Human Fibrosarcoma HT1080 Cells Research Articles

Selective Synergy of Recombinant Methioninase Plus Docetaxel Against Docetaxel-resistant and -sensitive Fibrosarcoma Cells Compared to Normal Fibroblasts.

Docetaxel combined with gemcitabine is a second-line treatment for soft-tissue sarcoma; however, its effectiveness is limited because of docetaxel resistance. The objective of the present study was to determine the potential of recombinant methioninase (rMETase) to enhance the efficacy of docetaxel on high-docetaxel-resistant human fibrosarcoma cells in vitro. Docetaxel-resistant HT1080 (DTR-HT1080) human fibrosarcoma cells were established by culturing them in by progressively increasing concentrations of docetaxel from 0.02 to 9 nM in vitro. The IC50 values for docetaxel and rMETase, as well as the efficacy of their combination, in inhibiting HT1080 human fibrosarcoma cells, DTR-HT1080 cells, and Hs27 normal human fibroblasts were determined. Four experimental groups were examined in vitro: control group without treatment; docetaxel alone; rMETase alone; docetaxel combined with rMETase. The IC50 of docetaxel for DTR-HT1080 cells was 7.57 nM, compared to the parental HT1080 cells with an IC50 of 1.68 nM, a 4.5-fold increase. The IC50 of docetaxel on Hs27 fibroblasts was 4.46 nM. The IC50 of rMETase on HT1080 cells was 0.75 U/ml (data from [6]). The IC50 of rMETase on DTR-HT1080 cells was 0.55 U/ml. The IC50 of rMETase on Hs27 fibroblasts was 0.93 U/ml (data from [6]). Docetaxel (1.68 nM [IC50]) plus rMETase (0.75 U/ml [IC50]) synergistically reduced the viability of HT1080 cells (p<0.05). In contrast, docetaxel (4.46 nM) plus rMETase (0.93 U/ml) did not reduce the viability of Hs27 fibroblasts, compared to either agent alone. The combination of rMETase (0.55 U/ml [IC50]) and docetaxel (1.68 nM [IC50 of the parental cells]) overcame docetaxel resistance of DTR-HT1080 cells, resulting in an inhibition of 48.1% compared to docetaxel alone (6.8%) or rMETase alone (37.5%) (p<0.05). rMETase thus increased the efficacy of docetaxel 7-fold on docetaxel-resistant human fibrosarcoma cells. The combination of docetaxel and rMETase was synergistic on HT1080 fibrosarcoma cells, but not normal fibroblasts. rMETase plus docetaxel synergistically reduced the high docetaxel resistance of DTR-HT1080 cells. The present results indicate the clinical potential of rMETase to reduce docetaxel resistance in soft-tissue sarcoma patients in the future.

Unraveling the role of integrating signal peptides into natural collagen on modulating cancer cell adhesion

The signal peptides GVMGFO and GFOGER exhibit differential binding affinities towards Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and HT-1080 human fibrosarcoma cells, respectively, which in turn modulate the cell adhesion properties of natural collagen. GVMGFO demonstrates a more potent interaction with discoidin domain receptor 1(DDR1)-expressing MCF-7 cells, whereas GFOGER preferentially binds to the integrin α2β1 present on HT-1080 cells. The integration of GVMGFO into natural collagen through direct doping or crosslinking markedly enhances its association with MCF-7 cells, especially when optimal peptide concentrations and blending ratios are utilized, indicating a synergistic effect. This augmented adhesion is attributed to specific binding at the DDR1-collagen interface, facilitated by a constellation of amino acids within the collagen scaffold engaging with the DDR1 discoidin (DS) domain through polar interactions and hydrogen bonding. Conversely, the incorporation of GFOGER into natural collagen through co-assembling or crosslinking leads to a progressive increase in adherence to HT-1080 cells, as evidenced by the peptide's affinity for integrin α2β1. These findings advance the design of collagen-based biomaterials for targeted cellular interactions in the medical, pharmaceutical, and enhance our understanding of the molecular mechanisms governing peptide-collagen mediated cell adhesion processes.

