Murine Fibrosarcoma Cell Line Research Articles

Theoretical, DNA/BSA Binding, Molecular Docking and In Vitro Cytotoxicity Studies of Homoleptic Metal(II) Complexes Derived from 2‐Formylpyridine‐Based Ligands

AbstractA new class of homoleptic metal(II) complexes of general formula [M(L1–4)2]2Cl in which L1=1‐([1,1′‐biphenyl]‐4‐yl)‐N‐(2‐((‐pyridin‐2‐ylmethylene)amino)ethyl)methanimine, L2=1‐([1,1′‐biphenyl]‐4‐yl)‐N‐(2‐((‐pyridin‐2‐ylmethylene)amino)phenyl)methanimine, L3=1‐([1,1′‐biphenyl]‐4‐yl)‐N‐(2‐((‐pyridin‐2‐ylmethylene)amino)‐4‐nitrophenyl)methanimine and L4=4‐(([1,1′‐biphenyl]‐4‐ylmethylene)amino)‐3‐((−pyridin‐2‐ylmethylene)amino)benzophenone, and M=Ni(II) (1–4), Cu(II) (5–8) and Zn(II) (9–12) have been synthesized and characterized. The IR spectra reveal the involvement of azomethine and 2‐formylpyridine ring nitrogen atoms in chelation. The UV‐Vis and EPR spectral studies envisage distorted octahedral geometry around metal(II) ion. All the complexes were stable for 72 h in biologically relevant solutions, and copper(II) complexes were reduced to Cu+ center in the presence of ascorbic acid solution. The binding studies of complexes with calf thymus DNA (CT‐DNA) indicate groove mode of binding, and the complexes 3, 7 and 11 containing the 4‐nitrophenyl substituent exhibit the highest binding affinity than the other complexes. Furthermore, the spectral studies also revealed the interaction of complexes with BSA through static mechanism. In vitro cytotoxicity activity of the complexes was screened against human lung adenocarcinoma (A549) and breast adenocarcinoma (MCF‐7) cancerous, and normal murine fibrosarcoma (L929) cell lines. The molecular docking studies of the homoleptic metal(II) complexes show hydrophobic, π–π, σ–π and hydrogen bonding interactions with EGFR/VEGFR2 kinase receptors.

The A179L Gene of African Swine Fever Virus Suppresses Virus-Induced Apoptosis but Enhances Necroptosis.

A179L, a non-structural protein of African swine fever virus (ASFV), is capable of suppressing apoptosis by binding the BH3 domain of the pro-apoptotic Bcl-2 family proteins via a conserved ligand binding groove. Our present study aims to determine if A179L affects necroptosis, the second form of programmed cell death induced by DNA and RNA viruses. Here we report that A179L enhanced TNF-α or TSZ (TNF-α, Smac, and Z-Vad)-induced receptor-interacting protein kinase (RIPK1), RIPK3, and mixed lineage kinase domain like peudokinase (MLKL) phosphorylation in L929 cells, a murine fibrosarcoma cell line. Sytox green staining revealed that A179L significantly increased the number of necroptotic cells in TSZ-treated L929 cells. Using human herpes simplex virus 1 (HSV-1) to model DNA virus-induced cell death, we found that A179L blocked the HSV-1-induced cleavage of poly (ADP-ribose) polymerase (PARP), caspase 8, and caspase 3 and decreased the number of apoptotic cells in HSV-1-infected IPEC-DQ cells, a porcine intestinal epithelial cell line. In contrast, A179L transfection of IPEC-DQ cells enhanced HSV-1-induced RIPK1, RIPK3, and MLKL phosphorylation and increased the number of necroptotic cells. Consistently, A179L also suppressed apoptosis but enhanced the necroptosis induced by two RNA viruses, Sendai virus (SeV) and influenza virus (IAV). Our study uncovers a previously unrecognized role of A179L in regulating cell death and suggests that A179L re-directs its anti-apoptotic activity to necroptosis.

