Human Fibrosarcoma HT1080 Cells Research Articles

Therapeutic angiogenesis using tumor cell-conditioned medium.

Stem cell-conditioned medium (CM), which contains angiogenic factors that are secreted by stem cells, represents a potential therapy for ischemic diseases. Along with stem cells, tumor cells also secrete various angiogenic factors. Here, tumor cells as a cell source of CM for therapeutic angiogenesis was evaluated and the therapeutic efficacy of tumor cell CM in mouse hindlimb ischemia models was demonstrated. CM obtained from a human fibrosarcoma HT1080 cell line culture was compared with CM obtained from a human bone marrow-derived mesenchymal stem cell (MSC) culture. HT1080 CM contained higher concentrations of angiogenic factors compared with MSC CM, which was attributable to the higher cell density that resulted from a much faster growth rate of HT1080 cells compared with MSCs. For use in in vitro and in vivo angiogenesis studies, HT1080 CM was diluted such that HT1080 CM and MSC CM would have the same cell number basis. The two types of CMs induced the same extent of human umbilical vein endothelial cell (HUVEC) proliferation in vitro. The injection of HT1080 CM into mouse ischemic limbs significantly improved capillary density and blood perfusion compared with the injection of fresh medium. Although the therapeutic outcome of HT1080 CM was similar to that of MSC CM, the preparation of CM by tumor cell line culture would be much more efficient due to the faster growth and unlimited life-time of the tumor cell line. These data suggest the potential application of tumor cell CM as a therapeutic modality for angiogenesis and ischemic diseases. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:456-464, 2016.

Evaluation of the safety and efficacy of TRAIL and taurolidine use on human fibrosarcoma xenografts in vivo.

Fibrosarcomas are rare malignant soft tissue tumours that exhibit a poor response to current therapeutic regimens. Previously, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and taurolidine were observed to induce apoptosis synergistically in HT1080 human fibrosarcoma cells in vitro. Consequently, the present study aimed to assess the safety and efficacy of TRAIL in combination with taurolidine on the local growth of fibrosarcoma xenografts in vivo. HT1080 fibrosarcoma cells were inoculated subcutaneously into both flanks of 49 athymic nude mice in order to establish tumour xenografts. TRAIL and taurolidine were applied intraperitoneally at various single and cumulative treatment doses. After 12 days, the experiment was terminated and surviving animals were euthanised. Tumour progression was determined during and following treatment. To assess the potential toxic effects of the two compounds, the organs (lung, liver, kidney and heart) of all animals were examined histologically. The results revealed that combined treatment with TRAIL and taurolidine significantly inhibited the growth of HT1080 xenografts, whereas untreated animals had steadily increasing tumours. The most effective combination was TRAIL at 2 µg per application (cumulative dose, 16 µg) and taurolidine at 30/15 mg per application (cumulative dose, 180 mg), reducing the mean size of implanted xenografts to 10.9 mm2 following treatment (vs. 48.9 mm2 in the control group; P=0.0100). Despite distinct tumour mass reduction, the rate of mortality was significantly increased in animals treated with TRAIL and taurolidine in a taurolidine dose-dependent manner; however, histological examinations of relevant organs revealed no evidence of systemic toxicity (mean survival time, 7.9 days in the treated groups vs. 12 days in the control group; P<0.0010). In summary, whilst the combination of TRAIL and taurolidine synergistically inhibited the growth of fibrosarcoma xenografts in vivo, it was also accompanied by significantly increased mortality rate. Thus, although taurolidine is assumed to be a compound with an acceptable toxicity profile, and therefore increasingly used in clinical trials, the current findings raise concerns with regard to its safety and therapeutic index, and indicate the requirement for further detailed toxicity tests.