Tailoring carrier-free nanoparticles based on natural small molecule assembly for synergistic anti-tumor efficacy

Interfacial modular assemblies of versatile polyphenols have attracted widespread interest in surface and materials engineering. In this study, natural polyphenol (tannic acid, TA) and nobiletin (NOB) can directly form binary carrier-free spherical nanoparticles (NT NPs) through synergistically driven by a variety of interactions (such as hydrogen bonding, oxidative reactions, etc.). The synthesis involves polyphenolic deposition on hydrophobic NOB nanoaggregates, followed by in situ oxidative self-polymerization. Interestingly, the assembled NT NPs exhibit controllable and dynamic changes in particle size during the initial stage. Ultimately, uniform and spherical NT NPs appear stable, with high loading capability, enabling incorporated NOB to preserve their function. Furthermore, in vitro evaluations demonstrate that the rational combination of polyphenol module and NOB can induce apoptosis and inhibit tumor metastasis for both lung cancer H1299 and human fibrosarcoma HT1080 cell lines. Notably, the optimized NT48 NPs were then verified in vivo experiments to achieve a promising synergistic anti-tumor efficacy. These findings not only provide new opportunities for the streamlined and sensible engineering of future polyphenol-based biomaterials, but also open up new prospects for the design of small-molecule nature phytochemicals.

Frequency-Dependent Antioxidant Responses in HT-1080 Human Fibrosarcoma Cells Exposed to Weak Radio Frequency Fields.

This study explores the complex relationship between radio frequency (RF) exposure and cancer cells, focusing on the HT-1080 human fibrosarcoma cell line. We investigated the modulation of reactive oxygen species (ROS) and key antioxidant enzymes, including superoxide dismutase (SOD), peroxidase, and glutathione (GSH), as well as mitochondrial superoxide levels and cell viability. Exposure to RF fields in the 2-5 MHz range at very weak intensities (20 nT) over 4 days resulted in distinct, frequency-specific cellular effects. Significant increases in SOD and GSH levels were observed at 4 and 4.5 MHz, accompanied by reduced mitochondrial superoxide levels and enhanced cell viability, suggesting improved mitochondrial function. In contrast, lower frequencies like 2.5 MHz induced oxidative stress, evidenced by GSH depletion and increased mitochondrial superoxide levels. The findings demonstrate that cancer cells exhibit frequency-specific sensitivity to RF fields even at intensities significantly below current safety standards, highlighting the need to reassess exposure limits. Additionally, our analysis of the radical pair mechanism (RPM) offers deeper insight into RF-induced cellular responses. The modulation of ROS and antioxidant enzyme activities is significant for cancer treatment and has broader implications for age-related diseases, where oxidative stress is a central factor in cellular degeneration. The findings propose that RF fields may serve as a therapeutic tool to selectively modulate oxidative stress and mitochondrial function in cancer cells, with antioxidants playing a key role in mitigating potential adverse effects.

Recombinant Methioninase Increases Eribulin Efficacy 16-fold in Highly Eribulin-resistant HT1080 Fibrosarcoma Cells, Demonstrating Potential to Overcome the Clinical Challenge of Drug-resistant Soft-tissue Sarcoma.