Cytotoxic effect of Montivipera bornmuelleri's venom on cancer cell lines: in vitro and in vivo studies.

Background Montivipera bornmuelleri’s venom has shown immunomodulation of cytokines release in mice and selective cytotoxicity on cancer cells in a dose-dependent manner, highlighting an anticancer potential. Here, we extend these findings by elucidating the sensitivity of murine B16 skin melanoma and 3-MCA-induced murine fibrosarcoma cell lines to M. bornmuelleri’s venom and its effect on tumor growth in vivo.MethodsThe toxicity of the venom on B16 and MCA cells was assessed using flow cytometry and xCELLigence assays. For in vivo testing, tumor growth was followed in mice after intratumoral venom injection.ResultsThe venom toxicity showed a dose-dependent cell death on both B16 and MCA cells. Interestingly, overexpression of ovalbumin increased the sensitivity of the cells to the venom. However, the venom was not able to eradicate induced-tumor growth when injected at 100 µg/kg. Our study demonstrates a cytotoxic effect of M. bornmuelleri’s venom in vitro which, however, does not translate to an anticancer action in vivo.

Abstract 2326: Time-resolved studies of necroptosis reveal distinct regulatory functions for NFκB

Abstract Resistance to regulated cell death (RCD) is an important hallmark of cancer and reason why chemotherapy fails. Efficient reprogramming of cancer cells requires a molecular and dynamic understanding of regulatory circuits. Research has uncovered several resistance mechanisms for apoptosis with nuclear factor κB (NFκB) as a key inhibitory transcription factor. Targeted induction of necroptosis, which was recently discovered as an alternative form of RCD, could be a promising strategy to overcome apoptosis resistance. However, whether NFκB has a regulatory role in necroptosis is less clear. Here, we develop a molecular and time-resolved understanding of how NFκB regulates necroptosis. We present a real-time experimental system to quantify necroptosis in single cancer cells to study the cytotoxic effect of substances while avoiding the bias of proliferation in endpoint protocols. Using live-cell microscopy, computational cell tracking and automated image analysis we quantitate necroptotic death rates in L929 cells, a murine fibrosarcoma cell line, treated with tumor necrosis factor (TNF). While it is known that L929 cells predominantly respond with necroptosis despite rapid activation of NFκB, our experiments reveal an unsuspected temporal phasing of necroptosis over a time course of 24 hours. By perturbing NFκB activation dynamics using targeted CRISPR/Cas9 of IκB and/or Rel proteins, we find that NFκB has at least two distinct roles regulating early and late phases of TNF-induced necroptosis. Further, we will present our molecular analysis that identified NFκB-responsive candidate genes and mapped them into a functional temporo-spatial context within the NFκB-necroptosis signaling network. Our combined approach may inform novel personalized anticancer strategies to exploit targeted necroptosis while avoiding NFκB-induced drug resistance. Citation Format: Marie Oliver Metzig, Simon Mitchell, Brooks Taylor, Alexander Hoffmann. Time-resolved studies of necroptosis reveal distinct regulatory functions for NFκB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2326.

PO-163 Identification of candidate genes underlying soft tissue sarcoma progression using a progression series of murine fibrosarcoma cell lines

