Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose.

Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets (Pcsk9, eGFP, mdx exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC50 concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent in vivo administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated in vivo editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue.

Comparison of the efficiency of the Sec and Tat secretory pathways in the secretion of recombinant neurturin protein using de novo designed signal peptides.

Since cytoplasmic expression of heterologous proteins with disulfide bonds leads to the formation of inclusion bodies in E. coli, periplasmic production is preferable. The N-terminal signal peptide attached to the secreted protein determines the type of secretory pathway through which the target protein is secreted; Sec, Tat, or SRP. The aim of this study was to design and compare two novel signal peptides for the secretion of recombinant neurturin (as a model) via the Sec and Tat pathways. For this purpose, we aligned the natural signal peptides from E. coli and Bacillus subtilis to identify the conserved amino acids and those with the highest repetition. The SignalP4.1 and TatP1.0 software were used to determine the secretion efficiency of the new signal peptides. The efficiency of new signal peptides was then evaluated and compared experimentally with two naturally used signal peptides. Quantitative analysis of Western blot bands showed that approximately 80% of the expressed neurturin was secreted into the periplasmic space by new signal peptides. Circular dichroism spectroscopy also confirmed the correct secondary structure of the secreted neurturin. In conclusion, these novel signal peptides can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Crosstalk Between NK Cell Receptors and Tumor Membrane Hsp70-Derived Peptide: A Combined Computational and Experimental Study.

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.

Lipoamino bundle LNPs for efficient mRNA transfection of dendritic cells and macrophages show high spleen selectivity.

Messenger RNA (mRNA) is a powerful tool for nucleic acid-based therapies and vaccination, but efficient and specific delivery to target tissues remains a significant challenge. In this study, we demonstrate lipoamino xenopeptide carriers as components of highly efficient mRNA LNPs. These lipo-xenopeptides are defined as 2D sequences in different 3D topologies (bundles or different U-shapes). The polar artificial amino acid tetraethylene pentamino succinic acid (Stp) and various lipophilic tertiary lipoamino fatty acids (LAFs) act as ionizable amphiphilic units, connected in different ratios via bisamidated lysines as branching units. A series of more lipophilic LAF4-Stp1 carriers with bundle topology is especially well suited for efficient encapsulation of mRNA into LNPs, facilitated cellular uptake and strongly enhanced endosomal escape. These LNPs display improved, faster transfection kinetics compared to standard LNP formulations, with high potency in a variety of tumor cell lines (including N2a neuroblastoma, HepG2 and Huh7 hepatocellular, and HeLa cervical carcinoma cells), J774A.1 macrophages, and DC2.4 dendritic cells. High transfection levels were obtained even in the presence of serum at very low sub-microgram mRNA doses. Upon intravenous application of only 3 µg mRNA per mouse, in vivo mRNA expression is found with a high selectivity for dendritic cells and macrophages, resulting in a particularly high overall preferred expression in the spleen.

Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot between Lipoplexes and Polyplexes.

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3 µg luciferase-encoding mRNA in mice.

Receptor-Targeted Dual pH-Triggered Intracellular Protein Transfer.

Protein therapeutics are of widespread interest due to their successful performance in the current pharmaceutical and medical fields, even though their broad applications have been hindered by the lack of an efficient intracellular delivery approach. Herein, we fabricated an active-targeted dual pH-responsive delivery system with favorable tumor cell entry augmented by extracellular pH-triggered charge reversal and tumor receptor targeting and pH-controlled endosomal release in a traceless fashion. As a traceable model protein, the enhanced green fluorescent protein (eGFP) bearing a nuclear localization signal was covalently coupled with a pH-labile traceless azidomethyl-methylmaleic anhydride (AzMMMan) linker followed by functionalization with different molar equivalents of two dibenzocyclooctyne-octa-arginine-cysteine (DBCO-R8C)-modified moieties: polyethylene glycol (PEG)-GE11 peptide for epidermal growth factor receptor-mediated targeting and melittin for endosomal escape. The cationic melittin domain was masked with tetrahydrophthalic anhydride revertible at mild acidic pH 6.5. At the optimally balanced ratio of functional units, the on-demand charge conversion at tumoral extracellular pH 6.5 in combination with GE11-mediated targeting triggered enhanced electrostatic cellular attraction by the R8C cell-penetrating peptides and melittin, as demonstrated by strongly enhanced cellular uptake. Successful endosomal release followed by nuclear localization of the eGFP cargo was obtained by taking advantage of melittin-mediated endosomal escape and rapid traceless release from the AzMMMan linker. The effectiveness of this multifunctional bioresponsive system suggests a promising strategy for delivery of protein drugs toward intracellular targets. A possible therapeutic relevance was indicated by an example of cytosolic delivery of cytochrome c initiating the apoptosis pathway to kill cancer cells.