A major challenge in treating soft-tissue sarcoma is the development of drug resistance. Eribulin, an anti-tubulin agent, is used as a second-line chemotherapy for patients with unresectable or metastatic soft-tissue sarcoma. However, most patients with advanced soft-tissue sarcoma are resistant to eribulin and do not survive. Recombinant methioninase (rMETase) targets the fundamental and general hallmark of cancer, methionine addiction, termed the Hoffman Effect. The present study aimed to show how much rMETase could increase the efficacy of eribulin on eribulin-resistant fibrosarcoma cells in vitro. HT1080 human fibrosarcoma cells were exposed to step-wise increasing concentrations of eribulin from 0.15-0.4 nM to establish eribulin-resistant HT1080 (ER-HT1080). ER-HT1080 cells were cultured in vitro and divided into four groups: untreated control; eribulin treated (0.15 nM); rMETase treated (0.75 U/ml); and eribulin (0.15 nM) plus rMETase (0.75 U/ml) treated. The IC50 of eribulin on ER-HT1080 cells was 0.95 nM compared to the IC50 of 0.15 nM on HT1080 cells, a 6-fold increase. The IC50 of rMETase on ER-HT1080 and HT1080 was 0.87 U/ml and 0.75 U/ml, respectively. The combination of rMETase (0.75 U/ml) and eribulin (0.15 nM) was synergistic on ER-HT1080 cells resulting in an inhibition of 80.1% compared to eribulin alone (5.0%) or rMETase alone (47.1%) (p<0.05). rMETase thus increased the efficacy of eribulin 16-fold on eribulin-resistant fibrosarcoma cells. The present study showed that the combination of eribulin and rMETase can overcome high eribulin resistance of fibrosarcoma. The present results demonstrate that combining rMETase with first- or second-line therapy for soft-tissue sarcoma has the potential to overcome the intractable clinical problem of drug-resistant soft-tissue sarcoma.

Overcoming High Trabectedin Resistance of Soft-tissue Sarcoma With Recombinant Methioninase: A Potential Solution of a Recalcitrant Clinical Problem.

Drug resistance has been a recalcitrant problem for sarcoma patients for many decades. Trabectedin is a second-line chemotherapy for soft-tissue sarcoma that often leads to resistance and death of the patients. The objective of the present study was to address the issue of trabectedin-chemoresistance in HT1080 fibrosarcoma cells by combining recombinant methioninase (rMETase) with trabectedin and examining their efficacy on trabectedin-resistant fibrosarcoma cells in vitro. Trabectedin-resistant HT1080 (TR-HT1080) cells were generated by subjecting HT1080 human fibrosarcoma cells to increasing trabectedin concentrations (3.3-8 nM). IC50 values for trabectedin and rMETase were compared for HT1080 and TR-HT1080 cells. TR-HT 1080 cells were placed into four groups to determine synergy of rMETase and trabectedin on TR-HT1080 cells: a control group with no treatment; a group treated with trabectedin (3.3 nM); a group treated with rMETase (0.75 U/ml); and a group treated with both trabectedin (3.3 nM) and rMETase (0.75 U/ml). The IC50 value of trabectedin- on TR-HT1080 cells was 42.9 nM, whereas the IC50 value of trabectedin on the parental HT1080 cells was 3.3 nM, indicating a 13-fold increase. The combination of rMETase (0.75 U/ml) and trabectedin (3.3 nM) was synergistic on TR-HT1080 cells resulting in an inhibition of 64.2% compared to trabectedin alone (5.7%) or rMETase alone (50.5%) (p<0.05). rMETase increased the efficacy of trabectedin 11-fold on trabectedin-resistant fibrosarcoma cells. The combined administration of trabectedin and rMETase was synergistic on the viability of TR-HT1080 cells in vitro. The combination of rMETase and trabectedin has promising clinical potential for overcoming chemo-resistance of soft-tissue sarcoma.

Recombinant Methioninase Is Selectively Synergistic With Doxorubicin Against Wild-type Fibrosarcoma Cells Compared to Normal Cells and Overcomes Highly-Doxorubicin-resistant Fibrosarcoma.

Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro. The 50% inhibitory concentrations (IC50) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope. The IC50 for doxorubicin was 3.3 μM for HT1080 cells, 12.4 μM for DR-HT1080 cells, and 7.25 μM for Hs27 cells. The IC50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells. The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma.