IntroductionThe transcription factor Sall4 is a well-known developmental regulator involved in embryonic patterning and stem cell maintenance. More recently Sall4 has been suggested to be an oncofetal gene due to being expressed in many fetal tissues and malignant tumours but only rarely in normal adult tissues. The re-expression of such genes in cancer might reflect crucial processes during development, which are reactivated in neoplasias such as increased migratory and invasive capacities. Sall4 is discussed as novel marker and target in some solid tumours and haematological malignancies, however its role in melanoma is unknown.Material and methodsTo address a putative role of Sall4 in melanoma, we used the Tyr::NrasQ61KINK4a−/− murine melanoma model, which spontaneously develops metastatic melanoma. Those mice were crossed with Tyr::CreERT2Sall4lox/loxR26R::GFP mice, allowing us to conditionally ablate Sall4 in melanocytes and to trace Cre activity via GFP expression. We further used human melanoma patient-derived primary cell cultures for in vitro experiments and the same cell lines as xenographs in immunocompromised mice.Results and discussionsMice lacking Sall4 did not form primary tumours compared to the control animals however strikingly had more GFP +melanoma metastases in the lungs. Sall4lox/wt animals formed tumours like control animals but also showed more lung metastases. Through RNA Sequencing on siSALL4-treated human melanoma cell lines we found that SALL4 decrease correlates with upregulation of melanoma invasiveness genes and functionally increased invasiveness in vitro. We further found by Co-IP that SALL4 binds to members of the histone deacetylase family (HDACs) and by running a ChIP-Seq for H3K27ac, we could indeed verify that knocking down SALL4 lead to increased histone acetylation of enhancer regions of the mentioned invasiveness genes. Interestingly, HDAC inhibitor (HDACi) treatment in vitro resulted in an invasiveness gene expression pattern very similar to SALL4 knock down and HDACi treatment of xenograph tumours also lead to an expression profile hinting towards increased invasiveness in vivo.ConclusionAll in all we found that downregulation of Sall4 in melanoma leads to increased acetylation and expression of invasiveness genes in vitro and increased metastasis in vivo, with both phenotypes being closely mimicked by HDAC inhibitor application.

An in vitro investigation of immunomodulatory properties of Lactobacillus plantarum and L. delbrueckii cells and their extracellular polysaccharides.

Many probiotic lactobacilli and their extracellular polysaccharides (EPS) have beneficial immunological properties. However, it is unclear how they elicit the host immune response. We thus investigated the immunological properties of UV-killed Lactobacillus delbrueckii TU-1 and L. plantarum KM-9 cells as well as their extracellular polysaccharides (EPSs). High-performance liquid chromatography and ion exchange chromatography analyses showed that their EPSs differ in sugar composition and sugar fractionation. The immunological properties were evaluated in a semi-intestinal model using a Transwell co-culture system that employed human intestinal epithelial (Caco-2) cells on the apical side and murine macrophage (RAW264.7) cells on the basolateral side. The UV-killed cells and EPSs were added to the apical side to allow direct contact with Caco-2 cells and incubated for 6 hr. After incubation, the amounts of tumor necrosis factor-α and several cytokines released by RAW264.7 or Caco-2 cells were quantified by cytotoxic activity on L929 cells (murine fibrosarcoma cell line) and quantitative reverse-transcriptase PCR. We found that the UV-killed cells and their EPSs had immunological effects on RAW264.7 cells via Caco-2 cells. The RAW264.7 cells showed different cytokine production profiles when treated with UV-killed cells and EPSs. The UV-killed cells and EPSs promoted a Th1-type cellular response. Furthermore, we found that the UV-killed cells sent positive signals through Toll-like receptor (TLR) 2. Meanwhile, neither EPS sent a positive signal through TLR4 and TLR2. This evidence suggests that both UV-killed cells of the lactobacillus strains and their EPSs trigger a Th1-type immune response in a human host, with the former triggering the response via the TLRs expressed on its epithelium and the latter employing a mechanism yet to be determined, possibly involving a novel receptor that is designed to recognize specific patterns of repeating sugar in the EPSs.

Induction of Apoptosis in TNF-Treated L929 Cells in the Presence of Necrostatin-1.

