Line T24 Research Articles

General Direct Anticancer Effects of Deer Growing Antler Extract in Several Tumour Cell Lines, and Immune System-Mediated Effects in Xenograft Glioblastoma.

Deer antlers are the fastest growing tissue. Because they are based on proto-oncogenes, to avoid the risk of cancer, antlers evolved strong anticancer mechanisms, and thus their extract (DVA) is effective also against the few human tumours studied so far. We assessed whether DVA is a general anticancer compound by testing the direct effects in cells of different tumours: glioblastoma (GBM; lines U87MG and U251), colorectal (CRC; lines DLD-1, HT-29, SW480, and SW620), breast cancer (BRCA; lines MCF7, SKBR3, and PA00), and leukaemia (THP-1). DVA reduced the viability of tumours but not healthy cells (NHC; lines 293T and HaCaT). Mobility decreased at least for the longest test (72 h). Intraperitoneal/oral 200 mg DVA/kg administration in GBM xenograft mice for 28 d reduced tumour weight by 66.3% and 61.4% respectively, and it also reduced spleen weight (43.8%). In addition, tumours treated with DVA showed symptoms of liquefactive necrosis. Serum cytokines showed DVA up-regulated factors related to tumour fighting and down-regulated those related to inducing immune tolerance to the tumour. DVA shows general anticancer effects in the lines tested and, in GBM mice, also strong indirect effects apparently mediated by the immune system. DVA may contain a future anticancer medicine without secondary effects.

Comprehensive evaluation of morphology-property relationship in porous zirconia bioceramics at different levels of scale hierarchy

The success of replacement of damaged bone tissue by porous ceramic scaffolds is determined not only by biochemical compatibility of the artificial material, but also by a set of interrelated morphological and mechanical characteristics. But even if all parameters are optimized, the implant can be rejected on account of the low cell adhesion. Cell adhesion is the first step towards the survival of the implant material. Therefore, a challenging issue is still the search for mechanisms of controlling the behavior of cells on the surface of the implant. According to new ideas, the cellular response is influenced no only by the chemical composition and surface energy of the implant but also by the material structure at the micro- and nanoscale levels. To date, the only way to take into account structural features of a material is to determine the fractal dimension. The paper is devoted to the study of structure-property-adhesion relationships at different scale levels of ZrO2 and ZrO2-Al2O3 bioceramics with pore structures of different morphology. Physical regularities are revealed for the relationship between the geometry of pore-forming particles, crystal structure parameters, strength, and fracture toughness of the ceramics. The correlation between the fractal dimension as an integral morphological characteristic of the material and the cellular activity of the 3T3 line is evaluated for the first time. This can be a universal method for assessing the relationship between the structure and osteoreplacement efficiency of ceramics.

Pyrrolidinedione-thiazolidinone hybrid molecules with potent cytotoxic effect in squamous cell carcinoma SCC-15 cells

The hybrid heterocyclic molecules are perspective materials in the development of anticancer drugs. Here, the pyrrolidinedione-thiazolidinone hybrid molecules were designed as potent anticancer agents. This study aimed to investigate the cytotoxic effect of three derivatives 1-(4-hydroxyphenyl)-, 1-(4-chlorophenyl)- and 1-(4-bromophenyl)-3-[5-[2-chloro-3-(4-nitrophenyl)prop-2-enylidene]-4-oxo-2-thioxothiazolidine-3-yl]pyrrolidine-2,5-diones (Les-6287, Les-6294, and Les-6328, respectively), their effect on the production of the reactive oxygen species (ROS), apoptosis induction, and expression of genes - PPARγ, AHR, and NRFL2 – whose products are important in metabolism in human tongue squamous cell carcinoma cells of SCC-15 line. The results of resazurin reduction and lactate dehydrogenase (LDH) release assays proved the toxicity of the tested derivatives for the SCC-15 cells. Les-6287, Les-6294, and Les-6328 inhibited the viability of SCC-15 cells with the half-maximal effective concentration (EC50) in the range of 10.18–32.75 µM at 24 and 48 h treatment. These derivatives reduced the metabolism of SCC-15 cells with the half-maximal inhibitory concentration (IC50) of 6.72–39.85 µM at 24 and 48 h treatment. Les-6287, Les-6294, and Les-6328 reduced the metabolism of normal human keratinocytes of HaCaT line murine fibroblasts of Balb/c 3T3 line to a lesser extent. The compounds used in a range from 50 to 100 µM concentrations decreased ROS production in the SCC-15 cells. The derivatives Les-6287 and Les-6328 decreased the level of expression of mRNA of PPARγ, AHR, and NRFL2 genes in these cells at PPARγ siRNA knockdown and without it. Thus, the anticancer effect of studied hybrid pyrrolidinedione-thiazolidinones in the SCC-15 carcinoma cells is accompanied by a reduction of their metabolic activity and ROS level, and increase in caspase 3 activity. However, these changes are not the result of direct interaction of Les-6287, Les-6294, and Les-6328 with the PPARγ molecule.