Novel Antitumor Invasive Actions of p-Cymene by Decreasing MMP-9/TIMP-1 Expression Ratio in Human Fibrosarcoma HT-1080 Cells.

p-Cymene (4-isopropyltoluene) has been reported to have beneficial actions such as anti-inflammatory and antinociceptive activities. To evaluate whether p-cymene exhibits antitumor invasive actions, we examined the effects of p-cymene on the production of matrix metalloproteinase 9 (MMP-9)/gelatinase B and tissue inhibitor of metalloproteinases-1 (TIMP-1) in human fibrosarcoma HT-1080 cells. p-Cymene was found to dose-dependently inhibit the 12-O-tetradecanoylphorbol 13-acetate (TPA)-augmented production and gene expression of MMP-9 in HT-1080 cells. In contrast, p-cymene enhanced the TPA-augmented production and gene expression of TIMP-1 in HT-1080 cells. However, there was no change in the constitutive level of MMP-9 and TIMP-1 mRNAs and TIMP-1 protein in p-cymene-treated cells. In addition, we found that the in-vitro TPA-augmented invasiveness of HT-1080 cells was inhibited by p-cymene in a dose-dependent manner. Furthermore, p-cymene was found to suppress the constitutive and/or TPA-augmented phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in HT-1080 cells. Thus, these results provide novel evidence that p-cymene is an effective candidate for the prevention of tumor invasion and metastasis through mechanisms that include the inhibition of MMP-9 expression and the augmentation of TIMP-1 production along with the suppression of ERK1/2 and p38 MAPK signal pathways in tumor cells.

An Innovative Cell Microincubator for Drug Discovery Based on 3D Silicon Structures

We recently employed three-dimensional (3D) silicon microstructures (SMSs) consisting in arrays of 3 μm-thick silicon walls separated by 50 μm-deep, 5 μm-wide gaps, as microincubators for monitoring the biomechanical properties of tumor cells. They were here applied to investigate the in vitro behavior of HT1080 human fibrosarcoma cells driven to apoptosis by the chemotherapeutic drug Bleomycin. Our results, obtained by fluorescence microscopy, demonstrated that HT1080 cells exhibited a great ability to colonize the narrow gaps. Remarkably, HT1080 cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and were prone to leave the gaps. Finally, we performed label-free detection of cells adherent to the vertical silicon wall, inside the gap of 3D-SMS, by exploiting optical low coherence reflectometry using infrared, low power radiation. This kind of approach may become a new tool for increasing automation in the drug discovery area. Our results open new perspectives in view of future applications of the 3D-SMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells.

Abstract B131: Chemical array screen identifies a novel MMP inhibitor NPDK-18 that suppresses tumor cell migration

Abstract Background and Purpose: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade extracellular matrix components and that have been implicated in a wide range of pathological processes including tumor metastasis. Although many small-molecule MMP inhibitors have been developed, there are some problems related to specificity and side effects. In addition, recent studies have shown that several MMPs have nonproteolytic and tumor-suppressing functions depending upon the stage of cancer progression. Thus, much work remains for the elucidation of each MMP function and the development of new MMP inhibitors. Small-molecule microarrays represent a powerful new platform for facilitating target-based screens. Thousands of small molecules are arrayed on a slide glass and used to screen for protein-small molecule interactions in a high-throughput manner. We have developed unique photo-cross-linked chemical arrays, which enable the immobilization of a variety of small molecules on a solid support in a functional group-independent manner using a photoaffinity reaction. In this study, we screened MMP-9 inhibitors using the photo-cross-linked chemical arrays. Methods and Results: The array slides with compounds of the RIKEN NPDepo chemical library were probed sequentially with His-Myc-tagged human recombinant MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody, and then scanned with a microarray scanner. The fluorescence signals from two slides treated with and without His-Myc-tagged MMP-9 were quantified. We obtained 27 hit compounds among 24,275 compounds; 2 of the identified compounds (isoxazole compound NPDK-1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of NPDK-1 and found that NPDK-18 showed the strongest inhibitory activity against MMP-9, with an IC50 value of 3.8 μM. NPDK-18 inhibited the enzyme activities of MMP-2, MMP-12 and MMP-13, as well as MMP-9 and MMP-9/ΔHemopexin, with almost identical potency, but showed no inhibition against MMP-1 and MMP-7. The biological potency of NPDK-18 was confirmed in a wound-healing scratch assay, where NPDK-18 significantly inhibited cell migration of human fibrosarcoma HT1080 cells with no cell toxicity at 10 μM. Conclusion: These results suggest that NPDK-18 is a broad-spectrum MMP inhibitor. Our study has shown that the photo-cross-linked chemical arrays are useful for high-throughput drug screening. Citation Format: Makoto Kawatani, Yasumitsu Kondoh, Kaori Honda, Tomomi Sekine, Hiroyuki Osada. Chemical array screen identifies a novel MMP inhibitor NPDK-18 that suppresses tumor cell migration. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B131.

Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds.

BackgroundExportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330.MethodsUsing the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays.ResultsTreatment of HT1080 cells with gradually increasing concentrations of SINE resulted in > 100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental and resistant cells, the extent of response was more robust in the parental cells.ConclusionsThese results suggest that SINE resistance is conferred by alterations in signaling pathways downstream of XPO1 inhibition. Modulation of these pathways could potentially overcome the resistance to nuclear export inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1790-z) contains supplementary material, which is available to authorized users.

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.

Morphological modulation of human fibrosarcoma HT-1080 cells by hydroxybenzoate compounds during apoptosis

&lt;p class="BodyText1"&gt;Hydroxybenzoate (HB) compounds have shown to modulate the morphology in human fibrosarcoma HT-1080 cells. The changes in HT-1080 cells showed marker signs of apoptosis, which included the condensation of nucleus, cell round, blebbing and the formation of apoptotic bodies. The different stages of apoptosis were assessed microscopically using different staining and immunohistochemical techniques, as well as scanning electron microscopy. In addition, HB compounds increased the expression of caspase-3, which is closely associated with the development of the modulation in HT-1080 cells that are undergoing the programmed cell death. Both acetyl salicylic acid (ASA) and HBZn compounds were dose and treatment duration dependent.&lt;/p&gt;

Identification of matrix metalloproteinase inhibitors by chemical arrays.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade many extracellular matrix components and that have been implicated in the pathogenesis of various human diseases including cancer metastasis. Here, we screened MMP-9 inhibitors using photo-cross-linked chemical arrays, which can detect small-molecule ligand-protein interactions on a chip in a high-throughput manner. The array slides were probed sequentially with His-MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody and then scanned with a microarray scanner. We obtained 27 hits among 24,275 compounds from the NPDepo library; 2 of the identified compounds (isoxazole compound 1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of 1 and found that compound 18 had the strongest inhibitory activity. Compound 18 also inhibited other MMPs, including MMP-2, MMP-12, and MMP-13 and significantly inhibited cell migration in human fibrosarcoma HT1080 cells. These results suggest that 18 is a broad-spectrum MMP inhibitor.

Evaluation of Effective MMP Inhibitors from Eight Different Brown Algae in Human Fibrosarcoma HT1080 Cells.

Matrix metalloproteinases (MMPs) are crucial extracellular matrices degrading enzymes that have important roles in metastasis of cancer progression as well as other significant conditions such as oxidative stress and hepatic fibrosis. Marine plants are on the rise for their potential to provide natural products that exhibit remarkable health benefits. In this context, brown algae species have been of much interest in the pharmaceutical field with reported instances of isolation of bioactive compounds against tumor growth and MMP activity. In this study, eight different brown algae species were harvested, and their extracts were compared in regard to their anti-MMP effects. According to gelatin zymography results, Ecklonia cava, Ecklonia bicyclis, and Ishige okamurae showed higher inhibitory effects than the other samples on MMP-2 and -9 activity at the concentrations of 10, 50, and 100 μg/mL. However, only I. okamurae was able to regulate the MMP activity through the expression of MMP and tissue inhibitor of MMP observed by mRNA levels. Overall, brown algae species showed to be good sources for anti-MMP agents, while I. okamurae needs to be further studied for its potential to yield pharmaceutical molecules that can regulate MMP-activity through cellular pathways as well as enzymatic inhibition.

