of UVB or UVA had almost no effect on the cancer cells. UV-induced cancer cell death varied among the cell lines. Cell death began about 4 h after irradiation and continued until 10 h after irradiation. UVC exposure also suppressed cancer cell growth in nude mice in a model of minimal residual cancer (MRC). No apparent side effects of UVC exposure were observed. This study opens up the possibility of UVC treatment for MRC after surgical resection.

3-Caffeoyl, 4-dihydrocaffeoylquinic acid from Salicornia herbacea inhibits tumor cell invasion by regulating protein kinase C-δ-dependent matrix metalloproteinase-9 expression

In this study, we determined the effects of a novel chlorogenic acid, 3-caffeoyl, 4-dicaffeoylquinic acid (CDCQ) isolated from Salicornia herbacea, on tumor invasion and migration in human fibrosarcoma HT-1080 cells and investigated the possible mechanism(s) involved. CDCQ reduced the phorbol myristate acetate (PMA)-induced activation of matrix metalloproteinase (MMP)-9 and MMP-2 and inhibited cell invasion and migration. CDCQ suppressed PMA-induced expression of MMP-9 mRNA and protein by suppressing the transcription factor AP-1, without changing the level of tissue inhibitor of metalloproteinase (TIMP)-1. CDCQ-inhibited PMA-induced MMP-2 expression by suppressing membrane-type 1 MMP (MT1-MMP), but did not alter the TIMP-2 level. CDCQ also inhibited the PMA-induced nuclear translocation of c-Jun and c-Fos, which are upstream of PMA-induced MMP-9 expression. Furthermore, CDCQ strongly repressed PMA-induced phosphorylation of ERK, p38 MAPK, and JNK, which are dependent on the PKCδ pathway. In conclusion, we demonstrated that the anti-invasive effects of CDCQ occur through the inhibition of AP-1 and signaling pathways involving PKCδ and three MAPKs, leading to the downregulation of MMP-9 expression. Thus, CDCQ is an effective anti-metastatic agent that functions by downregulating MMP-9 gene expression.

A novel MEK1/2 inhibitor induces G1/S cell cycle arrest in human fibrosarcoma cells

Blockade of the ERK pathway has antitumor effects against malignant tumor cells. In this study, we investigated the antitumor activity of JTP-70902, a novel specific MEK inhibitor, against human fibrosarcoma cells in which the ERK pathway is constitutively activated. JTP-70902 was synthesized at Japan Tabacco. Human fibrosarcoma HT1080 cells were cultured. JTP-70902 was added at various concentrations. The number of viable cells was counted employing a trypan blue dye exclusion test. Unsynchronized cells were exposed to JTP-70902 for 24 h. The nuclei were stained with propidium iodide. The DNA content was measured using a FACSCalibur flow cytometer. Protein extraction and Western blot analysis were performed. (1) A dose-dependent inhibition of cell growth was observed at concentrations of 10 nM or more. Forty-eight hours after the treatment, the growth of HT1080 cells was completely inhibited by 200 nM JTP-70902. (2) FACS analysis revealed that a 24-h exposure to JTP-70902 increased the population of G1/S phase cells in a dose-dependent manner. (3) The phosphorylation of ERK was inhibited by JTP-70902. Furthermore, after the treatment with JTP-70902, p21WAF1/CIP1 and p27KIP1 protein expression increased and the phosphorylation of RB was reduced. Our results showed that JTP-70902 inhibits cell growth and induces cell cycle arrest in human Ras mutant fibrosarcoma cells. These results indicate that JTP-70902 might be an attractive compound for molecular-targeting chemotherapy for malignant soft tissue tumors with the activation of the Ras-MEK-ERK pathway.

Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase‐9 activation through a calcium and protein kinase Cα‐dependent pathway: phorbol‐12‐myristate‐13‐acetate‐induced/extracellular signal‐regulated kinase/activator protein‐1

Population studies have revealed that treatment with the anti-diabetic drug metformin is significantly associated with reduced cancer risk, but the underlying mode of action has not been elucidated. The aim of our study was to determine the effect of metformin on tumour invasion and migration, and the possible mechanisms, using human fibrosarcoma HT-1080 cells. We employed invasion, migration and gelatin zymography assays to characterize the effect of metformin on HT-1080 cells. Transient transfection assays were performed to gene promoter activities, and immunoblot analysis to study its molecular mechanisms of action. Metformin inhibited migration and invasion by HT-1080 cells at sub-toxic concentrations. In these cells, metformin also suppressed phorbol-12-myristate-13-acetate (PMA)-enhanced levels of matrix metalloproteinases-9 (MMP-9) protein, mRNA and transcription activity through suppression of activator protein-1 (AP-1) activation. In addition, metformin strongly repressed the PMA-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and protein kinase C(PKC)alpha, whereas the phosphorylation of p38 mitogen-activated protein kinase was not affected by metformin. Metformin decreased the PMA-induced Ca(2+) influx. Furthermore, treatment with an intracellular Ca(2+) chelator (BAPTA-AM) or a selective calmodulin antagonist (W7) markedly decreased PMA-induced MMP-9 secretion and cell migration, as well as activation of ERK and JNK/AP-1. Metformin inhibited PMA-induced invasion and migration of human fibrosarcoma cells via Ca(2+)-dependent PKCalpha/ERK and JNK/AP-1-signalling pathways. Metformin therefore has the potential to be a potent anti-cancer drug in therapeutic strategies for fibrosarcoma metastasis.

Abstract 521: Impaired migration of human fibrosarcoma cells HT1080 on glycated collagen type I

Abstract Although a strict link does exist between cancer and aging, elderly patients present less invasive tumors than their younger counterparts. During aging, the fibrillar collagen type I, which is an abundant component of the extracellular matrix, becomes a prominent target of post-translational modifications such as glycation. Our present study is mainly designed to investigate the effect of non-enzymatic glycation of this long-lived protein on the proliferation and migration of human fibrosarcoma cells HT1080. For this purpose, collagen type I extracted from rat tail tendons was used for in vitro glycoxydation with ribose. Tumor cell behaviour was then evaluated on 2-dimensional plastic surfaces coated with native or glycated collagen type I. Results showed that the proliferation rate of HT1080 cells on glycated collagen type I did not differ from that on native collagen I after 1, 2 and 3 days of culture. Furthermore, the migration ability of HT1080 cells was studied by quantifying cell speed and trajectories via time-lapse videomicroscopy. At day 1, glycated collagen type I distinctly slowed down the cell locomotion by decreasing the mean cell speed by 47% (20.2 μm/h for glycated versus 38.3 μm/h for its native form). Continuous single cell tracking also permitted the investigation of cell trajectories. Cells grown on glycated collagen exhibited shorter or circular trajectories around their starting point in accordance with the cell speed inhibition. Because actin and vinculin appeared as key proteins involved in cell locomotion, we next investigated the effect of glycated collagen on the organization of these proteins in individual cells in motion. Cells exhibited a dramatic disorganization of F-actin cytoskeleton with loss of stress fibers as compared to native collagen. Actin was principally localized at the rim of the cell. Disturbance of actin integrity was accompanied with a disorganization of vinculin that also localized at the cell periphery. The impact of glycated collagen on the non-receptor protein tyrosine kinase FAK, which plays a crucial role in focal adhesion formation and cell locomotion, was also evaluated. We found that at day 1, glycated collagen significantly inhibits FAK expression by 39% but did not affect their phosphorylation state. These data clearly suggest that age-related changes of extracellular matrix proteins may contribute to a less invasive behaviour of tumor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 521.