DNA-Binding Agent Trabectedin Combined With Recombinant Methioninase Is Synergistic to Decrease Fibrosarcoma Cell Viability and Induce Nuclear Fragmentation But Not Synergistic on Normal Fibroblasts.

The alkylating agent trabectedin, which binds the minor groove of DNA, is second-line therapy for soft-tissue sarcoma but has only moderate efficacy. The aim of the present study was to determine the synergistic efficacy of recombinant methioninase (rMETase) and trabectedin on fibrosarcoma cells in vitro, compared with normal fibroblasts. HT1080 human fibrosarcoma cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and Hs27 normal human fibroblasts, were used. Each cell line was cultured in vitro and divided into four groups: no-treatment control; trabectedin treated; rMETase treated; and trabectedin plus rMETase treated. The dual-color HT1080 cells were used to quantitate nuclear fragmentation in each treatment group. The combination of rMETase and trabectedin was highly synergistic to decrease HT1080 cell viability. In contrast, there was no synergy on Hs27 cells. Moreover, nuclear fragmentation occurred synergistically with the combination of trabectedin and rMETase on dual-color HT1080 cells. The combination treatment of trabectedin plus rMETase was highly synergistic on fibrosarcoma cells in vitro suggesting that the combination can improve the outcome of trabectedin alone in future clinical studies. The lack of synergy of rMETase and trabectedin on normal fibroblasts suggests the combination is not toxic to normal cells. Synergy of the two drugs may be due to the high rate of nuclear fragmentation on treated HT1080 cells, and the late-S/G2 cell-cycle block of cancer cells by rMETase, which is a target for trabectedin. The results of the present study suggest the future clinical potential of the combination of rMETase and trabectedin for soft-tissue sarcoma.

Pacidusin B isolated from Phyllanthus acidus triggers ferroptotic cell death in HT1080 cells

Cancer cells generally exhibit ‘iron addiction’ phenotypes, which contribute to their vulnerability to ferroptosis inducers. Ferroptosis is a newly discovered form of programmed cell death caused by iron-dependent lipid peroxidation. In the present study, pacidusin B, a dichapetalin-type triterpenoid from Phyllanthus acidus (L.) Skeels (Euphorbiaceae), induces ferroptosis in the HT1080 human fibrosarcoma cell line. Cells treated with pacidusin B exhibited the morphological characteristic ‘ballooning’ phenotype of ferroptosis. The biochemical hallmarks of ferroptosis were also observed in pacidusin B-treated cells. Both oxidative stress and ER stress play significant roles in pacidusin B-induced ferroptosis. The activation of the PERK-Nrf2-HO-1 signaling pathway led to iron overload, while inhibition of GPX4 further sensitized cancer cells to ferroptosis. Furthermore, the molecular docking study showed that pacidusin B docked in the same pocket in xCT as the ferroptosis inducer erastin. These results revealed that pacidusin B exerts anticancer effects via inducing ER-mediated ferroptotic cell death.Graphical

Application of copper (I) selective ligands for PET imaging of reactive oxygen species through metabolic trapping