It has been shown that necroptosis—caspase-independent programmed necrotic cell death—can be induced by treatment with tumor necrosis factor (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of a caspase inhibitor. Although it was reported that necrostatin-1—a specific inhibitor of necroptosis—inhibited TNF-induced necroptosis in L929 cells, it has not been elucidated whether the cells eventually die by apoptosis in the presence of necrostatin-1. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of necrostatin-1. Co-treatment with cycloheximide expedited apoptosis induction in necrostatin-1/TNF-treated L929 cells: typical apoptotic morphological changes, including membrane blebbing and nuclear fragmentation, induction of caspase-3 activity, proteolytic activation of caspases-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) (a well-known substrate of caspase-3) were observed. Moreover, co-treatment with Z-VAD-fmk (a pan-caspase inhibitor) inhibited apoptosis by completely inhibiting caspases, resulting in a shift from apoptosis to necroptosis. In contrast, co-treatment with Z-Asp-CH2-DCB (a caspase inhibitor preferential to caspase-3) inhibited apoptosis without expediting necroptosis. These results indicate that apoptosis can be induced in TNF-treated L929 cells when the cells are protected from necroptosis, and support the notion that partial activation of caspase-8 in the presence of a caspase inhibitor preferential to caspase-3 suppresses both apoptosis and necroptosis.

Clnk plays a role in TNF-alpha-induced cell death in murine fibrosarcoma cell line L929

Clnk, as a third member of the Blnk/SLP-76 adapter family, is involved in the positive regulation of immunoreceptor signaling. Here we provide findings that Clnk may be is required for TNF induced cell death, it functions downstream of RIP3 and promotes TNF- induced ROS generation and MLKL tetramer formation and subsequent necrosis of L929 cells. Therefore, Clnk, as an adaptor protein, may take part in the other cellular processes.

Cytotoxic Compounds from Endemic Arnebia purpurea

Phytochemical studies of the roots and aerial parts of endemic Arnebia purpurea S. Erik & H. Sumbul resulted in the isolation and characterization of four naphthoquinones [isovalerylalkannin (1), α-methyl-n-butanoyl alkannin (2), acetylalkannin (3), and alkannin (4)], a triterpene derivative [3-O-acetyl-oleanolic acid (5)], a steroid [β-sitosterol (6)], three flavonoid glycosides [isorhamnetin-3-O-rutinoside (7), kaempferol-3-O-rutinoside (8), kaempferol 3-O-(5"-acetyl) apiofuranoside 7-O-rhamnopyranoside (9)] and a phenolic acid [rosmarinic acid (10)]. 3-O-Acetyl-oleanolic acid, isorhamnetin-3-O-rutinoside, kaempferol-3-O-mrutinoside, and kaempferol 3-O-(5"-acetyl) apiofuranoside 7-O-rhamnopyranoside are reported from an Arnebia species for the first time. Cytotoxic activities on L929 murine fibrosarcoma cell line of the isolated compounds were investigated using MTT assay. Naphthoquinones (1-4) showed intermediate cytotoxic activity in comparison with the standard, doxorubicin.

3-Keto umbilicagenin A and B, new sapogenins from Allium umbilicatum Boiss

Two sapogenins, named 3-keto umbilicagenin A and B (1 and 2), possessing a novel chemical structure with a 3-keto group on the spirostane skeleton, have been isolated from Allium umbilicatum Boiss. Their chemical structure has been established through a combination of extensive spectroscopic analysis, mainly nuclear magnetic resonance and mass spectrometry, and chemical methods as (25R)-3-keto-spirostan-2α,5α,6β-triol (1) and (25R)-3-keto-spirostan-2α,5α-diol (2). The isolated compounds were tested for cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma cell lines.

Synergistic antitumor effect of recombinant adeno-associated virus-mediated pigment epithelium-derived factor with hyperthermia on solid tumor.