Comparison of NIH 3T3 Cellular Adhesion on Fibrous Scaffolds Constructed from Natural and Synthetic Polymers.

Polymer scaffolds are increasingly ubiquitous in the field of tissue engineering in improving the repair and regeneration of damaged tissue. Natural polymers exhibit better cellular adhesion and proliferation than biodegradable synthetics but exhibit inferior mechanical properties, among other disadvantages. Synthetic polymers are highly tunable but lack key binding motifs that are present in natural polymers. Using collagen and poly(lactic acid) (PLA) as models for natural and synthetic polymers, respectively, an evaluation of the cellular response of embryonic mouse fibroblasts (NIH 3T3 line) to the different polymer types was conducted. The samples were analyzed using LIVE/DEAD™, alamarBlue™, and phalloidin staining to compare cell proliferation on, interaction with, and adhesion to the scaffolds. The results indicated that NIH3T3 cells prefer collagen-based scaffolds. PLA samples had adhesion at the initial seeding but failed to sustain long-term adhesion, indicating an unsuitable microenvironment. Structural differences between collagen and PLA are responsible for this difference. Incorporating cellular binding mechanisms (i.e., peptide motifs) utilized by natural polymers into biodegradable synthetics offers a promising direction for biomaterials to become biomimetic by combining the advantages of synthetic and natural polymers while minimizing their disadvantages.

Resorbable Nanomatrices from Microbial Polyhydroxyalkanoates: Design Strategy and Characterization.

From a series of biodegradable natural polymers of polyhydroxyalkanoates (PHAs)-poly-3-hydroxybutyrate (P(3HB) and copolymers containing, in addition to 3HB monomers, monomers of 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), and 4-hydroxybutyrate (4HB), with different ratios of monomers poured-solvent casting films and nanomembranes with oriented and non-oriented ultrathin fibers were obtained by electrostatic molding. With the use of SEM, AFM, and measurement of contact angles and energy characteristics, the surface properties and mechanical and biological properties of the polymer products were studied depending on the method of production and the composition of PHAs. It has been shown in cultures of mouse fibroblasts of the NIH 3T3 line and diploid human embryonic cells of the M22 line that elastic films and nanomembranes composed of P(3HB-co-4HB) copolymers have high biocompatibility and provide adhesion, proliferation and preservation of the high physiological activity of cells for up to 7 days. Polymer films, namely oriented and non-oriented nanomembranes coated with type 1 collagen, are positively evaluated as experimental wound dressings in experiments on laboratory animals with model and surgical skin lesions. The results of planimetric measurements of the dynamics of wound healing and analysis of histological sections showed the regeneration of model skin defects in groups of animals using experimental wound dressings from P(3HB-co-4HB) of all types, but most actively when using non-oriented nanomembranes obtained by electrospinning. The study highlights the importance of nonwoven nanomembranes obtained by electrospinning from degradable low-crystalline copolymers P(3HB-co-4HB) in the effectiveness of the skin wound healing process.