IntroductionReactive oxygen species (ROS) are attractive targets for clinical PET imaging. In this study, we hypothesized that PET imaging of ROS would be possible by using chelating ligands (L) that form stable complexes with copper (I) but not with copper (II), based on metabolic trapping. Namely, when [64Cu][CuI(L)2]+ is oxidized by ROS, the oxidized complex will release [64Cu]Cu2+. Then, the released [64Cu]Cu2+ will be trapped inside the cell, resulting in PET signal depending on the redox potential of ROS. To examine the potential of this novel molecular design for ROS imaging, we synthesized copper (I) complexes with bicinchoninic acid (BCA) disodium salt and bathocuproinedisulfonic acid (BCS) disodium salt and evaluated their reactivity with several kinds of ROS. In addition, the cellular uptake of [64Cu][CuI(BCS)2]3− and the stability of [64Cu][CuI(BCS)2]3− in a biological condition were also evaluated. Methods[64Cu]Cu2+ was reduced to [64Cu]Cu+ by ascorbic acid and coordinated with BCA and BCS in the acetate buffer to synthesize [64Cu][CuI(BCA)2]3− and [64Cu][CuI(BCS)2]3−. The radiochemical yields were determined by thin-layer chromatography (TLC). After [64Cu][CuI(BCS)2]3− was incubated with hydroxyl radical, lipid peroxide, superoxide, and hydrogen peroxide, the percentage of released [64Cu]Cu2+ from the parent complex was evaluated by TLC. HT-1080 human fibrosarcoma cells were treated with 0.1 % Dimethyl sulfoxide (control), imidazole ketone erastin (IKE), or IKE + ferrostatin-1 (Fer-1). Then, the uptake of [64Cu][CuI(BCS)2]3− to HT-1080 cells in each group was evaluated as %Dose/mg protein. Lastly, [64Cu][CuI(BCS)2]3− was incubated in human plasma, and its intact ratio was determined by TLC. ResultsThe radiochemical yield of [64Cu][CuI(BCS)2]3− (86 ± 1 %) was higher than that of [64Cu][CuI(BCA)2]3− (44 ± 3 %). [64Cu][CuI(BCA)2]3− was unstable and partially decomposed on TLC. After [64Cu][CuI(BCS)2]3− was reacted with hydroxyl radical, lipid peroxide, and superoxide, 67 ± 2 %, 44 ± 13 %, and 22 ± 3 % of total radioactivity was detected as [64Cu]Cu2+, respectively. On the other hand, the reaction with hydrogen peroxide did not significantly increase the ratio of [64Cu]Cu2+ (4 ± 1 %). These results suggest that [64Cu][CuI(BCS)2]3− could be used for detecting high-redox-potential ROS such as hydroxyl radical and lipid peroxide with high selectivity. The cellular uptake values of [64Cu][CuI(BCS)2]3− in the control, IKE, and Fer-1 group were 42 ± 2, 54 ± 2, and 47 ± 5 %Dose/mg protein (n = 3), respectively, suggesting the ROS specific uptake of [64Cu][CuI(BCS)2]3−. On the other hand, the intact ratio after the incubation of [64Cu][CuI(BCS)2]3− in human plasma was 9 ± 5 %. ConclusionPET imaging of ROS would be possible by using a copper (I) selective ligand, based on metabolic trapping. Although improvement of the membrane permeability and the stability of copper (I) complexes is required, the present results pave the way for the development of novel 64Cu-labeled complexes for PET imaging of ROS.

Extensive Synergy Between Recombinant Methioninase and Eribulin Against Fibrosarcoma Cells But Not Normal Fibroblasts.

The aim of the present study was to determine the synergy of recombinant methioninase (rMETase) and the anti-tubulin agent eribulin on fibrosarcoma cells, in comparison to normal fibroblasts, in vitro. HT1080 human fibrosarcoma cells and HS27 human fibroblasts were used for in vitro experiments. Four groups were analyzed in vitro: No-treatment control; eribulin; rMETase; eribulin plus rMETase. Dual-color HT1080 cells which express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize cytoplasmic and nuclear dynamics during treatment. Eribulin combined with rMETase greatly decreased the viability of HT 1080 cells. In contrast, eribulin combined with rMETase did not show synergy on Hs27 normal fibroblasts. Eribulin combined with rMETase also caused more fragmentation of the nucleus than all other treatments. The combination treatment of eribulin plus rMETase demonstrated efficacy on fibrosarcoma cells in vitro. In contrast, normal fibroblasts were resistant to this combination, indicating the potential clinical applicability of the treatment.

Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S.

