Cell Blebbing Research Articles

Focal adhesion kinase is activated by microtubule-depolymerizing agents and regulates membrane blebbing in human endothelial cells.

Microtubule‐depolymerizing agents can selectively disrupt tumor vessels via inducing endothelial membrane blebbing. However, the mechanism regulating blebbing is largely unknown. IMB5046 is a newly discovered microtubule‐depolymerizing agent. Here, the functions of focal adhesion kinase (FAK) during IMB5046‐induced blebbing and the relevant mechanism are studied. We found that IMB5046 induced membrane blebbing and reassembly of focal adhesions in human vascular endothelial cells. Both FAK inhibitor and knock‐down expression of FAK inhibited IMB5046‐induced blebbing. Mechanism study revealed that IMB5046 induced the activation of FAK via GEF‐H1/ Rho/ ROCK/ MLC2 pathway. cRGD peptide, a ligand of integrin, also blocked IMB5046‐induced blebbing. After activation, FAK further promoted the phosphorylation of MLC2. This positive feedback loop caused more intensive actomyosin contraction and continuous membrane blebbing. FAK inhibitor blocked membrane blebbing via inhibiting actomyosin contraction, and stimulated stress fibre formation via promoting the phosphorylation of HSP27. Conclusively, these results demonstrate that FAK is a molecular switch controlling endothelial blebbing and stress fibre formation. Our study provides a new molecular mechanism for microtubule‐depolymerizing agents to be used as vascular disrupting agents.

Lotusine, an alkaloid from Nelumbo nucifera (Gaertn.), attenuates doxorubicin-induced toxicity in embryonically derived H9c2 cells.

Cardiotoxicity is the major challenge in chemotherapy with doxorubicin (DOX) or adriamycin. Doxorubicin manifests oxidative stress via an uncontrolled progression of reactive oxygen species in cardiomyocytes; thereby, dysregulation and dysfunction of myocardium thus lead to apoptosis. Several attempts have been made to overcome this side effect in patients with antioxidant-rich supplements to control the free radicals. Plant-based or plant-derived compounds pay more attention to cure such complications in patients for supporting the treatment, revitalizing or regulating the normal metabolism. Hence, our study focused on pretreatment of embryonically derived rat cardiomyocytes (H9c2) with phytocompound lotusine to prevent DOX-mediated oxidative stress. From the experiment, the DOX-exposed cells have shown morphological abnormalities such as reduced cell size, shrinkage, blebbing, and chromatin condensation, whereas no such deformities were observed in lotusine-pretreated cells even after the exposure to DOX. Increased endogenous antioxidants with reduced lipid peroxidation were observed in lotusine-pretreated cells, whereas the antioxidants were reduced along with increased lipid peroxidation in doxorubicin-exposed cells. A decreased reactive oxygen species generation was evidenced with the 2',7'-dichlorofluorescein diacetate (DCF-DA) staining method. In qPCR analysis, the lotusine-pretreated cells have mitigated doxorubicin-mediated apoptosis by downregulating the pro-apoptotic gene Bax and apoptotic executor caspase-3. It was further confirmed with the luminometric assay, which resulted in lesser luminescence in lotusine-pretreated cells, whereas higher luminescence was recorded in doxorubicin-alone-treated cells. In conclusion, the present study revealed that the lotusine pretreatment has exhibited potential cardioprotective activity against DOX-induced oxidative stress by increasing the intracellular antioxidant defense.

Hop-derived Xanthohumol Induces HL-60 Leukemia Cells Death

Background: Acute promyelocytic leukemia (APL) affects both kids and adults, however it is more prevalent in younger population. Although APL has a favorable prognostic, patients that relapse often do not respond positively to additional chemotherapy. Therefore, there is a need to further identify ways to overcome these challenges. 
 Hypothesis: In this study, we examined antileukemic effects of xanthohumol (XN), a prenylated flavonoid derived from hops (Humulus lupulus L), on human promyelocytic HL-60 cells. 
 Materials and Methods: HL-60 cells were exposed to different concentrations of XN (μM) for 24 h. Cell viability, cell morphology, chromatin condensation, cPARP-1 level, and caspase-3 activation, and the expression of p21WAF1/Cip1 were analyzed.
 Results: XN reduced HL-60 cell viability in a dose-dependent manner. XN induced a dose-dependent morphological changes including cell shrinkage and blebbing, and significantly increased the number of cells with condensed chromatin. XN significantly increased the level of cPARP-1, active caspase-3, and the expression of p21WAF/CIP mRNA.
 Conclusion: These data indicate that XN induces HL-60 cell death by regulating cell cycle progression and apoptosis. This study suggests that XN may have antileukemic preventive effects.