Abstract 3378: Discoidin domain receptor 2 differentially controls HT-1080 cell proliferation in young-adult and old type I collagen 3D matrices

Abstract Type I collagen plays a key role in tumor proliferation, adhesion and migration. We have recently demonstrated that this fibrillar and life-long protein undergoes detrimental changes in biochemical and biomechanical properties during aging (1). In addition to a high Advanced Glycation End-products (AGE) and cross-links content, old collagen exhibits a significant loss in fibrillar properties. In this study, we analyzed the effect of collagen aging on human HT-1080 fibrosarcoma cell proliferation in 3D matrix model. For this, type I collagen was extracted from tail tendons of young-adult (2 months) and old (2 years) rats. In 3D matrix, the rate of HT-1080 cell growth was significantly lower in young-adult collagen. This was accompanied by a down-regulation of ERK1/2 phosphorylation and an increase in the cyclin-dependent kinase inhibitor p21 expression. Blocking antibodies and siRNA strategy against β1 integrin and AGEs receptor (RAGE) did not increase cell growth in young-adult collagen. Accumulating evidence suggest that Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase with the unique ability to be activated by fibrillar collagen. To determine whether differential DDR2 responses are elicited by the two collagens, DDR2 was immunoprecipitated from cell lysates, and its phosphorylated form was detected by immunoblotting. DDR2 phosphorylation level was increased in young-adult collagen. This was accompanied by an increase in the tyrosine phosphatase SHP2 phosphorylation, the primary target of DDR2. Interestingly, the DDR2 inhibitor nilotinib was able to restore cell proliferation in young-adult collagen. In addition to an increase in ERK1/2 phosphorylation, nilotinib decreased SHP2 phosphorylation and p21 expression. Taken together, these data show that age-associated changes of type I collagen organization, especially fibrillar properties, are critical for tumor cell proliferation control by DDR2.

DNA-hypomethylating agent, 5'-azacytidine, induces cyclooxygenase-2 expression via the PI3-kinase/Akt and extracellular signal-regulated kinase-1/2 pathways in human HT1080 fibrosarcoma cells.

The cytosine analogue 5'-azacytidine (5'-aza) induces DNA hypomethylation by inhibiting DNA methyltransferase. In clinical trials, 5'-aza is widely used in epigenetic anticancer treatments. Accumulated evidence shows that cyclooxygenase-2 (COX-2) is overexpressed in various cancers, indicating that it may play a critical role in carcinogenesis. However, few studies have been performed to explore the molecular mechanism underlying the increased COX-2 expression. Therefore, we tested the hypothesis that 5'-aza regulates COX-2 expression and prostaglandin E2 (PGE2) production. The human fibrosarcoma cell line HT1080, was treated with various concentrations of 5'-aza for different time periods. Protein expressions of COX-2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), pAkt, Akt, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (pERK) were determined using western blot analysis, and COX-2 mRNA expression was determined using RT-PCR. PGE2 production was evaluated using the PGE2 assay kit. The localization and expression of COX-2 were determined using immunofluorescence staining. Treatment with 5'-aza induces protein and mRNA expression of COX-2. We also observed that 5'-aza-induced COX-2 expression and PGE2 production were inhibited by S-adenosylmethionine (SAM), a methyl donor. Treatment with 5'-aza phosphorylates PI3-kinase/Akt and ERK-1/2; inhibition of these pathways by LY294002, an inhibitor of PI3-kinase/Akt, or PD98059, an inhibitor of ERK-1/2, respectively, prevents 5'-aza-induced COX-2 expression and PGE2 production. Overall, these observations indicate that the hypomethylating agent 5'-aza modulates COX-2 expression via the PI3-kinase/Akt and ERK-1/2 pathways in human HT1080 fibrosarcoma cells.

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.

In vitro and in vivo bioactivities of aqueous and ethanol extracts from Helicteres angustifolia L. root