CAR T Cells Targeting Membrane-Bound Hsp70 on Tumor Cells Mimic Hsp70-Primed NK Cells.

Strategies to boost anti-tumor immunity are urgently needed to treat therapy-resistant late-stage cancers, including colorectal cancers (CRCs). Cytokine stimulation and genetic modifications with chimeric antigen receptors (CAR) represent promising strategies to more specifically redirect anti-tumor activities of effector cells like natural killer (NK) and T cells. However, these approaches are critically dependent on tumor-specific antigens while circumventing the suppressive power of the solid tumor microenvironment and avoiding off-tumor toxicities. Previously, we have shown that the stress-inducible heat shock protein 70 (Hsp70) is frequently and specifically expressed on the cell surface of many different, highly aggressive tumors but not normal tissues. We could take advantage of tumors expressing Hsp70 on their membrane ('mHsp70') to attract and engage NK cells after in vitro stimulation with the 14-mer Hsp70 peptide TKDNNLLGRFELSG (TKD) plus low dose interleukin (IL)-2. However, a potential limitation of activated primary NK cells after adoptive transfer is their comparably short life span. T cells are typically long-lived but do not recognize mHsp70 on tumor cells, even after stimulation with TKD/IL-2. To combine the advantages of mHsp70-specificity with longevity, we constructed a CAR having specificity for mHsp70 and retrovirally transduced it into primary T cells. Co-culture of anti-Hsp70 CAR-transduced T cells with mHsp70-positive tumor cells stimulates their functional responsiveness. Herein, we demonstrated that human CRCs with a high mHsp70 expression similarly attract TKD/IL-2 stimulated NK cells and anti-Hsp70 CAR T cells, triggering the release of their lytic effector protein granzyme B (GrB) and the pro-inflammatory cytokine interferon (IFN)-γ, after 4 and 24 hours, respectively. In sum, stimulated NK cells and anti-Hsp70 CAR T cells demonstrated comparable anti-tumor effects, albeit with somewhat differing kinetics. These findings, together with the fact that mHsp70 is expressed on a large variety of different cancer entities, highlight the potential of TKD/IL-2 pre-stimulated NK, as well as anti-Hsp70 CAR T cells to provide a promising direction in the field of targeted, cell-based immunotherapies which can address significant unmet clinical needs in a wide range of cancer settings.

Direct Regulation of Hyperpolarization-Activated Cyclic-Nucleotide Gated (HCN1) Channels by Cannabinoids.

Cannabinoids are a broad class of molecules that act primarily on neurons, affecting pain sensation, appetite, mood, learning, and memory. In addition to interacting with specific cannabinoid receptors (CBRs), cannabinoids can directly modulate the function of various ion channels. Here, we examine whether cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the most prevalent phytocannabinoids in Cannabis sativa, can regulate the function of hyperpolarization-activated cyclic-nucleotide-gated (HCN1) channels independently of CBRs. HCN1 channels were expressed in Xenopus oocytes since they do not express CBRs, and the effects of cannabinoid treatment on HCN1 currents were examined by a two-electrode voltage clamp. We observe opposing effects of CBD and THC on HCN1 current, with CBD acting to stimulate HCN1 function, while THC inhibited current. These effects persist in HCN1 channels lacking the cyclic-nucleotide binding domain (HCN1ΔCNBD). However, changes to membrane fluidity, examined by treating cells with TX-100, inhibited HCN1 current had more pronounced effects on the voltage-dependence and kinetics of activation than THC, suggesting this is not the primary mechanism of HCN1 regulation by cannabinoids. Our findings may contribute to the overall understanding of how cannabinoids may act as promising therapeutic molecules for the treatment of several neurological disorders in which HCN function is disturbed.

Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

The control of RNA metabolism is an important aspect of molecular biology with wide-ranging impacts on cells. Central to processing of coding RNAs is the addition of the methyl-7 guanosine (m7G) "cap" on their 5' end. The eukaryotic translation initiation factor eIF4E directly binds the m7G cap and through this interaction plays key roles in many steps of RNA metabolism including nuclear RNA export and translation. eIF4E also stimulates capping of many transcripts through its ability to drive the production of the enzyme RNMT which methylates the G-cap to form the mature m7G cap. Here, we found that eIF4E also physically associated with RNMT in human cells. Moreover, eIF4E directly interacted with RNMT in vitro. eIF4E is only the second protein reported to directly bind the methyltransferase domain of RNMT, the first being its co-factor RAM. We combined high-resolution NMR methods with biochemical studies to define the binding interfaces for the RNMT-eIF4E complex. Further, we found that eIF4E competes for RAM binding to RNMT and conversely, RNMT competes for binding of well-established eIF4E-binding partners such as the 4E-BPs. RNMT uses novel structural means to engage eIF4E. Finally, we observed that m7G cap-eIF4E-RNMT trimeric complexes form, and thus RNMT-eIF4E complexes may be employed so that eIF4E captures newly capped RNA. In all, we show for the first time that the cap-binding protein eIF4E directly binds to the cap-maturation enzyme RNMT.

Cross-Linkable Polyion Complex Micelles from Polypept(o)ide-Based ABC-Triblock Copolymers for siRNA Delivery.

ABC-type triblock copolymers are a rising platform especially for oligonucleotide delivery as they offer an additional functionality besides the anyhow needed functions of shielding and complexation. The authors present a polypept(o)ide-based triblock copolymer synthesized by amine-initiated ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs), comprising a shielding block A of polysarcosine (pSar), a poly(S-ethylsulfonyl-l-cystein) (pCys(SO2 Et)) block B for bioreversible and chemo-selective cross-linking and a poly(l-lysine) (pLys) block C for complexation to construct polyion complex (PIC) micelles as vehicle for small interfering RNA (siRNA) delivery. The self-assembly behavior of ABC-type triblocks is investigated to derive correlations between block lengths of the polymer and PIC micelle structure, showing an enormous effect of the ß-sheet forming pCys(SO2 Et) block. Moreover, the block enables the introduction of disulfide cross-links by reaction with multifunctional thiols to increase stability against dilution. The right content of the additional block leads to well-defined cross-linked 50-60 nm PIC micelles purified from production impurities and determinable siRNA loading. These PIC micelles can deliver functional siRNA into Neuro2A and KB cells evaluated by cellular uptake and specific gene knockdown assays.

Transferrin Receptor Targeted Polyplexes Completely Comprised of Sequence-Defined Components.

Human transferrin protein (Tf) modified polyplexes have already displayed encouraging potential for receptor-mediated nucleic acid delivery into tumors. The use of a blood-derived targeting protein and polydisperse macromolecular cationic subunits however presents a practical challenge for pharmaceutical grade production. Here, Tf receptor (TfR) targeted small interfering RNA (siRNA) polyplexes are designed that are completely composed of synthetic, monodisperse, and sequence-defined subunits generated by solid-phase supported synthesis. An optimized cationizable lipo-oligoaminoamide (lipo-OAA) is used for siRNA core polyplex formation, and a retro-enantio peptide (reTfR) attached via a monodisperse polyethylene glycol (PEG) spacer via click chemistry is applied for targeting. Improved gene silencing is demonstrated in TfR-expressing KB and DU145 cells. Analogous plasmid DNA (pDNA) polyplexes are successfully used for receptor-mediated gene delivery in TfR-rich K562 cells and Neuro2a cells. Six lipo-OAAs differing in their lipidic domain and redox-sensitive attachment of lipid residues are tested in order to evaluate the impact of core polyplex stability on receptor-dependent gene transfer.

NK Cells Armed with Chimeric Antigen Receptors (CAR): Roadblocks to Successful Development.