During progression of rheumatoid arthritis (RA), angiogenesis provides oxygen and nutrients for the cells' increased metabolic demands and number. To turn on angiogenesis, pro-angiogenic factors must outweigh anti-angiogenic factors. We have previously shown that CD147/extracellular matrix metalloproteinase inducer (EMMPRIN) can induce the expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) in a co-culture of the human HT1080 fibrosarcoma and U937 monocytic-like cell lines. However, whether CD147 influences anti-angiogenic factors was not known. We now show that relative to single cultures, the co-culture of these cells not only enhanced pro-angiogenic factors but also decreased the anti-angiogenic factors endostatin and thrombospondin-1 (Tsp-1), generally increasing the angiogenic potential as measured by a wound assay. Using anti-CD147 antibody, CD147 small interfering RNA (siRNA), and recombinant CD147, we demonstrate that CD147 hormetically regulates the generation of endostatin but has no effect on Tsp-1. Since endostatin is cleaved from collagen XVIII (Col18A), we applied different protease inhibitors and established that MMP-9 and proteasome 20S, but not cathepsins, are responsible for endostatin generation. MMP-9 and proteasome 20S collaborate to synergistically enhance endostatin generation, and in a non-cellular system, CD147 enhanced MMP-9 activity and hormetically regulated proteasome 20S activity. Serum samples obtained from RA patients and healthy controls mostly corroborated these findings, indicating clinical relevance. Cumulatively, these findings suggest that secreted CD147 mediates a possibly allosteric effect on MMP-9 and proteasome 20S activities and can serve as a switch that turns angiogenesis on or off, depending on its ambient concentrations in the microenvironment.

Design, Synthesis, and Biological Evaluation of Hydrophobic-Tagged Glutathione Peroxidase 4 (GPX4) Degraders

Ferroptosis is an iron-dependent form of oxidative cell death induced by lipid peroxidation accumulation. Glutathione peroxidase 4 (GPX4) plays a key role in the regulation of ferroptosis and is considered to be a promising therapeutic target for cancer and other human diseases. Herein, we describe our design, synthesis, and biological evaluation of a series of HyT-based degraders of the GPX4. One of the most promising compounds, 7b (ZX782), effectively induces dose- and time-dependent degradation of GPX4 protein and potently suppresses the growth of human fibrosarcoma HT1080 cells, which are highly sensitive to ferroptosis and widely used for evaluating compound specificity in ferroptosis. Mechanism investigation indicated that 7b depletes GPX4 through both the ubiquitin–proteasome and the autophagy-lysosome. Furthermore, the degradation of GPX4 induced by 7b could significantly increase the accumulation of lipid reactive oxygen species (ROS) in HT1080 cells, ultimately leading to ferroptosis. Overall, compound 7b exhibits robust potency in depleting endogenous GPX4, thereby modulating ferroptosis in cancer cells.

DPY19L3 promotes vasculogenic mimicry by its C-mannosyltransferase activity.

C-mannosylation is a post-translational modification that occurs intracellularly in the endoplasmic reticulum. In humans, biosynthesis of C-mannosylation in proteins containing thrombospondin type 1 repeat is catalyzed by the DPY19 family; nonetheless, biological functions of protein C-mannosylation are not yet fully understood, especially in tumor progression. Vasculogenic mimicry (VM) is the formation of fluid-conducting channels by highly invasive and genetically deregulated tumor cells, enabling the tumors to form matrix-embedded vasculogenic structures, containing plasma and blood cells to meet the metabolic demands of rapidly growing tumors. In this study, we focused on DPY19L3, a C-mannosyltransferase, and aimed to unravel its role in VM. Knockout of DPY19L3 inhibited the formation of VM in HT1080 human fibrosarcoma cells. Re-expression of wild-type DPY19L3 recovered VM formation; however, DPY19L3 isoform2, an enzymatic activity-defect mutant, did not restore it, suggesting that the C-mannosyltransferase activity of DPY19L3 is crucial to its function. Furthermore, the knockdown of DPY19L3 in MDA-MB-231 breast cancer cells hindered its network formation ability. Altogether, our findings suggest that DPY19L3 is required for VM formation and stipulate the relevance of C-mannosylation in oncogenesis.