Adeno-associated virus (AAV) is an ideal choice for gene delivery; however, its further development has been limited owing to its low transduction efficiency. DNA-damaging agents can improve AAV-mediated transgene expression. Hyperthermia, as one of the oldest documented tumor treatment modalities, can cause DNA damage as well. However, combined treatment consisting of hyperthermia and AAV-mediated gene therapy has not been reported yet. In this work we investigated whether therapy consisting of AAV-mediated pigment epithelium-derived factor (PEDF) delivery combined with hyperthermia has synergistic antitumor effect on established solid tumors. We produced the recombinant AAV encoding PEDF (rAAV-PEDF). The therapeutic effect of rAAV-PEDF plus hyperthermia was evaluated in a subcutaneous fibrosarcoma mouse model, and the possible mechanism of antitumor effect was investigated. We found that rAAV-PEDF could infect a murine fibrosarcoma cell line (Meth-A) and express PEDF protein with bioactivity in vitro. In addition, in vivo experiments suggested that the combination of rAAV-PEDF with hyperthermia could significantly suppress tumor growth and prolong survival time of treated mice. Immunofluorescence studies indicated that the combination therapy could inhibit angiogenesis and induce apoptosis in tumor tissues. An immunohistochemistry assay of tumor tissue showed that PEDF expression in the combined treatment group was significantly higher than in the rAAV-PEDF group, which implied that hyperthermia could improve the expression of PEDF protein in vivo. No significant differences were observed in each group by hematoxylin-eosin staining of major organs, serum chemistry test, and complete blood assay. These results indicate that the combination of rAAV-PEDF with hyperthermia has synergistic therapeutic effects on established solid tumors, with no side effects. In addition, hyperthermia could improve AAV-mediated transgene expression, which suggests that hyperthermia could serve as a promising adjunctive therapy for AAV-mediated gene therapy.

Modulation of activity of known cytotoxic ruthenium(III) compound (KP418) with hampered transmembrane transport in electrochemotherapy in vitro and in vivo.

To increase electrochemotherapy (ECT) applicability, the effectiveness of new drugs is being tested in combination with electroporation. Among them two ruthenium(III) compounds, (imH)[trans-RuCl4(im)(DMSO-S)] (NAMI-A) and Na[trans-RuCl4(ind)2] (KP1339), proved to possess increased antitumor effectiveness when combined with electroporation. The objective of our experimental work was to determine influence of electroporation on the cytotoxic and antitumor effect of a ruthenium(III) compound with hampered transmembrane transport, (imH)[trans-RuCl4(im)2] (KP418) in vitro and in vivo and to determine changes in metastatic potential of cells after ECT with KP418 in vitro. In addition, platinum compound cisplatin (CDDP) and ruthenium(III) compound NAMI-A were included in the experiments as reference compounds. Our results show that electroporation leads to increased cellular accumulation and cytotoxicity of KP418 in murine melanoma cell lines with low and high metastatic potential, B16-F1 and B16-F10, but not in murine fibrosarcoma cell line SA-1 in vitro which is probably due to variable effectiveness of ECT in different cell lines and tumors. Electroporation does not potentiate the cytotoxicity of KP418 as prominently as the cytotoxicity of CDDP. We also showed that the metastatic potential of cells which survived ECT with KP418 or NAMI-A does not change in vitro: resistance to detachment, invasiveness, and re-adhesion of cells after ECT is not affected. Experiments in murine tumor models B16-F1 and SA-1 showed that ECT with KP418 does not have any antitumor effect while ECT with CDDP induces significant dose-dependent tumor growth delay in the two tumor models used in vivo.

PEG-g-chitosan thermosensitive hydrogel for implant drug delivery: cytotoxicity, in vivo degradation and drug release

Thermosensitive hydrogels based on chitosan are of great interests for injectable implant drug delivery. The poly(ethylene glycol)-grafted-chitosan (PEG-g-CS) hydrogel was reported as a potential thermosensitive system. The objective of the present study is to evaluate the cytotoxicity, in vivo degradation and drug release of PEG-g-CS hydrogel. Cytotoxicity was evaluated using L929 murine fibrosarcoma cell line. Degradation and drug release in vivo were investigated by subcutaneous injection of the hydrogel into Sprague-Dawley rats. PEG-g-CS polymer exhibits no significant cytotoxicity when its concentration is less than 3 mg mL−1. After being implanted, PEG-g-CS hydrogel maintains its integrity for two weeks and collapses, merging into the tissue, in the third week. It causes moderate inflammatory response but no fibrous encapsulation around the hydrogel is found. The hydrogel presents a three-week sustained release of cyclosporine A with no significant burst release in vitro and produces the effective drug concentration in blood for more than five weeks in vivo, performing almost the same bioavailability to chitosan/glycerophosphate hydrogel. Further modifications of PEG-g-CS hydrogel might be necessary to modulate the degradation and to mitigate the fluctuations in blood drug concentration.