Ethnopharmacological relevanceHelicteres angustifolia L. (H. angustifolia L.) has been used as traditional medicine in the treatment of cancer in China and Laos. Its medical benefits, however, are still lacking of scientific evidence. Two extracts successively obtained from the root of H. angustifolia L., namely the aqueous root extract (ARE) and the ethanolic root extract (ERE), were used to evaluate the antioxidant and anticancer activities in vitro, and the antitumor efficacy of ARE was examined in vivo, respectively. Materials and methodsARE and ERE were extracted successively from H. angustifolia L. root with water and ethanol. In vitro antioxidant activities were assessed by radicals scavenging assay, ferrous chelating assay and reducing power assay. In vitro anticancer activities of ARE and ERE were evaluated by their cytotoxic effects against three human cancer cell lines. In addition, the anti-tumor activities of ARE in vivo were assessed by using Ht1080 (human fibrosarcoma cell line Ht1080) tumor xenografts mice. BALB/c nude mice were orally administrated with 200mg/kg/d of ARE. The tumor inhibition rate was determined on day 42 after treatment by using histopathology analysis of the tumor tissues. Furthermore, relevant biochemical parameters in blood were analyzed to monitor their cytotoxic effect. ResultsIn vitro assays indicated that ARE possessed relatively higher antioxidant and anticancer activities than ERE, with IC50 values of 82.31±9.62, 62.50±6.99, and 127.49±2.9μg/mL against DLD-1, A549, and HepG2 cells, respectively. In vivo tumor inhibition experiments suggested that ARE possessed significant antitumor efficacy in BALB/c nude mice with a tumor inhibition rate of 49.83±14.38% (p<0.05) and little toxicity was observed to the host. ConclusionARE from H. angustifolia L. possessed high antioxidant activities is active against liver cancer HepG2, lung cancer A549 and colon cancer DLD-1 cells in vitro and tumor xenografts bearing BALB/c nude mice in vivo. Further studies on elucidation of the mechanisms involved and isolation of the active components may provide more valuable information for the development of functional products from H. angustifolia L. and their application in cancer treatment.

Jaeumganghwa-Tang Induces Apoptosis via the Mitochondrial Pathway and Lactobacillus Fermentation Enhances Its Anti-Cancer Activity in HT1080 Human Fibrosarcoma Cells.

Jaeumganghwa-tang (JGT, Zi-yin-jiang-huo-tang in Chinese and Jiin-koka-to in Japanese) is an oriental herbal formula that has long been used as a traditional medicine to treat respiratory and kidney diseases. Recent studies revealed that JGT exhibited potent inhibitory effects on allergies, inflammation, pain, convulsions, and prostate hyperplasia. Several constituent herbs in JGT induce apoptotic cancer cell death. However, the anti-cancer activity of JGT has not been examined. In this study, we investigated the anti-cancer effects of JGT using highly tumorigenic HT1080 human fibrosarcoma cells and elucidated the underlying mechanisms. In addition, we examined whether the Lactobacillus fermentation of JGT enhanced its anti-cancer activity using an in vivo xenograft model because fermentation of herbal extracts is thought to strengthen their therapeutic effects. Data revealed that JGT suppressed the growth of cancer cells efficiently by stimulating G1 cell cycle arrest and then inducing apoptotic cell death by causing mitochondrial damage and activating caspases. The phosphorylation of p38 and ERK also played a role in JGT-induced cell death. In vitro experiments demonstrated that JGT fermented with Lactobacillus acidophilus, designated fJGT162, elicited similar patterns of cell death as did non-fermented JGT. Meanwhile, the daily oral administration of 120 mg/kg fJGT162 to HT1080-bearing BALB/c nude mice suppressed tumor growth dramatically (up to 90%) compared with saline treatment, whereas the administration of non-fermented JGT suppressed tumor growth by ~70%. Collectively, these results suggest that JGT and fJGT162 are safe and useful complementary and alternative anti-cancer herbal therapies, and that Lactobacillus fermentation improves the in vivo anti-cancer efficacy of JGT significantly.

Cellular uptake and in vivo distribution of polyhistidine peptides

Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.