Endoplasmic Reticulum Stress Inducer Dithiothreitol Affects the Morphology and Motility of Cultured Human Dermal Fibroblasts and Fibrosarcoma HT1080 Cell Line

Endoplasmic Reticulum Stress Inducer Dithiothreitol Affects the Morphology and Motility of Cultured Human Dermal Fibroblasts and Fibrosarcoma HT1080 Cell Line

Poly(2-n-propyl-2-oxazoline)/tannic acid nanocapsules with in-situ synthesized gold nanorods for cancer theranostics

The concept of hollow polymeric capsules fabricated with thermoresponsive polymers has revealed immense possibilities in the field of nanomedicine due to their tunable temperature and pH responsiveness caused by lower critical solution temperature (LCST) behavior. Herein, we report a new methodology to in-situ synthesize gold nanorods (AuNRs) within the pores of mesoporous silica nanoparticles (NPs) by trapping gold seed crystals and growing them into NRs in the presence of a weak reducing agent. The mesoporous silica NPs with incorporated NRs, i.e., modified silica NPs (SiO2-NRs) were used as sacrificial template for the fabrication of hollow polymeric multilayer capsules via layer-by-layer (LbL) assembly using poly(2-n-propyl-2-oxazoline) (PnPrOx) and tannic acid (TA) as layer components. The NRs of 25 ± 5 nm in size were uniformly distributed in the interior structure of PnPrOx/TA nanocapsules. The photo-thermal conversion efficiency of the PnPrOx/TA nanocapsules was found to be 47.5%. The release of encapsulated doxorubicin was found to be 55%, which increased to 68% when the incubation time was increased from 1 to 2 h. An on-demand burst release of 56% of the drug occurred within 2 min of NIR light exposure, and it reached 72% within 30 min, which inflicted major damage to cancer cells. Notably, the capsule suspension temperature increase from room temperature to 47 °C under laser light exposure, which is not only playing a key role in rupturing the capsules to enhance the drug release but also induces a hyperthermia effect in the tumor environment. Further, the in-vitro cytotoxicity experiments with human epithelial HEp-2 cells and fibrosarcoma HT1080 cells revealed an enhanced anti-cancer activity due to a synergistic chemo-photothermal effect. Hence, the proposed nanocapsule system can provide a unique, cheap, and efficient way of fabricating cancer theranostics for in-vivo applications.

Endoplasmic Reticulum Stress Inducer Dithiothreitol Affects the Morphology and Motility of Cultured Human Dermal Fibroblasts and Fibrosarcome HT1080 Cell Line

Some inducers of endoplasmic reticulum (ER) stress can affect the motility of normal and tumor cells. However, it is unknown what mechanisms mediate this effect and whether it is a consequence of ER stress. The aim of our work was to study the effect of the ER stress inducer dithiothreitol (DTT) on morphological features reflecting the locomotor properties of cells, as well as directly on the migratory properties of cultured human dermal fibroblasts and fibrosarcoma HT1080 cells. We have shown that DTT causes disruption of the organization of actin cytoskeleton in both types of cells, which is accompanied by a change in the cell surface and shape of cells, as well as a decrease in their spreading area. In addition, a decrease in the number of focal contacts was observed in dermal fibroblasts. DTT also reduced the motility of dermal fibroblasts and fibrosarcoma cells. To analyze cell motility and determine the moment of its change, we developed a method which showed that the change in the migratory properties of fibrosarcoma cells cultured with DTT began earlier than in dermal fibroblasts. Thus, activation of ER stress by DTT is accompanied by a change in the organization of the actin cytoskeleton and motility in normal and tumor human cells. Consequently, ER stress triggered by various inducers with different mechanisms of action affects the motility of normal and tumor cells, which must be taken into account when developing antitumor drugs that cause cell death through activation of ER stress.