In recent years, cell-based immunotherapies have demonstrated promising results in the treatment of cancer. Chimeric antigen receptors (CARs) arm effector cells with a weapon for targeting tumor antigens, licensing engineered cells to recognize and kill cancer cells. The quality of the CAR-antigen interaction strongly depends on the selected tumor antigen and its expression density on cancer cells. CD19 CAR-engineered T cells approved by the Food and Drug Administration have been most frequently applied in the treatment of hematological malignancies. Clinical challenges in their application primarily include cytokine release syndrome, neurological symptoms, severe inflammatory responses, and/or other off-target effects most likely mediated by cytotoxic T cells. As a consequence, there remains a significant medical need for more potent technology platforms leveraging cell-based approaches with enhanced safety profiles. A promising population that has been advanced is the natural killer (NK) cell, which can also be engineered with CARs. NK cells which belong to the innate arm of the immune system recognize and kill virally infected cells as well as (stressed) cancer cells in a major histocompatibility complex I independent manner. NK cells play an important role in the host's immune defense against cancer due to their specialized lytic mechanisms which include death receptor (i.e., Fas)/death receptor ligand (i.e., Fas ligand) and granzyme B/perforin-mediated apoptosis, and antibody-dependent cellular cytotoxicity, as well as their immunoregulatory potential via cytokine/chemokine release. To develop and implement a highly effective CAR NK cell-based therapy with low side effects, the following three principles which are specifically addressed in this review have to be considered: unique target selection, well-designed CAR, and optimized gene delivery.

Coronavirus Disease 2019 (COVID-19) in Children: Prevalence, Diagnosis, Clinical Symptoms, and Treatment.

In this article, we have reviewed the prevalence, diagnosis, symptoms, and treatment of COVID-19 in children. The incidence of COVID-19 among children under 18 years was 2.1% based on the reported studies, where the mortality rate in the same age group was 0.2%. No death has been reported in children under 9-years old. There are some articles that report children with COVID-19 having symptoms similar to Kawasaki's disease. In these cases, heart complications were observed. The best markers for diagnosing the severity of the disease in children are the levels of bilirubin and hepatic enzymes. Large number of angiotensin converting enzyme 2 (ACE2) receptors on cell surfaces, effective innate immune system, and high level of blood lymphocyte have been reported to be the potent reasons for lower incidence of severe symptoms of COVID-19 among children. Children can very well be the carriers of this virus. Children with severe COVID-19 clinical symptoms, especially those suffering from pneumonia, must be hospitalized similar to adults, while quarantine is required for those having mild symptoms. Antiviral medication (lopinavir, darunavir, favipiravir, remdesivir, ribavirin, oseltamivir, tocilizumab, and umifenovir), ACE inhibitors, interferon-α2b, co-therapy with azithromycin, inhaling iNO, and oxygen therapy can be used for treatment. For the treatment of children without any clinical and infection symptoms, home isolation protocol has been recommended.

A new whole-cell biocatalyst for sulfur dioxide filtering and degradation.

In this study, the interaction of the magnetotactic bacterium with sulfite compounds and their potential to degrade SO2 was investigated using cyclic voltammetry (CV), molecular emission cavity analysis (MECA) and ion-exchange chromatography (IEC). This biofilter was able to degrade SO2 up to 22281 mg m-3 by disproportionation reaction and the formation of S2- and SO42- with ≥99% efficiency. Designed biofilter was able to restart the initial performance at least after seven cycles if it was used at 14-day intervals. According to theoretical studies, the value of mean free energy (E) obtained using the Dubinin-Radushkevich isotherm model was 0.02 kJ mol-1, which is in the range expected for physical adsorption. Designed biofilter can be considered as a powerful tool to degrade SO2 in diverse urban and industrial centers.

Introducing a bioelectrochemical method for highly selective enumeration of magnetotactic bacteria.