Abstract 533: Critical role of MMP-9 dimers in cell migration: design of inhibitory peptides

Abstract Non-proteolytic activities of matrix metalloproteinases (MMPs) have recently been shown to impact cell migration, but the precise mechanism remains to be understood. We previously demonstrated that the hemopexin (PEX) domain of MMP-9 is a prerequisite for enhanced cell migration. Using a biochemical approach, we now report that dimerization of MMP-9 through the PEX domain is necessary for MMP-9-enhanced cell migration. Following a series of substitution mutations within the MMP-9 PEX domain, two critical motifs in the outermost strands of the first and fourth blades of MMP-9 PEX domain were found to be involved in MMP-9 homo- and hetero- dimer formations. Peptides mimicking these motifs efficiently blocked MMP-9 dimer formations and inhibited MMP-9-mediated cell migration, but not membrane type 1- MMP (MT1-MMP)-induced cell migration. In addition, these peptides demonstrate inhibitory effects on migration of human fibrosarcoma HT1080 cells, which express high level of endogenous MMP-9. Using a shRNA approach, CD44 was found to be a critical molecule in MMP-9-mediated cell migration. Furthermore, a novel axis involving an MMP-9-CD44-EGFR signaling pathway in cell migration was identified using antibody microarray analysis and specific receptor tyrosine kinase inhibitors. In conclusion, we dissected the mechanism of proMMP-9-enhanced cell migration and developed structure-based inhibitory peptides specific targeting of MMP-9-mediated cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 533.

Suppression of PMA-induced tumor cell invasion by dihydroartemisinin via inhibition of PKCα/Raf/MAPKs and NF-κB/AP-1-dependent mechanisms

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently been shown to possess antitumor activity in various cancer cells. However, the effects of DHA in preventing the invasion of cancer cells have not been studied. In the present study, we investigated the inhibitory effects of DHA on tumor invasion and migration and the possible mechanisms involved using human fibrosarcoma HT-1080 cells. DHA reduced PMA-induced activation of MMP-9 and MMP-2 and further inhibited cell invasion and migration. DHA suppressed PMA-enhanced expression of MMP-9 protein, mRNA, and transcriptional activity through suppressing NF-κB and AP-1 activation without changing the level of tissue inhibitor of metalloproteinase (TIMP)-1. DHA also reduced PMA-enhanced MMP-2 expression by suppressing membrane-type 1 MMP (MT1-MMP), but did not alter TIMP-2 levels. DHA-inhibited PMA-induced NF-κB and c-Jun nuclear translocation, which are upstream of PMA-induced MMP-9 expression and invasion. Furthermore, DHA strongly repressed the PMA-induced phosphorylation of Raf/ERK and JNK, which are dependent on the PKCα pathway. In conclusion, we demonstrated that the anti-invasive effects of DHA may occur through inhibition of PKCα/Raf/ERK and JNK phosphorylation and reduction of NF-κB and AP-1 activation, leading to down-regulation of MMP-9 expression. The data presented show that DHA is an effective anti-metastatic agent that functions by down-regulating MMP-9 gene expression.

Canthin-6-one Alkaloids and a Tirucallanoid from Eurycoma longifolia and Their Cytotoxic Activity against a Human HT-1080 Fibrosarcoma Cell Line

Phytochemical investigation of the stems of Eurycoma longifolia Jack led to the isolation of two new canthin-6-one alkaloids, 4,9-dimethoxycanthin-6-one (1) and 10-hydroxy-11-methoxycanthin-6-one (2), and a new tirucallane-type triterpenoid, 23,24,25-trihydroxytirucall-7-en-3,6-dione (3), along with 37 known compounds. Among these, an oxasqualenoid (4) was isolated as a natural product for the first time. The structures of the isolates were elucidated by spectroscopic and mass spectrometric means. All the isolates were evaluated for their cytotoxic activity against a HT-1080 human fibrosarcoma cell line. Among them, 9,10-dimethoxycanthin-6-one (14, IC50 = 5.0 microM), 10-hydroxy-9-methoxycanthin-6-one (15, IC50 = 7.2 microM), dihydroniloticin (18, IC50 = 8.2 microM), and 14-deacetyleurylene (34, IC50 = 3.2 microM) displayed stronger activity than the positive control 5-FU (IC50 = 9.2 microM).