Abstract 5296: The role of VEGF receptors 1 and 2 on tumor vascularization and progression in single VEGF isoform expressing tumors

Abstract Background Murine fibrosarcoma cell lines that express single VEGF isoforms (VEGF120 VEGF165 and VEGF188) and a wild-type control (VEGFWT) have been developed. The most extreme differences in vascular properties are observed between VEGF120 and VEGF188 tumors, with VEGF120 tumors developing more immature vessels. These differences impact on therapeutic outcome. We hypothesised that differential signaling through VEGF receptors 1 and 2 underlie the isoform-specific differences. Methods VEGFWT, VEGF120 and VEGF164 tumors were implanted sub-cutaneously (s/c), and VEGF188 tumors s/c or in dorsal skin-flap window-chambers (WC) in SCID mice. Mice were treated with ImClone neutralizing antibodies against VEGFR-1 (MF-1) or VEGFR-2 (DC101). Excised s/c tumors were stained for blood vessels (CD31), pericytes (αSMA), necrosis (H&E) or apoptosis (Caspase III). Tumor vascular development in WCs was imaged using intra-vital microscopy. Results VEGFWT and VEGF188 tumors were most sensitive to growth inhibition following treatment with DC101, whereas MF-1 had no effect on growth of any tumor type. In vitro, DC101 had no effect on proliferation of any tumor cell types. Immunohistochemical studies showed no changes in necrosis or apoptosis in VEGF120 tumors, but a significant decrease in vascular area with DC101. VEGF188 tumors showed no change in apoptosis, but both treatments increased necrosis. Vascular area was significantly reduced post DC101 treatment but significantly increased post MF-1 treatment. Both antibodies significantly increased αSMA staining, primarily due to myofibroblast infiltration. WC studies using VEGF188 tumors supported the effects observed with DC101, showing a significantly reduced blood vessel number and length. Furthermore, MF-1 treatment (either immediately post tumor implantation or to established tumors) resulted in more hemorrhagic tumors with large peripheral feeder vessels. Early DC101 treatment impaired tumor development, and once tumors developed on cessation of treatment, the vessels were highly chaotic. Conclusions DC101-induced tumor growth retardation, and corresponding vascular effects in VEGF188 tumors, confirms a role for VEGFR2 signaling in tumor progression and vascular development. However, the lack of growth effect in VEGF120 and VEGF164 tumors following treatment with either antibody suggests a level of receptor redundancy. MF-1 increased angiogenesis in VEGF188 tumors, despite no change in growth rate, and these neo-vessels appeared hemorrhagic, implying increased permeability. These data suggest a role for VEGFR-1 in tumor angiogenesis, involving vascular maturation, at least where VEGF188 is highly expressed. This may occur via specific VEGFR-1 signaling and/or sequestration of VEGF to control signaling via VEGFR-2. Acknowledgements Supported by CR-UK We thank ImClone Systems for providing MF-1 and DC101. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5296. doi:1538-7445.AM2012-5296

RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation

BackgroundProgrammed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis. Necrostatin-1 (Nec-1) and its derivatives were previously shown to target the pro-necrotic kinase RIP1/RIPK1. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function.Methodology/Principal FindingsWe compared the effect of Nec-1 and siRNA-mediated silencing of RIP1 in the murine fibrosarcoma cell line L929. Treatment of L929 cells with the pan-caspase inhibitor zVAD-fmk or exogenous TNF induces necrosis. Strikingly, we found that siRNA-mediated silencing of RIP1 inhibited zVAD-fmk induced necrosis, but not TNF-induced necrosis. TNF-induced cell death in RIP1 knocked down L929 cells was inhibited by Nec-1, but not the caspase inhibitor zVAD-fmk. We found that PKA-C§ expression, but not Jnk or Erk activation, was moderately inhibited by Nec-1. Moreover, we found that Nec-1 inhibits proximal T cell receptor signaling independent of RIP1, leading to inhibition of T cell proliferation.Conclusions/SignificanceOur results reveal that besides RIP1, Nec-1 also targets other factors crucial for necrosis induction in L929 cells. In addition, high doses of Nec-1 inhibit other signal transduction pathways such as that for T cell receptor activation. These results highlight the importance to independently validate results obtained using Nec-1 with other approaches such as siRNA-mediated gene silencing. We propose that some of the previous published results obtained using Nec-1 should be re-evaluated in light of our findings.