344. Targeting piggyBac Transposition Into a User-Defined Chromosomal Locus in Human Cells

The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into the genomes of human cells. We have previously demonstrated biased PB integration into an artificial target site engineered into human cells (Kettlun et al., Molecular Therapy, 2011). We undertook the current investigation to see if we could engineer PB to target integration into a user-defined chromosomal locus in human cells. We chose to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on chromosome X. HPRT-deficient cells can be selected by growth in 6-thioguanine (6TG), so PB-mediated targeted knockout of HPRT can be used to isolate cells with targeted integration events. We used HT-1080 human fibrosarcoma cells which harbor one × chromosome for our studies. We engineered a series of 4 zinc finger proteins (ZFP) and 4 transcription activator-like (TAL) proteins fused to the PB transposase. The transposon used contained a splice-acceptor sequence followed by a beta-galactosidase-neomycin resistance fusion gene (b-Geo) which allowed measurement of gene transfer activity (measuring neomycin resistant colonies after transposition) as well as HPRT knockout efficiency (due to the splice-acceptor sequence). All HPRT-targeted ZFP-PB and TAL-PB proteins exhibited transposition activity. Chromatin immunoprecipitation assays revealed varied targeting-binding of the different PB chimeras to the HPRT locus. One ZFP-PB and one TAL-PB chimera demonstrated higher HPRT gene targeting via reproducible production of a higher number of 6TG resistant cells. Deep sequencing analysis revealed integration efficiency of 0.42% and 1% for the ZFP-PB and TAL-PB chimeric transposases in 6TG-resistant HT-1080 cells. Our study provides new insights into engineering PB to target locations in the human genome which will permit further refinements to improve targeting efficiency. Additionally, selection of cells with targeted integration events should allow isolation of “targeted-only” cells for consideration of therapeutic purposes.ss

Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

Indocyanine Green Derivative Covalently Conjugated with Gold Nanorods for Multimodal Phototherapy of Fibrosarcoma Cells.

A hydrophilic indocyanine green derivative (ICG-Der-02) was covalently doped into mesoporous silica-coated gold nanorods (AuNRs/mSiO2). The self-synthesized derivative offers one carboxyl functional group on a side chain, which enables ICG-Der-02 to be covalently linked to nanomaterials and reduces the probability of leakage/desorption of the dye. The detection of infrared luminescence around 1270 nm confirmed that 102 is efficiently generated by the nanocomposite (AuNRs/mSiO2-ICG-Der-02). Furthermore, a second layer of silica was coated onto the nanocomposite, which then was conjugated with the α(v) integrin-targeting cyclic peptide (RGD-4C). The cell tests showed that the resulting nanoconjugate (AuNRs/mSiO2-ICG-Der-02/RGD-4C) was able to bind preferentially to HT-1080 human fibrosarcoma cells. Due to the synergistic effect of the produced nanoconjugates, a dual-modality photothermal and photochemical therapy was successfully achieved by 808 nm irradiation. Compared to using photothermal or photochemical therapy alone, the dual-modality photothermal/photochemical therapeutic strategy proved to be more damaging to HT-1080 cells and enhanced the effectiveness of photodestruction. Our work presents a novel approach to the multimodal treatment of fibrosarcoma and shows promise for future use in cancer theranostics.

THE DUAL ROLE OF ANDROGEN RECEPTOR IN MESENCHYMAL CELLS

The androgen receptor (AR) mediates differentiation, proliferation and transformation of target tissues. These processes require a crosstalk between epithelial and stromal cells. Prostate cancer (PCa) represents a major cause of cancer-related mortality in men, and is often associated with deregulation of androgen/AR axis. Clinical and molecular findings have highlighted the role of epithelial AR in PCa progression. In contrast, the functions of AR in mesenchymal cells are still unclear. We previously reported that low androgen concentration (1 pM) triggers interaction of AR with the Src tyrosine kinase and PI3-K, thus driving cell cycle progression in fibroblasts. In contrast, stimulation of fibroblasts and fibrosarcoma cells with physiological (10 nM) androgen concentration leads to interaction of AR with full-length filamin A (FLNa) and does not trigger DNA synthesis. On the basis of these findings, we re-examined the role of androgen/AR axis in fibroblasts and human fibrosarcoma HT1080 cells. Recently, we obtained two original and integrated findings on the decision of mesenchymal cells to undergo reversible quiescence and migrate upon stimulation with 10 nM androgens (Castoria et al. 2011 and 2014). This decision is dependent upon the interaction of AR with FLNa. Once assembled, the bipartite AR/FLNa complex recruits a1-integrin and triggers Rac1 activation, thereby enhancing on the one hand cell motility. On the other, Rac 1 activation triggers its downstream effector DYRK 1B, which phosphorylates Ser10 of p27. Stabilization of p27 and cell quiescence then follow. These results strengthen and extend our studies, adding a new and exciting piece to the complex puzzle of signaling networks activated by androgens in target cells. Our findings might have implications for current approaches to AR-related diseases.