Synthesis of the novel cage amides and imides and evaluation of their antibacterial and antifungal activity

Cage amides and imides bearing bicyclo[2.2.1]- and bicyclo[2.2.2]-subunits were synthesized and evaluated both for antimicrobial activity toward five key ESKAPE pathogenic bacteria: one Gram‐positive bacteria methicillin‐resistant Staphylococcus aureus (ATCC 43300), four Gram‐negative bacteria Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Acinetobacter baumannii (ATCC 19606) and Pseudomonas aeruginosa (ATCC 27853) and for antifungal activity towards pathogenic fungal strains Candida albicans (ATCC 90028) and Cryptococcus neoformans var. Grubii (H99; ATCC 208821). Compound VP-4539 with bicyclo[2.2.2]octene motif demonstrated the highest cytotoxic activity towards C. neoformans, while human keratinocytes of HaCaT line, murine fibroblasts of Balb/c 3T3 line and mitogen-activated lymphocytes of peripheral human blood were found to be tolerant to its action. VP-4539 compound did not intercalate into salmon sperm DNA indicating that its cytotoxicity is not related to intercalation into nucleic acid. Keywords: antifungal, antimicrobial, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octene, cytotoxicity, DNA intercalation, human keratinocytes, lymphocytes, сage compounds

English

Initial myeloid and lymphoid differentiation of hematopoietic and non-hematopoietic stem/progenitor cells by appropriate ex vivo- and in vitro-incubation was evaluated. So, hematopoietic stem/progenitor cells from mouse spleen were ex vivo-incubated in the presence of different interleukins, as well as of various combinations of them. Non-hematopoietic mouse embryonic stem cells (mESCs) were in vitro-incubated in the presence of GM-CSF and malignant antigens of human cervical carcinoma cells HeLa. Analogically, mouse embryonic fibroblasts from line 3T3 were pre-incubated in the presence of malignant antigens from mouse myeloma cells. Possibilities for derivation of myeloid and lymphoid cells by hematopoietic and non-hematopoietic cellular progenitors were proposed. The literature ability for production of immune molecules, including membrane glycoprotein receptors and antibodies/immunoglobulins by initial myeloid and lymphoid cells, derived from hematopoietic, as well as by non-hematopoietic cellular progenitors was accepted when appropriate factors are available. Because the produced antibodies are out of the germinative centers of the specialized lymphoid tissues and organs, control of their function is very important, for escape of chronic inflammatory processes. Realistically, this control proved the role of small ions and molecules, by direct and/or indirect influence on different intra- and extra-cellular inter-molecular interactions in various cascade regulatory pathways. Key words: Hematopoietic and non-hematopoietic stem/progenitor cells, cellular differentiation, immune molecules, incubation conditions.

Synthesis of novel indole-thiazolidinone hybrid structures as promising scaffold with anticancer potential

A series of novel indole-azolidinone hybrids has been synthesized via Knoevenagel reaction of 5-fluoro-3-formyl-1H-indole-2-carboxylic acid methyl ester and some azolidinones differing in heteroatoms in positions 1, 2 and 4. Their anticancer activity in vitro was screened towards MCF-7 (breast cancer), HCT116 (colon cancer), HepG2 (hepatoma), HeLa (cervical cancer), A549 (lung cancer), WM793 (melanoma) and THP-1 (leukemia) cell lines, and a highly active 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester (3a) was identified and subjected to in-depth investigation of cytotoxicity mechanisms. This compound was found to possess the highest cytotoxic action towards tumor cells comparing with the action of other derivatives (1, 3b, 3c, 3d, 3e). Compound 3a exhibited toxicity toward MCF-7, HCT116, and A549, HepG2 cancer cells, while the non-malignant cells (human keratinocytes of HaCaT line and murine embryonic fibroblasts of Balb/c 3T3 line) possessed moderate sensitivity to it. The compound 3a induced apoptosis in studied tumor cells via caspase 3-, PARP1-, and Bax-dependent mechanisms; however, it did not affect the G1/S transition in HepG2 cells. The compound 3a impaired nuclear DNA in HepG2, HCT116, and MCF-7 cells without intercalating this biomolecule, but much less DNA damage events were induced by 3a in normal Balb/c 3T3 fibroblasts compared with HepG2 carcinoma cells. Thus, 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester 3a was shown to trigger DNA damage and induce apoptosis of human tumor cells and it might be considered as an anticancer agent perspective for in-depth studies.