Synthesis, 18F-radiolabeling and apoptosis inducing studies of novel 4, 7-disubstituted coumarins.

In present study, a new series of 4, 7-disubstituted coumarin derivatives (7a-y) have been synthesized as galectin-1 targeting apoptosis inducing agents and evaluated for their in vitro cytotoxic potentials against a panel of selected human cancer cell lines namely, Brest (MCF7), Ovarian (SKOV3), Prostate (PC-3 & DU145) and normal embryonic kidney (HEK293T) cells, using MTT assay. Most of the compounds exhibited potent growth inhibitory action against the treated cancer cell lines with an IC50 range of 10-30µM. Compound 7q exhibited a significant growth inhibition against prostate cancer (PC-3 & DU145) cell lines with an IC50 value of 7.45±0.03µM, 8.95±0.17µM respectively. Further, the target compound 7q was radiolabeled with fluorine-18 [18F] to be used as a novel PET radiotracer for imaging of tumors via targeting galectin-1, using appropriate reaction conditions in the GE Tracer-lab FX2N synthesis module. The purification of the [18F] radiolabeled compound [18F]-7q was successfully achieved with 60% ethanol. The radiochemical purity was>85% and residual solvent limits of DMF was 65±3ppm as analysed by HPLC, TLC & GC analytical methods. The apoptosis studies confirm the inhibition of cell proliferation with morphological changes like cell shrinkage, blebbing and cell wall deformation, increasing the ROS levels, and loss of mitochondrial membrane potential by Acridine orange/Ethidium bromide staining, Hoechst-33342 staining, H2DCFDA staining, annexin V-FITC/PI, and JC-1 staining methods. In flow cytometric analysis, 7q selectively arrested the sub-G1 phase of the cell cycle in a dose-dependent manner. In Gal-1 ELISA studies, compound 7q efficiently reduced the levels of Gal-1 protein in dose-dependent manner with an IC50 value of 100µM. The binding constant (Ka) of 7q with Gal-1 was observed as 1.3×104 M-1 by fluorescence spectroscopy. The molecular docking studies clearly showed possible interactions and the pharmacokinetic (ADMET) properties of compound 7q with Gal-1. Hence, the novel 4, 7-disubstituted coumarins could be a potential cytotoxic and PET imaging agents via Gal-1.

Aquaporin expression in breast cancer and their involvement in bleb formation, cell motility and invasion in endocrine resistant variant cells.

Estrogen receptor (ER)‑silenced breast cancer cell lines exhibit endocrine resistance and morphological changes from an epithelial to a mesenchymal phenotype. These cells also display increased motility and invasive properties that are further accentuated by exposure to an alkaline pH, exhibiting dynamic plasma membrane blebbing and cytoplasmic streaming. These latter morphological changes are hypothesized to involve substantial water movement across the plasma membrane, contributing to bleb formation; this may involve aquaporin channel proteins(AQPs). AQP1, 3, 4 and5 expression/localization was examined via reverse transcription‑quantitative PCR, western blotting and confocal microscopy in endocrine‑sensitive(YS1.2) and ‑resistant (pII and MDA‑MB‑231) breast cancer cells, as well as normal breast epithelial cells(MCF10A). The effects of osmotic changes on bleb formation were examined via live cell imaging. AQP3 protein expression was knocked down by small interfering RNA(siRNA) transfection, and the effect of its reduced expression on bleb formation, cell motility and invasion were determined via immunofluorescence, scratch and Cultrex assays, respectively. Expression of the four AQPs varied across the different cell lines, and exhibited nuclear, cytoplasmic and membranous localization. Osmotic changes affected the formation of blebs. In pIIcells exposed to alkaline pH, AQP3 was observed to be redistributed from the nucleus into the newly formed blebs. siRNA‑mediated knockdown of AQP3 in pIIcells significantly reduced cellular blebbing induced by alkaline pH, as well as motility and invasion. These data suggested that AQP3, and potentially other aquaporins, may participate in the processes leading to blebbing of endocrine‑resistant cells which is proposed to be a mechanism that drives tumor metastasis.