Proliferation, migration, and resistance to oxidative and thermal stresses of HT1080 cells with knocked out genes encoding Hsp90α and Hsp90β

Heat shock protein 90 (Hsp90) fulfils essential housekeeping functions in the cell associated with the folding, stabilization, and turnover of various proteins. In mammals, there exist two Hsp90 isoforms, stress-inducible Hsp90α and constitutively expressed Hsp90β. In an attempt to identify cellular processes dependent on Hsp90α and Hsp90β, we generated a panel of clones of human fibrosarcoma HT1080 cells with the knocked out HSP90AA1 or HSP90AB1 genes encoding, respectively, Hsp90α and Hsp90β. The knockout of the HSP90AA1 and HSP90AB1 genes practically did not affect cell proliferation and resistance to thermal shock and oxidative stress. The loss of Hsp90α in Hsp90α-null cell clones also did not impair cell migration, while the migration of the Hsp90β-null cell clones was prominently reduced as compared to parent HT1080 cells. This indicated the necessity of Hsp90β for efficient basal migration of HT1080 cells whereas Hsp90α seems to be dispensable for this process. The knockout of one Hsp90 isoform was invariably accompanied by an increase in the level of the other Hsp90 isoform by 30–50%, which partly or fully compensated for a decrease in the total level of Hsp90. Thus, we demonstrated the dispensability of Hsp90α and Hsp90β for HT1080 cells in several cellular processes under normal and stress conditions, which suggested the participation of the two Hsp90 isoforms in the same biological processes and full or at least partial functional substitution of one Hsp90 isoform by the other.

Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with roles in cellular stress responses, differentiation, and viral defense. It functions in both quality control and post-transcriptional regulation of gene expression. NMD has also emerged as a modulator of cancer progression, although available evidence supports both a tumor suppressor and a pro-tumorigenic role, depending on the model. To further investigate the role of NMD in cancer, we knocked out the NMD factor SMG7 in the HT1080 human fibrosarcoma cell line, resulting in suppression of NMD function. We then compared the oncogenic properties of the parental cell line, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. We also tested the effect of a drug inhibiting the NMD factor SMG1 to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays and a mouse xenograft tumor model, we showed that suppression of NMD function severely compromises the oncogenic phenotype. Molecular pathway analysis revealed that NMD suppression strongly reduces matrix metalloprotease 9 (MMP9) expression and that MMP9 re-expression partially rescues the oncogenic phenotype. Since MMP9 promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation may contribute to the reduced tumorigenicity of NMD-suppressed cells. Collectively, our results highlight the potential value of NMD inhibition as a therapeutic approach.

Danthron, an Anthraquinone Isolated from a Marine Fungus, Is a New Inhibitor of Angiogenesis Exhibiting Interesting Antitumor and Antioxidant Properties.

The role played by a sustained angiogenesis in cancer and other diseases stimulates the interest in the search for new antiangiogenic drugs. In this manuscript, we provide evidence that 1,8- dihydroxy-9,10-anthraquinone (danthron), isolated from the fermentation broth of the marine fungus Chromolaenicola sp. (HL-114-33-R04), is a new inhibitor of angiogenesis. The results obtained with the in vivo CAM assay indicate that danthron is a potent antiangiogenic compound. In vitro studies with human umbilical endothelial cells (HUVEC) reveal that this anthraquinone inhibits certain key functions of activated endothelial cells, including proliferation, proteolytic and invasive capabilities and tube formation. In vitro studies with human breast carcinoma MDA-MB231 and fibrosarcoma HT1080 cell lines suggest a moderate antitumor and antimetastatic activity of this compound. Antioxidant properties of danthron are evidenced by the observation that it reduces the intracellular reactive oxygen species production and increases the amount of intracellular sulfhydryl groups in endothelial and tumor cells. These results support a putative role of danthron as a new antiangiogenic drug with potential application in the treatment and angioprevention of cancer and other angiogenesis-dependent diseases.