In this study, we employed an electrochemical (potentiometric) method to enumerate magnetotactic bacteria (MTB) during its coupling with iodometric titration to obtain a selective, precise and rapid counting system. Oxygen was considered as an important factor for the orientation and movement of MTB towards the magnet-modified indicator electrode. In the direct potentiometry, a linear correlation was detected between potentiometric response and dissolved oxygen (DO) concentrations. By the increase of the DO concentration, potential difference would increase in the range of 4.0 to 20.0 parts per million (ppm) at different pressure conditions. The reliability of the O2 bio-sensing feature provides a selective MTB-based cell enumeration methodology based on indirect potentiometric titration. Furthermore, a five-minute H2-purging resulted in an increase of potentiometric response sensitivity arising from the decrease in DO concentration of the electrolyte solution. Results were also investigated by zeta potential difference, which show the effect of charge density of MTB in presence of DO. Zeta potential was increased proportionally by addition of the MTB population. Regarding the reliability of the suggested method, data obtained by the designed system showed no statistical difference from those obtained by the most common procedure in microbiology for enumeration of bacteria, known as colony forming unit (CFU) method.

Purification, Characterization and Mechanistic Evaluation of Angiotensin Converting Enzyme Inhibitory Peptides Derived from Zizyphus Jujuba Fruit.

The synthetic Angiotensin Converting Enzyme (ACE) inhibitors have side effects and hence demands for natural ACE inhibitors have been rising. The aim of this study is to purify and introduce natural ACE inhibitors extracted from Zizyphus jujuba fruits. Proteins from Zizyphus jujuba were lysed by trypsin, papain and their combination. Acquired peptides were purified and evaluated for ACE inhibitory activity. Peptide fractions with inhibitory activity were sequenced using tandem mass spectrometry. To elucidate the mode of peptide binding to ACE, homology modeling, molecular docking and molecular dynamics simulation were performed. Amino acid sequence of F2 and F4 peptides, which were the most active hydrolysates, were determined to be IER and IGK with the IC50 values of 0.060 and 0.072 mg/ml, respectively. Results obtained by computational analysis revealed that similar to the common ACE competitive inhibitors such as captopril, IER tripeptide binds to the enzyme active site, in vicinity of the zinc binding site, and occupies the S1 and S2' subsites. Binding occurs through hydrogen bonding with Gln293, Lys522, His524, Tyr531 and also several hydrophobic interactions. Collectively, these findings indicate that IER tripeptide inhibits the rabbit ACE enzyme through a competitive mechanism of inhibition with IC50 values in the millimolar range.

Evaluation of the Bioactive Potential of Secondary Metabolites Produced by a New Marine Micrococcus Species Isolated from the Persian Gulf.

BACKGROUND: In the present work, a newly isolated marine bacterium, Micrococcus sp. MP76, from coastal area of Persian Gulf around Bushehr province, Iran, was identified with the ability to produce bioactive compounds. METHODS: The pigment production was optimized by changing carbon and nitrogen sources in bacterial growth media at 28°C and 220 rpm for 5 days. Partial purification of the pigment was carried out using suitable solvents. RESULTS: Maximum pigment extract was achieved (1.4 g/l) when cultured in the medium containing 0.5% (v/v) molasses, 0.5% (w/v) peptone, 1% (w/v) sea salt, 0.01% (w/v) potassium phosphate, and 0.05% (w/v) yeast extract, pH=7.0. Antibacterial effect assessment of the extract against pathogenic bacteria revealed the MIC values in the range of 4.2-7.5 mg/ml depending on different pathogens. The pigment extracted from medium supplemented by molasses and ammonium sulfate had 81% radical scavenging activity, and its IC50 value was 0.28 mg/ml. CONCLUSION: The newly isolated strain of Micrococcus genus from the Persian Gulf revealed a valuable source to access worth medicinal ingredients when cultured under optimized conditions.

Doxorubicin-induced senescence through NF-κB affected by the age of mouse mesenchymal stem cells.