Suppression of hepatitis B virus replication in Tupaia hepatocytes by tumor necrosis factor alpha of Tupaia belangeri

Recently, Tupaia belangeri was used to study the full replication cycle of hepatitis B virus (HBV) in the primary hepatocyte cultures. Thus, the Tupaia model represents a suitable model to study the effects of cytokines on HBV infection. Here, Tupaia tumor necrosis factor-alpha (TNF-α) was molecularly cloned and expressed in mammalian cells. A test system for the biological activity of Tupaia TNF-α was established on the basis of its cytotoxic effect to the murine fibrosarcoma cell line L929. Recombinant Tupaia TNF-α was able to suppress HBV replication in primary Tupaia hepatocytes (PTH). However, the formation of HBV covalently closed circular DNA (cccDNA) and viral RNA was not completely prevented. Therefore, Tupaia TNF-α may contribute significantly to the control of HBV infection though it is not able to completely inhibit HBV replication alone. The characterization of this important cytokine allows further studies on its antiviral actions in the Tupaia model.

Gadolinium‐conjugated folate–poly(ethylene glycol)–polyamidoamine dendrimer–carboxyl nanoparticles as potential tumor‐targeted, circulation‐prolonged macromolecular magnetic resonance imaging contrast agents. II

AbstractA new macromolecular gadolinium‐based magnetic resonance imaging (MRI) contrast agent, folic acid (FA)–poly(ethylene glycol) (PEG)–polyamidoamine dendrimer (PAMAM)–Gd, was synthesized with a core of PAMAM, which was modified with conjugates of FA and PEG and then chelated by gadolinium ions. The final products, with FA as the targeting moiety, were evaluated for their tumor‐targeting MRI contrast‐agent potential. The concentration detection limits in vitro; contrast‐enhanced MRI in the heart, kidney, and liver of mice; and metabolism of FA–PEG–PAMAM–Gd were measured by a Siemens Tim Trio human MRI scanner at 3 T. Transmission electron microscopy was used to determine the targeting of FA–PEG–PAMAM–Gd to human epidermoid carcinomas cell lines (KB) or murine aneuploid fibrosarcoma cell lines (L929). The toxicity was also assayed to evaluate the biocompatibility of this contrast agent. The minimum detectable concentration of FA–PEG4K–PAMAM–Gd (where the subscript 4K indicates a molecular weight of 4000 Da) was 15‐fold lower than that of the commercially available contrast agent gadopentetate dimeglumine. The MRI images displayed a gradual persistent signal enhancement on tumors, and millimeter‐sized (∼ 3 mm) tumors were well visualized with FA–PEG4K–PAMAM–Gd. In conclusion, the dendritic complexes were well suited for use as an FA‐mediated targeting contrast agent for early diagnosis of tumors in mice. The dendritic contrast agents displayed lower concentration detection limits, which suggests their future use in molecular imaging. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Suppression of Soft Tissue Sarcoma Growth by a Host Defense-Like Lytic Peptide