Abstract 2495: SPARC: a bladder cancer tumor-suppressor via targeting metabolic programming

Abstract Bladder cancer (BC) is the sixth most common cancer in the United States. Approximately, 75% of BC patients present with non-muscle invasive bladder cancer (NMIBC), which is responsive to local treatment and surveillance. However, recurrence is common with progression to muscle-invasive bladder cancer (MIBC). Forty percent of MIBC patients develop distant metastases, with few treatment options and poor survival. Limited advances have been made in BC therapy with only a few FDA-approved new treatments for advanced disease since 2016 with response rates hovering around 15%. Thus, there is an unmet need for better understanding of the pathobiology of the disease to develop more effective therapeutics. Metabolic re-programming of cancer is increasingly recognized as a hallmark of cancer as cancer cells take advantage of metabolic products to meet the increasing demands of proliferating cells. Our lab has identified the Secreted Protein Acidic Rich in Cysteine (SPARC) as a multi-faceted tumor suppressor within the BC tumor microenvironment. Herein, we sought to elucidate the role of SPARC in regulating metabolic programming in BC and identify pathways that may serve as diagnostic and therapeutic targets. We used three BC cell lines UMUC3, T24 (SPARChigh) and its isogenic SPARClow T24T. We treated BC cells with recombinant SPARC protein and genetically manipulated SPARC expression in these cells. We performed real-time monitoring of the effect of SPARC on bioenergetics using Seahorse assays. In tandem, we profiled transcriptomes of T24, T24T, and T24shSP-cell lines to identify aggressive phenotype signatures using GSEA. We then compared signatures with survival data from BC cohorts from the GEO and TCGA. We found that SPARC inhibited ATP production, basal and maximal respiration, and spare respiratory capacity in BC cell lines. This effect is more pronounced in more aggressive SPARClow T24T cells compared to its isogenic SPARChigh T24 cells. Knockdown of SPARC in BC cell lines not only induced an aggressive phenotype, but also increased ATP production and mitochondrial respiration. Integrated transcriptomic profiling indicated that SPARC-loss is associated with enriched glycolytic, lipidomic, and mitochondrial metabolic pathways. Importantly, in patient samples, SPARC expression negatively correlated with rate limiting enzymes in metabolic pathways associated with lower survival. SPARC loss also associated with oncogenic signatures of EMT, PI3K-AKT-mTOR signaling, and G1/S cell cycle arrest. Our data reveals a novel function of SPARC inhibiting mitochondrial bioenergetics and ATP production that fuel growth and invasion in BC cells. The SPARC-regulated metabolic profile highlights potential vulnerabilities that can be exploited as therapeutic targets. Citation Format: Sameh Waleed Almousa, Alia Ghoneum, Hesham Afify, Neveen Said. SPARC: a bladder cancer tumor-suppressor via targeting metabolic programming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2495.

Chemical Modification of Films from Biosynthetic Poly‐3‐Hydroxybutyrate Aimed to Improvement of Their Surface Properties

AbstractFilms from biodegradable poly‐3‐hydroxybutyrate are treated with chemical reagents to improve their hydrophilicity and biocompatibility. Two approaches are tested: a single treatment with alkali, acids, oxidizing or reducing agents, and a step‐by step treatment of the alkali pre‐activated surface of polymer films with bromine water and amino‐compounds (ammonia or triethylamine). The maximal level of hydrophilicity (the lowest water contact angle and the highest polar component of the surface free energy) is registered after a single treatment with NaOH and after the step‐by‐step treatment. These samples also showed the best adhesion of mouse fibroblasts of NIH 3T3 line on the film surface. So, the proposed methods can be used to enhance hydropilicity and biocompatibility of biopolymer surface.