Essential role of the Crk family-dosage in DiGeorge-like anomaly and metabolic homeostasis.

CRK and CRKL (CRK-like) encode adapter proteins with similar biochemical properties. Here, we show that a 50% reduction of the family-combined dosage generates developmental defects, including aspects of DiGeorge/del22q11 syndrome in mice. Like the mouse homologs of two 22q11.21 genes CRKL and TBX1, Crk and Tbx1 also genetically interact, thus suggesting that pathways shared by the three genes participate in organogenesis affected in the syndrome. We also show that Crk and Crkl are required during mesoderm development, and Crk/Crkl deficiency results in small cell size and abnormal mesenchyme behavior in primary embryonic fibroblasts. Our systems-wide analyses reveal impaired glycolysis, associated with low Hif1a protein levels as well as reduced histone H3K27 acetylation in several key glycolysis genes. Furthermore, Crk/Crkl deficiency sensitizes MEFs to 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, to induce cell blebbing. Activated Rapgef1, a Crk/Crkl-downstream effector, rescues several aspects of the cell phenotype, including proliferation, cell size, focal adhesions, and phosphorylation of p70 S6k1 and ribosomal protein S6. Our investigations demonstrate that Crk/Crkl-shared pathways orchestrate metabolic homeostasis and cell behavior through widespread epigenetic controls.

ZnCl2 Enabled Synthesis of Highly Crystalline and Emissive Carbon Dots with Exceptional Capability to Generate O2⋅–

Summary Highly crystalline and emissive carbon dots (CDs) are firstly synthesized via an ionothermal method in organic solvents. In contrast to well-established approaches, ZnCl2 is utilized as the pyrolysis-promoting agent to prepare emissive CDs with tunable wavelengths, directly by carbonization from different O and N precursors under a mild reaction condition at 210°C. More than 50% synthetic yield and 39% photoluminescent quantum yield (blue CD) are obtained. XAFS measurements, in conjunction with TEM, HAADF-STEM, UPS, and ESR analyses, allow us to obtain energy diagram configuration and confirm the luminescent nature of these hard-core structured CDs. Importantly, the resulting CDs exhibit excellent capability to photogenerate superoxide radical anion (O2⋅–), which can be exemplified by both photodynamic therapy and photooxidative cyclization synthesis of tetrahydroquinolines. Representative pharmaceutical mifepristone can be derivatized in 90% yield within 10 min of irradiation of the CDs in an elegant flow reactor.

Role of Rho kinase pathway in diabetic retinopathy: from a diabetic rat model to human eye pathology

AbstractDiabetic macular oedema is the main cause of visual loss in diabetic retinopathy, particularly in type 2 diabetic patients. The pathogenic mechanisms are not fully elucidated. Rho/Rock1 pathway is activated in diabetes and is responsible for cellular contraction. ROCK1 activation induces actin cytoskeleton remodeling. We hypothesized that Rho/Rock1 diabetes‐induced activation could contribute to cytoskeleton remodelling especially in retinal vessels subsequently altering retinal perfusion.MethodsWe used 12 months old Goto‐Kakizaki (GK) type 2 diabetic rats. Rho/Rhock1 pathway activation effects on retinal vasculature was analysed and ROCK inhibition by Fasudil administered into the vitreous was tested in rats in vivo on vascular perfusion.ResultsIn diabetic rats, activation of ROCK‐1 induces focal vascular constrictions, endoluminal blebbing and subsequent retinal hypoxia. Intraocular injection of fasudil, significantly reduces retinal hypoxia. Diabetes‐induced cell blebbing may contribute to ischemic maculopathy and represent an intervention target.

Development of a new animal model of radiation retinopathy using an experimental radiation platform