BackgroundSoft tissue sarcoma (STS) is an anatomically and histologically heterogeneous neoplasia that shares a putative mesenchymal cell origin. The treatment with common chemotherapeutics is still unsatisfying because of association with poor response rates. Although evidence is accumulating for potent oncolytic activity of host defense peptides (HDPs), their potential therapeutic use is often limited by poor bioavailability and inactivation in serum. Therefore, we tested the designer host defense-like lytic D,L-amino acid peptide [D]-K3H3L9 on two STS cell lines in vitro and also in an athymic and syngeneic mouse model. In recent studies the peptide could show selectivity against prostate carcinoma cells and also an active state in serum.Methods In vitro the human synovial sarcoma cell line SW982, the murine fibrosarcoma cell line BFS-1 and primary human fibroblasts as a control were exposed to [D]-K3H3L9, a 15mer D,L-amino acid designer HDP. Cell vitality in physiological and acidic conditions (MTT-assay), cell growth (BrdU) and DNA-fragmentation (TUNEL) were investigated. Membrane damage at different time points could be analyzed with LDH assay. An antibody against the tested peptide and recordings using scanning electron microscopy could give an inside in the mode of action. In vivo [D]-K3H3L9 was administered intratumorally in an athymic and syngeneic (immunocompetent) mouse model with SW982 and BFS-1 cells, respectively. After three weeks tumor sections were histologically analyzed.ResultsThe peptide exerts rapid and high significant cytotoxicity and antiproliferating activity against the malignant cell lines, apparently via a membrane disrupting mode of action. The local intratumoral administration of [D]-K3H3L9 in the athymic and syngeneic mice models significantly inhibited tumor progression. The histological analyses of the tumor sections revealed a significant antiproliferative, antiangiogenic activity of the treatment group.ConclusionThese findings demonstrate the in vitro and in vivo oncolytic activity of [D]-K3H3L9 in athymic and syngeneic mouse models.

Formation of solid tumors by a single multinucleated cancer cell

Large multinucleated cells (MNCs) commonly exist in tumorigenic cancer cell lines that are used widely in research. However, the contributions of MNCs to tumorigenesis are unknown. In this study, MNCs were characterized in the murine fibrosarcoma cell line UV-2237 in vitro and in vivo at the single-cell level. The authors observed that MNCs originated from a rare subpopulation of mononuclear cells and were positive for a senescent marker, β-galactosidase. In addition, MNCs were responsible for the majority of clonogenic activity when cultured in hard agar; they were more resistant to chemotherapeutic agents than mononuclear cells; they could undergo asymmetric division (producing mononuclear cells) and self-renewal in vitro and in vivo; and, most important; a single MNC produced orthotopic, subcutaneous tumors (composed mainly of mononuclear cells) that gave rise to spontaneous lung metastases in nude mice. The current results indicated that the growth of MNCs may be arrested under stress and that MNCs are highly resistant to chemotherapy and can generate clonal, orthotopic, metastatic tumors.

In vitro biocompatibility of novel Au-Pt-based metal-ceramic alloys

The aim of this research was to evaluate the effect of individual metallic elements within experimental Au-Pt-based metal-ceramic alloys on in vitro biocompatibility. A binary Au-10 at.% Pt alloy (AP10) was designed as a parent alloy. Six ternary AP10-X (X = In/Fe/Sn/Zn) alloys and four quaternary (AP10-In2)-Y (Y = Fe/Sn/Zn) with different compositions were cast into square plates with size 10X10X0.5 mm(3) and subjected to porcelain-firing thermal cycling. A commercial alloy was used as a control. In vitro biocompatibility was investigated using L929 murine aneuploid fibrosarcoma cell line. The test samples and cells were incubated at 37°C in a 5% CO(2) atmosphere for 72 h. Alamar™ Blue Assay was carried out to determine the respiratory viability of cultures maintained in the presence of the different materials. The cell only control showed significantly higher levels of cell viability than all six of the ternary alloys and two of the four quaternary alloys, (AP10-In2)-Zn2.1 and (AP10-In2)-Sn1.0 (P < 0.05). The quaternary alloys showed slightly higher levels of cell viability than the ternary alloys, with the exception of AP10-Sn0.9. No statistical differences were seen between the ternary and quaternary alloy groups. Acceptable cell viability was observed on the surfaces of all the alloys.