Dynamics of 5-Aminolevulinic Acid-Induced Accumulation of Protoporphyrin IX in Three Cell Lines of Different Origin

The dynamics of synthesis and accumulation of protoporphyrin IX induced by 5-aminolevulinic acid (5-ALA) was studied using confocal fluorescence microscopy in three established cell lines: immortalized cells originated from human tumor tissues—epithelial carcinoma (HeLa cells) and lung adenocarcinoma (A549 cells)—and immortalized fibroblast-like mouse embryo cells (3T3 line). The accumulation of protoporphyrin IX in cell cultures was estimated by analyzing the intensity of its fluorescence. The dynamics of changes in the fluorescence intensity with the cell incubation duration in the presence of 5-ALA and with the 5-ALA concentration was studied.

Experimental in vitro Comparative Study of Chromovitectomy Agents Cyto- and Phototoxicity

Intravitreal use of vital dyes in combination with the action of endoillumination can induce a cyto- and phototoxic effect on posterior eye segment structures. The search for a staining agent with a maximum safety profile to retinal structures, intensively and selectively coloring vitreous body and vitreoretinal interface structure, remains relevant.Objective: to determine comparative viability of NIH / 3T3 mouse fibroblast cell culture with traditional agents for chromovitrectomy and “Vitreocontrast” suspension with and without endovitreal illumination.Materials and methods. NIH / 3T3 mouse fibroblast cultures contacted with agents for chromovitrectomy (MembraneBlue® Dual, Triamcinolone acetonide, “Vitreocontrast” suspension) and the corresponding controls in a volume of 50 μl / well. The test plate was irradiated with a Photon II illuminator (Synergetics, USA), working distance of 5 mm. The control tablet with the introduced preparations was not exposed to light. Next, the cells were washed and incubated, after which the morphology and lysis of the cells, as well as the number of proliferating relatively negative control of fibroblasts, were evaluated using the vital dye PrestoBlue Cell Viability Reagent. Negative control was the complete growth medium for the cultivation of mouse fibroblasts of the NIH / 3T3 line. The results of the cytotoxic reaction of a culture of mouse fibroblasts of the NIH / 3T3 line were interpreted using the table “The degree of cell response”.Results. Studies have shown that exposure to a source of endovitual illumination does not affect the cytotoxic effect of TA suspension and MembraneBlue® Dual dye. The TA suspension, both after light source and without it, has a moderate cytotoxic effect, and MembraneBlue® Dual has no cytotoxic effect on the culture of mouse fibroblasts of the NIH / 3T3 strain. Without light, “Vitreocontrast” suspension does not have cytotoxic effect on mouse fibroblasts culture NIH / 3T3 line. Light irradiation for 1 h increases the cytotoxicity of “Vitreocontrast” suspension to the level of unsharp cytotoxicity allowed by ISO Standard 10993-5-2011.Conclusion. The safety profile of MembraneBlue® Dual and “Vitreocontrast” suspension allows them to be recommended for use in endovitreal surgery. The cyto- and phototoxicity demonstrated in the experiment with TA suspension can reduce the functional outcomes of retinal surgery.

Establishing Correlations between Breast Tumor Response to Radio-Immunotherapy and Radiomics from Multi-Parametric Imaging: An Animal Study