AbstractPurposeChoroidal melanoma is the most frequent malignant intraocular tumor. Radiotherapy using various modalities is the standard conservative treatment for confined lesions. It allows globe preservation in most cases, but 50% to 60% of irradiated eyes develop radiation retinopathy, secondary to multiple retinal vascular occlusions, leading in the most severe cases to neovascular glaucoma and secondary enucleation. The purpose of this experimental study was to create and test a new model of radiation retinopathy in the rat retina, and to explore potential pathways involved in the early pathogenesis of radiation retinopathy.MethodsWe used the Small Animal Radiation Research Platform (SARRP, Xstrahl, UK) to build a dose‐response model of retinal irradiation. Long‐Evans rats were irradiated with an X‐ray source, after exorbiting the eye. Only one eye was irradiated for ethical reasons. There were 4 groups of 4 animals according to the radiation dose received. Group 1 received one 15‐Gray fraction of, Group 2 received two 15‐Gray fractions, Group 3 received three 15‐Gray fractions, and Group 4, the control group, received one sham irradiation fraction.Multimodal imaging with OCT, color fundus photograph, and fluoresceine angiography (Micron 4, Phoenix Researchlabs, USA) was performed at baseline and seven days after irradiation.Animals were sacrificed 8 days after irradiation. Eyes were either fixated for inclusion and sections, fixated for flat‐mounts of retina/RPE, or milled for Western Blot analysis.ResultsIrradiating of rat retinas using SARRP was successful. At day 7, there were no specific features on angiography, but OCT revealed inconsistent features in the peripheral retina of one animal (Group 3 fractions). In two animals, exorbitation was not possible due to variations of periocular morphology (Groups 2 and 3 fractions). Both animals developed corneal opacifications attributed to lacrymal gland damage.ConclusionsSARRP is a simple and efficient tool to model radiation retinopathy in small animals. Further work is required to improve our understanding of this potentially blinding complication of ocular irradiationReferencesRamos MS, Echegaray JJ, Kuhn‐Asif S, Wilkinson A, Yuan A, Singh AD, Browne AW.Animal models of radiation retinopathy ‐ From teletherapy to brachytherapy.Exp Eye Res. 2019 Apr;181:240‐251. Rothschild PR, Salah S, Berdugo M, Gélizé E, Delaunay K, Naud MC, Klein C, Moulin A, Savoldelli M, Bergin C, Jeanny JC, Jonet L, Arsenijevic Y, Behar‐Cohen F, Crisanti P. ROCK‐1 mediates diabetes‐induced retinal pigment epithelial and endothelial cell blebbing: Contribution to diabetic retinopathy. Sci Rep. 2017 Aug 18;7(1):8834.Rousseau M, Gaugler M‐H, Rodallec A, Bonnaud S, Paris F, Corre I. RhoA GTPase regulates radiation‐induced alterations in endothelial cell adhesion and migration. Biochem Biophys Res Commun. 2011;414(4):750‐755.

Post-Passage rock inhibition induces cytoskeletal aberrations and apoptosis in Human embryonic stem cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are prone to anoikis after single cell dissociation. The small molecule, Y-27632 is known to increase survival of hESCs and hiPSCs by inhibiting the Rho-associated protein kinase (ROCK). However, the underlying mechanisms are still unclear. Here, we thoroughly screened small molecules to investigate the adhesion and survival of hESCs in adherent culture. Y-27632 provided higher adhesion and survival of hESCs by increased cell migration and preventing cell blebbing in single dissociated cells. The combination of Matrigel with poly-d-lysine increased the attachment and survival of dissociated cells via actin filament and microtubule spreading in Y-27632-treated cells. Although Y-27632 prevented apoptosis by suppressing actin filament contraction, microtubule bundling, and blebbing, prolonged Y-27632 treatment presented a different morphology in the attached growing hESC colony. It induced apoptosis of cells by promoting cytoplasmic spread, E-cadherin structural change, and increased detachment. It also induced actin cytoskeleton disruption, combined with microtubule and intermediate filament elongation. For optimal hPSC culture, our research suggests that Y-27632 should be removed shortly after passaging.

Antagonistic Interaction of Staphylococcus aureus and Staphylococcus epidermidis with Rhizopus arrhizus Mediated by Phenol Soluble Modulins and Organic Acids.

Rhizopus arrhizus (R.arrhizus) is a common causative agent of mucormycosis that usually enters the human body through the respiratory tract and skin. Both these sites harbor staphylococci as a part of the normal microflora, indicating the possibility of interspecies interactions. We aimed to elucidate this interaction and identify the molecular mechanisms involved. Both Staphylococcus aureus (S.aureus) and Staphylococcus epidermidis (S.epidermidis) substantially hindered R.arrhizus radial growth, spore germination, and liquid culture biomass. Secreted components in the stationary-phase supernatant were responsible for this activity. The active components, based on molecular weight-based fractionation, mass spectrometry, and ion exclusion chromatography, were identified as a truncated version of phenol soluble modulin α2 (Δ1Δ2PSMα2) and PSMα3 in S.aureus, PSMδ in S.epidermidis, and organic acids in both the species. Exposure to the phenol soluble modulins (PSMs) extensively damaged the fungal spores and pre-existing hyphae, leading to bleb formation, shriveling, hyphal shrinkage, and cell distortion.