The senescence is proposed as a defense mechanism against many anticancer drugs. This complication is marked by differences in cell appearance and inner structures underlying the impairment in function. In this experiment, doxorubicin-induced senescence was assessed in mesenchymal stem cells (MSCs) isolated from the bone marrow of different-aged Balb/c mice (1, 8, and 16 months old). In addition, doxorubicin kinetics in culture medium were investigated to compare the drug absorption rate by different-aged MSCs. Several methods were exerted including Sandwich ELISA for NF-κB activation, propidium iodide staining for cell cycle analysis, Flow-fluorescent in-situ hybridization (Flow-FISH) assay for telomere length measurement, and specific staining for evaluation of ß-galactosidase. Determination of doxorubicin in a medium was performed by high-performance liquid chromatography technique. Following doxorubicin exposure, cells underwent substantial telomere shortening, cell cycle arresting in G2 phase, and increased ß-galactosidase activity. Interestingly, the enhanced level of NF-κB was observed in all age groups. The highest and lowest sensitivity to telomere shortening attributed to 1- and 8-month-old MSCs, respectively. In consistent with Flow-FISH results, the ß-galactosidase activity was higher in young-aged MSCs after treatment. Statistical analysis indicated a correlation between the reduction of telomere length and cessation in G2 phase. Regarding the obtained kinetics equations, the rate of doxorubicin absorption by all aged MSCs followed the same trend. In conclusion, the changing of some elements involved in doxorubicin-induced senescence can be affected by the age of the cells significantly in young MSCs than two other age groups. Hereupon, these changing patterns can open new insights to develop anticancer therapeutic strategies.

Induction of the Epithelial-to-Mesenchymal Transition of Human Colorectal Cancer by Human TNF-ß (Lymphotoxin) and its Reversal by Resveratrol.

OBJECTIVE: Tumor necrosis factor-beta (TNF-ß), as an inflammatory mediator that has been shown to promote tumorigenesis, induces NF-κB. Natural multi-targeted agent resveratrol in turn shows anti-inflammatory and anti-cancer properties. Epithelial-to-mesenchymal transition (EMT) allows cancer cells to turn into a motile state with invasive capacities and is associated with metastasis and development of cancer stem cells (CSC). However, TNF-ß-induced EMT and the anti-invasion mechanism of resveratrol on CRC are not yet completely understood. METHODS: We investigated the underlying molecular mechanisms of resveratrol on TNF-ß/TNF-ßR-induced EMT and migration of CRC cells (HCT116, RKO, SW480) in monolayer or 3D alginate cultures. RESULTS: TNF-ß, similar to TNF-α, induced significant cell proliferation, morphological change, from an epithelial to a spindle-like mesenchymal shape with the formation of filopodia and lamellipodia associated with the expression of EMT parameters (elevated vimentin and slug, reduced E-cadherin), increased migration/invasion, and formation of CSC in all CRC cells. Interestingly, these effects were dramatically decreased in the presence of resveratrol or anti-TNF-ßR with TNF-ß co-treatment, inducing biochemical changes to the mesenchymal-epithelial transition (MET), with a planar cell surface and suppressed formation of CSC cells. This was associated with a significant increase in apoptosis. Furthermore, we found that resveratrol suppressed TNF-ß-induced NF-κB and NF-κB-regulated gene biomarkers associated with growth, proliferation, and invasion. Finally, TNF-ßR interacts directly with focal adhesion kinase (FAK) and NF-κB. CONCLUSION: These results suggest that resveratrol down-regulates TNF-ß/TNF-ßR-induced EMT, at least in part via specific suppression of NF-κΒ and FAK in CRC cells.

Evidence that TNF-ß induces proliferation in colorectal cancer cells and resveratrol can down-modulate it.

IMPACT STATEMENT: The mechanism by which natural products such as resveratrol suppresses TNF-ß-promoted tumor cell proliferation, invasion, and colony formation is unknown. In this study, we explored for the first time the effect of resveratrol on the proinflammatory cytokine TNF-ß-, compared to TNF-α-stimulated proliferative and pro-inflammatory signaling in HCT116 cells. Our findings suggest that expression of TNF-ß and TNF-ß-receptor, like TNF-α, can lead to activation of inflammatory transcription factor (NF-κB) and NF-κB-regulated gene biomarkers, which are involved in the promotion of cancer proliferation, invasion, metastasis, and cell survival of tumor. Resveratrol can block TNF-ß/TNF-ß-receptor-induced activation of NF-κB, NF-κB-modulated gene products, and inhibition of caspase-3 cleavage. These results highlight the therapeutic effect of resveratrol-mediated anti-tumor activity by multitargeting cellular signaling pathways.