Triple-negative breast cancer (TNBC), which is a type of invasive breast cancer, is characterized by severe disease progression, poor prognosis, high recurrence rate, and short survival. We sought to gain new insight into TNBC by applying computed tomography (CT) and magnetic resonance (MR) quantitative imaging (radiomics) approaches to predict the outcome of radio-immunotherapy treatments in a syngeneic subcutaneous murine breast tumor model. Five Athymic Nude mice were implanted with breast cancer cell lines (4T1) tumors on the right flank. The animals were CT- and MRI-imaged, tumors were contoured, and radiomics features were extracted. All animals were treated with radiotherapy (RT), followed by the administration of PD1 inhibitor. Approximately 10 days later, the animals were sacrificed, tumor volumes were measured, and histopathology evaluation was performed through Ki-67 staining. Linear regression modeling between radiomics and Ki-67 results was performed to establish a correlation between quantitative imaging and post-treatment histochemistry. There was no correlation between tumor volumes and Ki-67 values. Multiple CT- and MRI-derived features, however, correlated with histopathology with correlation coefficients greater than 0.8. MRI imaging helps in tumor delineation as well as an additional orthogonal imaging modality for quantitative imaging purposes. This is the first investigation correlating simultaneously CT- and MRI-derived radiomics to histopathology outcomes of combined radio-immunotherapy treatments in a preclinical setting applied to treatment naïve tumors. The findings indicate that imaging can guide discrimination between responding and non-responding tumors for the combined RT and ImT treatment regimen in TNBC.

Abstract A16: A novel death receptor ligand fabclavine inhibits cancer and cancer stem cell proliferation by extrinsic apoptosis

Abstract Many antibiotics produced by bacteria also have antineoplastic activity. Examples of such bacterial compounds include actinomycin D, doxorubicin, mitoxantrone, bleomycin, mitomycin, etoposide, and others. Recently, it was identified that Xenorhabdus budapestnesis (Xbu) produces a secondary metabolite, fabclavine, that has antibiotic property. Here, for the first time we demonstrate that fabclavine exhibits potent cytotoxic activity against ovarian cancer cells and ovarian cancer stem cells and demonstrate its mechanism of action, Fabclavine was isolated and purified from cell-free supernatants of Xbu cultures and analyzed by mass spectrometry. Proliferation assays conducted on several ovarian cancer cell lines Ovcar3, OVCA433, ID8, and breast cancer cell lines MCF7 and 4T1 demonstrate that fabclavine inhibits the cancer cell proliferation at nanomolar (250-400nM) concentration. Fabclavine is ten times more potent than cisplatin in inhibiting cell proliferation of ovarian cancer cell line OVCA33 and breast cancer cell line MCF7. We also tested the effect of fabclavine in 3D culture of ovarian cancer cell lines. The results indicate, even in 3D culture, fabclavine killed 40-50% of the cells at 300nM concentration. In addition, fabclavine was also tested against ovarian cancer stem cells isolated from five ovarian cancer patients and grown in 3D culture. Fabclavine inhibited the growth of stem cells by 70-85%. In cancer cell lines fabclavine induced apoptosis in 20-30% cells after 24-hour treatment. Caspase activity assay for caspase3, caspase8, and caspase9 shows a significant increase in caspase3 and caspse8 activity with no change in caspase9 activity, suggesting an activation of the extrinsic apoptosis pathway in the fabclavine-treated cells. This observation was supported by the increase in phospho-FADD upon exposure to fabclavine. When conjugated to agarose beads, fabclavine efficiently precipitated two death receptors, DR4 and DR5, from ovarian and breast cancer cell extracts. These experiments indicate that fabclavine is a novel DR4/5 ligand and should therefore be considered as a death receptor-targeting agent for the treatment of ovarian and other tumors. We will present data on the comparison of DR4/5 binding to fabclavine versus its natural ligand, TRAIL, and will also present data from PDex and mouse xenograft models on the ex vivo and in vivo effects of fabclavine on the proliferation of ovarian tumors and cancer stem cells. Citation Format: Arvinder Kapur, Mayur Kajla, Susan Paskewitz, Allegra Cappuccini, Pooja Mehta, Geeta Mehta, Manish Patankar. A novel death receptor ligand fabclavine inhibits cancer and cancer stem cell proliferation by extrinsic apoptosis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A16.