Functional Validation of DownRegulated MicroRNAs in HeLa Cells Treated with Polyalthia longifolia Leaf Extract Using Different Microscopic Approaches: A Morphological Alteration-Based Validation.

Several microscopy methods have been developed to assess the morphological changes in cells in the investigations of the mode of cell death in response to a stimulus. Our recent finding on the treatment of the IC50 concentration (26.67 μg/mL) of Polyalthia longifolia leaf extract indicated the induction of apoptotic cell death via the regulation of miRNA in HeLa cells. Hence, the current study was conducted to validate the function of these downregulated microRNAs in P. longifolia-treated HeLa cells using microscopic approaches. These include scanning electron microscope (SEM), transmission electron microscope (TEM), and acridine orange/propidium iodide (AO/PI)-based fluorescent microscopy techniques by observing the morphological alterations to cells after transfection with mimic miRNA. Interestingly, the morphological changes observed in this study demonstrated the apoptotic hallmarks, for instance, cell blebbing, cell shrinkage, cytoplasmic and nuclear condensation, vacuolization, cytoplasmic extrusion, and the formation of apoptotic bodies, which proved the role of dysregulated miRNAs in apoptotic HeLa cell death after treatment with the P. longifolia leaf extract. Conclusively, the current study proved the crucial role of downregulated miR-484 and miR-221-5p in the induction of apoptotic cell death in P. longifolia-treated HeLa cells using three approaches-SEM, TEM, and AO/PI-based fluorescent microscope.

Dielectrophoretic deformation of breast cancer cells for lab on a chip applications.

This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA-MB-231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA-MB-231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA-MB-231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10Vpp at both 10kHz and 50MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

Synthesis of 1-benzyl-1H-benzimidazoles as galectin-1 mediated anticancer agents

In our pursuit to develop novel non-carbohydrate small molecule Galectin-1 Inhibitors, we have designed a series of 1-benzyl-1H-benzimidazole derivatives and demonstrated their anticancer activity. The compound 6g, 4-(1-benzyl-5-chloro-1H-benzo[d]imidazol-2-yl)-N-(4-hydroxyphenyl) benzamide was found to be most potent with an IC50 of 7.01 ± 0.20 µM and arresting MCF-7 cell growth at G2/M phase and S phase. Induction of apoptosis was confirmed by morphological changes like cell shrinkage, blebbing and cell wall deformation, dose dependent increase in the mitochondrial membrane potential (ΔΨm) and ROS levels. Further, dose dependent decrease in Gal-1 protein levels proves Gal-1 mediated apoptosis by 6g. Molecular docking studies were performed to understand the Gal-1 interaction with compound 6g. In addition, RP-HPLC studies showed 85.44% of 6g binding to Gal-1. Binding affinity studies by fluorescence spectroscopy and Surface Plasmon Resonance (SPR) showed that 6g binds to Gal-1 with binding constant (Ka) of 1.2 × 104 M−1 and equilibrium constant KD value of 5.76 × 10−4 M respectively.

Enhanced blebbing as a marker for metastatic prostate cancer.

Highly metastatic prostate cancer cells flowing through a microfluidic channel form plasma membrane blebs: they form 27% more than normal cells and have a lower stiffness (about 50%). Hypo-osmotic stress assays (with osmolarity) show 22% more blebbing of highly metastatic than moderately metastatic and 30% more than normal cells. Plasma membrane blebbing is known to provide important metastatic capabilities to cancer cells by aiding cell detachment from the primary tumor site and increasing cell deformability to promote cell migration through the extracellular matrix. Increased blebbing was attributed by others to decreased phosphorylated ezrin, radixin, and moesin (ERM) (p-ERM) protein expression-p-ERMs bind the plasma membrane to the actin cortex and reduced p-ERM expression can weaken membrane-cortex attachment. Myosin II also influences blebbing as myosin's natural contraction generates tension in the actin cortex. This increases cellular hydrostatic pressure, causes cortex rupture, cytoplasm flow out of the cortex, and hence blebbing. Highly metastatic cells are surprisingly found to express similar ezrin and myosin II levels but higher moesin levels in comparison with lowly metastatic or normal cells-suggesting that their levels, contrary to the literature [G. Charras and E. Paluch, Nat. Rev. Mol. Cell Biol. 9(9), 730-736 (2008); J.-Y. Tinevez, U. Schulze, G. Salbreux, J. Roensch, J.-F. Joanny, and E. Paluch, Proc. Natl. Acad. Sci. U.S.A. 106(44), 18581-18586 (2009); M. Bergert, S. D. Chandradoss, R. A. Desai, and E. Paluch, Proc. Natl. Acad. Sci. U.S.A. 109(36), 14434-14439 (2012); E. K. Paluch and E. Raz: Curr. Opin. Cell Biol. 25(5), 582-590 (2013)], are not important in metastatic prostate cell blebbing. Our results show that reduced F-actin is primarily responsible for increased blebbing in these metastatic cells. Blebbing can thus serve as a simple prognostic marker for the highly incident and lethal metastatic prostate cancer.

Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) against HepG2 cells

Naturally occurring polyamines like Putrescine, Spermidine, and Spermine are polycations which bind to the DNA, hence stabilizing it and promoting the essential cellular processes. Many synthetic polyamine analogues have been synthesized in the past few years, which have shown cytotoxic effects on different tumours. In the present study, we evaluated the antiproliferative effect of a novel, acylspermidine derivative, (N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide) (AAHD) on HepG2 cells. Fluorescence staining was performed with nuclear stain (Hoechst 33342) and acridine orange/ethidium bromide double staining. Dose and the time-dependent antiproliferative effect were observed by WST-1 assays, and radical scavenging activity was measured by ROS. Morphological changes such as cell shrinkage & blebbing were analyzed by fluorescent microscopy. It was found that AAHD markedly suppressed the growth of HepG2 cells in a dose- and time-dependent manner. It was also noted that the modulation of ROS levels confirmed the radical scavenging activity. In the near future, AAHD can be a promising drug candidate in chalking out a neoplastic strategy to control the proliferation of tumour cells. This study indicated that AAHD induced anti-proliferative and pro-apoptotic activities on HCC. Since AAHD was active at micromolar concentrations without any adverse effects on the healthy cells (Fibroblasts), it is worthy of further clinical investigations.

Syringic acid triggers reactive oxygen species-mediated cytotoxicity in HepG2 cells.

Hepatocellular carcinoma is the second most common cause of cancer death in the world and its incidence has dramatically increased worldwide in the past two decades. Syringic acid (SA) has been studied for its hepatoprotective, anti-inflammatory, immunomodulatory, free radical scavenging, and antioxidant activities. We aimed to evaluate the cytotoxic effect of SA against human hepatoma HepG2 cell line. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. HepG2 cells were treated with SA at concentration ranges of 25, 50, and 100 µM for 24 h. Reactive oxygen species (ROS) expression was investigated by dichlorofluorescein staining assay. Morphological changes of SA-treated HepG2 cells were evaluated by acridine orange (AO) and ethidium bromide (EB) dual staining. Apoptotic marker gene expressions were evaluated by qPCR. SA treatment caused significant cytotoxicity and liberation of ROS in HepG2 cells. AO and EB staining showed membrane blebbing and distortion in SA-treated cells. Apoptotic markers such as caspases 3 and 9, cytochrome c, Apaf-1, Bax, and p53 gene expressions were significantly increased upon SA treatment indicating the possibility of apoptosis induction in HepG2 cells. This treatment also caused significant downregulation of Bcl-2 gene expression. SA has a cytotoxic effect on human HepG2 cell line, and this might be a promising agent in anticancer research.

Advances in geometric techniques for analyzing blebbing in chemotaxing Dictyostelium cells.

We present a technical platform that allows us to monitor and measure cortex and membrane dynamics during bleb-based chemotaxis. Using D. discoideum cells expressing LifeAct-GFP and crawling under agarose containing RITC-dextran, we were able to simultaneously visualize the actin cortex and the cell membrane throughout bleb formation. Using these images, we then applied edge detect to generate points on the cell boundary with coordinates in a coordinate plane. Then we fitted these points to a curve with known x and y coordinate functions. The result was to parameterize the cell outline. With the parameterization, we demonstrate how to compute data for geometric features such as cell area, bleb area and edge curvature. This allows us to collect vital data for the analysis of blebbing.

Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA

BackgroundCarcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.MethodsEDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.ResultsWe demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.ConclusionsOur data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.