The Prostate Cancer-Associated Human Retrovirus XMRV Lacks Direct Transforming Activity but Can Induce Low Rates of Transformation in Cultured Cells

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5alpha (Ref1), showing that XMRV is resistant to TRIM5alpha restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.

Suppression of phorbol-12-myristate-13-acetate-induced tumor cell invasion by bergamottin via the inhibition of protein kinase Cδ/p38 mitogen-activated protein kinase and JNK/nuclear factor-κB-dependent matrix metalloproteinase-9 expression

Matrix metalloproteinase (MMP) plays an important role in the invasion and metastasis of cancer cells. The inhibitory effects of bergamottin, a cytochrome P450 inhibitor from Citrus paradis (grapefruit), on tumor invasion and migration and the possible mechanisms involved in this inhibition were investigated in human fibrosarcoma HT-1080 cells. Bergamottin reduced phorbol-12-myristate-13-acetate (PMA)-induced activation of MMP-9 and MMP-2 and further inhibited cell invasion and migration. Bergamottin suppressed PMA-enhanced expression of MMP-9 protein, mRNA and transcription activity levels through suppression of nuclear factor-kappaB (NF-kappaB) activation without changing the tissue inhibitor of metalloproteinase 1 level. Bergamottin also reduced PMA-enhanced MMP-2 expression through suppression of membrane-type 1 MMP, but did not alter tissue inhibitor of metalloproteinase 2 levels. Bergamottin inhibited PMA-induced NF-kappaB nuclear translocation and IkappaBalpha degradation, which are upstream of PMA-induced MMP-9 expression and invasion. Furthermore, bergamottin strongly repressed the PMA-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK), which are dependent on the protein kinase C-delta pathway. In conclusion, we demonstrated that the anti-invasive effects of bergamottin might occur through inhibition of protein kinase C-delta, p38 mitogen-activated protein kinase, and JNK phosphorylation and reduction of NF-kappaB activation, leading to downregulation of MMP-9 expression. These results suggest that the suppression of MMP expression contributes, at least in part, to the antitumor activity of bergamottin.

Sub-millimolar concentration of the novel phenol-based compound, 2-hydroxy benzoate zinc, induces apoptosis in human HT-1080 fibrosarcoma cells

To examine the effect of a novel phenolic-based compound, 2-hydroxy benzoate zinc (2HBZ), and acetylsalicylic acid (ASA) on human HT-1080 fibrosarcoma cells. MTT assay was used to assess cell proliferation while different methods were used to detect apoptosis morphologically and immunohistochemically in Human HT-1080 fibrosarcoma cells. Apoptosis was determined by Annexine-V labelling, and caspase-3 activation. In addition, western blot was used to analyse p21, p53 and Bax and flow cytometry was to analyse the cell cycle. 2HBZ exhibited a more than 5-fold increase in cytotoxic potency when compared with ASA with mean LD50 values of 210 and 1100 lM respectively (P < 0.0001). The cytotoxic effects of 2HBZ were both time- and dosedependent with marked apoptosis being evident only after 24 h at concentrations as low as 200 mM. In contrast, ASA-induced apoptosis was observed only at concentrations in excess of 1000 mM at the same time point. Both 2HBZ and ASA induced caspase-3 activation in the cells, which confirmed that their cytotoxic effects were the result of apoptotic cell death. These findings were further confirmed by immunomorphological studies for the detection of apoptosis including haematoxylineosin, methyl green/pyronin Y staining and scanning electron microscopy. In addition, 2HBZ caused a marked increase in p21, p53 and Bax protein expressions and these effects were associated with an increase in G1 and G2 arrest of the cell cycle and a reduction in S-phase. These results demonstrate that the novel phenolic compound 2HBZ is a potent apoptosis-inducing agent in HT-1080 cells and warrants further investigation as a potential chemotherapeutic agent in primary cancer cell models.