Electric Fields at Breast Cancer and Cancer Cell Collective Galvanotaxis

Cancer growth interferes with local ionic environments, membrane potentials, and transepithelial potentials, resulting in small electrical changes in the tumor microenvironment. Electrical fields (EFs) have significant effects on cancer cell migration (galvanotaxis/electrotaxis), however, their role as a regulator of cancer progression and metastasis is poorly understood. Here, we employed unique probe systems to characterize the electrical properties of cancer cells and their migratory ability under an EF. Subcutaneous tumors were established from a triple-negative murine breast cancer cell line (4T1), electric currents and potentials of tumors were measured using vibrating probe and glass microelectrodes, respectively. Steady outward and inward currents could be detected at different positions on the tumor surface and magnitudes of the electric currents on the tumor surface strongly correlated with tumor weights. Potential measurements also showed the non-homogeneous intratumor electric potentials. Cancer cell migration was then surveyed in the presence of EFs in vitro. Parental 4T1 cells and metastatic sublines in isolation showed random migration in EFs of physiological strength, whereas cells in monolayer migrated collectively to the anode. Our data contribute to an improved understanding of breast cancer metastasis, providing new evidence in support of an electrical mechanism that promotes this phenomenon.

Intracellular delivery of drugs by chitosan-based multi-liposomal complexes

Positively charged linear chitosan molecules were cross-linked with sulfate-anions to form chitosan nanoparticles which were used as a scaffold of negatively charged cardiolipin/egg lecithin liposomes loaded with doxorubicin (DOX). Thus formed multi-liposomal complexes (MLCs) containing 55 liposomes/chitosan and bearing a slight positive net charge effectively transmitted DOX into the cytoplasm of cells in culture. The efficiency of DOX delivery increased 4–5-fold upon drug incorporation in MLCs. Inside the cells, the penetrated MLCs released DOX thus enabling its accumulation in the nuclei and interaction with its intracellular target DNA leading to a decrease in cell survival. The effects were demonstrated on 3T3 line of mouse fibroblasts, drug sensitive tumor MCF-7 cells and drug resistant human ovarian carcinoma OVCAR-8 cells (formerly called MCF-7/ADR). Thus, MLC particles can be used for effective delivery of payloads in cells of different origin.

Erdafitinib exerts the anticancer effect on urothelial carcinoma via induction of authophagy.

Erdafitinib has recently been approved for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma (UC). The current study aimed to evaluate whether erdafitinib inhibits the proliferation in UC via inducing authophagy. Our data showed that erdafitinib demonstrated strong toxicity for the T24 and UMUC6 cell lines. Flow cytometry analysis showed that erdafitinib increased the proportion of G0/G1 phase in both T24 and UMUC6 cells. Additionally, Annexin V staining indicated that erdafitinib enhanced the apoptosis of UC cells. Meanwhile, the expression of apoptosis-related proteins was significantly elevated after treatment with erdafitinib. Transwell assay showed that erdafitinib significantly reduced T24 and UMUC6 cell migration and invasion. More importantly, western blot assay showed that erdafitinib induced the expression of autophagy-related proteins. Besides, GFP-LC3 transfection assay and electron microscopy examination showed that erdafitinib could partially diminish 3-methyladenine (3-MA) induced impairment of autophagy in UC cells. In summary, for the first time we showed novel data that erdafitinib inhibited UC cell malignant proliferation via inducing cell autophagy.

Development of Techniques about Production of Recombinant vaccines and cells with Activated Immunogenic potential

Methods about derivation of myeloid-like and lymphoid-like cells from normal embryonic fibroblasts were developed and tested. Balb/c mice normal mouse embryonic cells from line 3T3 were co-cultivated with mouse malignant myeloma cells, containing inserted. Murine Leukemia Virus (MuLV) (with RNA-genome, Retroviridae family). For this goal, separate subpopulation of normal mouse embryonic fibroblasts were pre-incubated in cultural fluid, supplemented by previous incubation of myeloma cells in it, after subsequent centrifugation and filtration. Other sub-populations of 3T3 cells were co-cultivated with myeloma cells, by addition of cultural fluids plus cellular suspensions of both types. Subsequently, all mixed cultures were freezed after addition of cryo-protector Dimethylsufoxide (DMSO), subsequently thawed and re-incubated in standard laboratory conditions. In the cultures, pre-incubated in cultural fluid, supplemented by previous incubation of mouse malignant myeloma cells, cells in different stages of myeloid/phagocyte and lymphoid/plasmatic cell differentiation were observed, but in addition of suspensions of cells from both types, appearance of hybrid cells of myeloid-like and phagocyte-like, as well as of lymphoid-like and plasmatic cells-like with mouse malignant myeloma cells were noted. The established changes could be explained with the eventual existence of capable to differentiate to various lineages stem-like cellular sub-populations in the general 3T3 cell line. Also, activated fusion between different cells, as well as between cells and viral particles on the influence of DMSO and of the drastic temperature changes was proposed, which could also lead to transfer of nucleotide sequences. On this principle, a possibility for production of recombinant viral vaccines by exchange of nucleotide sequences between cells and viral particles could be suggested. Furthermore, in confirmation of the literature findings, a capability of non-myeloid and non-lymphoid cells to produce membrane receptor glycoproteins was proposed.

EL4-derived Exosomes Carry Functional TNF-related Apoptosis-inducing Ligand that are Able to Induce Apoptosis and Necrosis in the Target Cells.

Exosomes released by tumor cells play critical roles in tumor progression, immune cell suppression, and cancer metastasis. The aim of the present study was to investigate whether the exosomes released by EL4 cells carry a functional TNF-related apoptosis-inducing ligand (TRAIL) molecule. Exosomes were harvested from the supernatants of EL4 cell culture, and the shape, size, and identity of EL4-derived exosomes were evaluated by utilizing scanning electron microscopy, dynamic light scattering, and dot-blot method. The expression of mRNA and TRAIL protein in EL4 cells and EL4-exosomes were investigated using real-time PCR method and dot-blot analysis. Moreover, the effects of EL4-derived exosomes on cell death in a TRAIL-sensitive cell line (4T1) were studied by using flow cytometry (annexin V/propidium iodide (PI) staining) and fluorescent microscopy analyses (acridine orange/ethidium bromide staining). The results showed that EL4 cells continuously and without the need for stimulation, produce exosomes that carry TRAIL protein. In addition, EL4-derived exosomes were capable to induce apoptosis as well as necrosis in 4T1 cells. It was ultimately revealed that EL4 cells express TRAIL protein and release exosomes containing functional TRAIL. Moreover, the released exosomes were able to induce apoptosis and necrosis in a TRAIL-sensitive cell line. Further studies are needed to reveal the potential roles of tumor-derived exosomes in the pathogenesis of cancers.

Chemo-treated 4T1 breast cancer cells radiation response measured by single and multiple cell ionization using infrared laser trap

We present a study that uses a laser trapping technique for measurement of radiation sensitivity of untreated and chemo-treated cancer cells. We used a human mammary tumor cell line (4T1) treated by an antitumor compound, 2-Dodecyl-6-methoxycyclohexa-2, 5-diene-1,4-dione (DMDD), which was extracted from the root of Averrhoa carambola L. The untreated control group, and both 2-hour and 24-hour treated groups of 4T1 cells were used in this study. The absorbed threshold ionization energy (TIE) and the threshold radiation dose (TRD) were determined using a high-power infrared laser (at 1064 nm) trap by single and multiple cells trapping and ionization. The results were analyzed using descriptive and t-statistics. The relation of the TIE and TRD to the mass of the individual cells were also analyzed for different hours of treatment in comparison with the control group. Both TIE and TRD decrease with increasing treatment periods. However, the TRD decreases with mass regardless of the treatment. Analyses of the TRD for single vs multiple cells ionizations within each group have also consistently showed this same behavior regardless of the treatment. The underlying factors for these observed relations are explained in terms of radiation, hyperthermia, and chemo effects.