BrdU Test Research Articles

Heterogeneous Cellular Response of Primary and Metastatic Human Gastric Adenocarcinoma Cell Lines to Magnoflorine and Its Additive Interaction with Docetaxel

Gastric cancer is the most common cancer and remains the leading cause of cancer death worldwide. In this study, the anticancer action of magnoflorine isolated via counter-current chromatography from the methanolic extract of Berberis vulgaris root against gastric cancer in models of primary ACC-201 and AGS and metastatic MKN-74 and NCI-N87 cell lines was analyzed. Cell viability and proliferation were tested through the use of MTT and BrdU tests, respectively. Cell cycle progression and apoptosis were evaluated using flow cytometry. The interaction of magnoflorine and docetaxel has been examined through isobolographic analysis. Moreover, potential toxicity was verified in zebrafish in an in vivo model. Gastric cancer cell lines revealed different responses to magnoflorine treatment with regard to viability/proliferation, apoptosis induction and cell cycle inhibition without any undesirable changes in the development of larval zebrafish at the tested concentrations. What is more, magnoflorine in combination with docetaxel produced an additive pharmacological interaction in all studied gastric cancer cell lines, which may suggest a complementary mechanism of action of both compounds. Taken together, these findings provide a foundation for the possibility of magnoflorine as a potential therapeutic approach for gastric cancer and merits further investigation, which may pave the way for clinical uses of magnoflorine.

Polyhydroxylated Fullerene C60(OH)40 Nanofilms Promote the Mesenchymal-Epithelial Transition of Human Liver Cancer Cells via the TGF-β1/Smad Pathway.

The various growth factors change the phenotype of neoplastic cells from sedentary (epithelial) to invasive (mesenchymal), which weaken intercellular connections and promote chemotaxis. It can be assumed that the use of anti-inflammatory polyhydroxyfull nanofilms will restore the sedentary phenotype of neoplastic cells in the primary site of the tumor and, consequently, increase the effectiveness of the therapy. The studies were carried out on liver cancer cells HepG2, C3A and SNU-449, and non-cancer hepatic cell line THLE-3. Transforming growth factor (TGF), epidermal growth factor and tumor necrosis factor were used to induce the epithelial-mesenchymal transition. C60(OH)40 nanofilm was used to induce the mesenchymal-epithelial transition. Obtaining an invasive phenotype was confirmed on the basis of changes in the morphology using inverted light microscopy. RT-PCR was used to confirm mesenchymal or epithelial phenotype based on e-cadherin, snail, vimentin expression or others. Water colloids at a concentration of 100 mg/L were used to create nanofilms of fullerene, fullerenol, diamond and graphene oxide. The ELISA test for the determination of TGF expression and growth factor antibody array were used to select the most anti-inflammatory carbon nanofilm. Mitochondrial activity and proliferation of cells were measured by XTT and BrdU tests. Cells lost their natural morphology of cells growing in clusters and resembled fibroblast cells after adding a cocktail of factors. Among the four allotropic forms of carbon tested, only the C60(OH)40 nanofilm inhibited the secretion of TGF in all the cell lines used and inhibited the secretion of other factors, including insulin-like growth factor system. Nanofilm C60(OH)40 was non-toxic to liver cells and inhibited the TGF-β1/Smad pathway of invasive cells treated with the growth factor cocktail. The introduction of an anti-inflammatory, nontoxic component that can induce the mesenchymal-epithelial transition of cancer cells may represent a future adjuvant therapy after tumor resection.

Syntheses, characterization of Imidazo[4,5-f][1,10]phenanthroline derivative and As (III) complex, In vitro evaluation, the determine of apoptosis mechanism; theoretical and quantum studies

In the present work, a novel ligand, (4-amino-3-methoxybenzaldehyde)-1H-imidazo[4,5-f][1,10] phenanthroline (AMIP) based on the formation of an imidazole ring and [As(AMIP)](Cl)]Cl2 was synthesized and characterized through Fourier-transform infrared (FT-IR) spectroscopy. In addition, the anticancer activity of [As(AMIP)](Cl)]Cl2 complex was tested against human acute promyelocytic leukemia cells (HL-60) and granulocytic differentiation of human promyelocytic leukemia cells (NB4). The MTT results showed IC50 values of 7.31 ± 15 and 8.4 ± 62 μM for As (III) complex against NB4 and HL-60, respectively. In a study on both cell lines, As (III) reduced gene expression Bcl-2 and Bcl-xl, increased Bax and Bad, and activated caspase-3, resulting in apoptosis. The dye Hoechst 33342 showed increased apoptosis induction in the NB4 cell line than in the HL-60 cell line. BrdU test exhibited an inhibitory effect on DNA synthesis. The protein levels of caspase-3 were enhanced in a dose-dependent manner in both HL-60 and NB-4 cell lines. Based on the Annexin V-FITC/PI assay, apoptosis was the primary mechanism of cell death. The real-time polymerase chain reaction (real-time PCR) results demonstrated that the expression level of Atg 12, Atg 7, Lc 3, and Beclin-1 genes, antiapoptotic genes including Bcl-2 and Bcl-xl, and apoptotic genes containing BAD and BAX was improved. Furthermore, the hemolysis assay showed the anti-hemolytic activity of the complex. Moreover, quantum chemical calculations were utilized to optimize all structures, and the output results were employed to study molecular docking of compounds with caspase-3 tethered to irreversible inhibitor (PDB ID: 1NMS) and DNA with sequence d(CCGTCGACGG) (PDB ID:423D). The results showed that As (III) complex had the lowest-energy conformation, suggesting the optimal interaction.

Cannabidiol Interacts Antagonistically with Cisplatin and Additively with Mitoxantrone in Various Melanoma Cell Lines-An Isobolographic Analysis.

The medical application of cannabidiol (CBD) has been gathering increasing attention in recent years. This non-psychotropic cannabis-derived compound possesses antiepileptic, antipsychotic, anti-inflammatory and anxiolytic properties. Recent studies report that it also exerts antineoplastic effects in multiple types of cancers, including melanoma. In this in vitro study we tried to reveal the anticancer properties of CBD in malignant melanoma cell lines (SK-MEL 28, A375, FM55P and FM55M2) administered alone, as well as in combination with mitoxantrone (MTX) or cisplatin (CDDP). The effects of CBD on the viability of melanoma cells were measured by the MTT assay; cytotoxicity was determined in the LDH test and proliferation in the BrdU test. Moreover, the safety of CBD was tested in human keratinocytes (HaCaT) in LDH and MTT tests. Results indicate that CBD reduces the viability and proliferation of melanoma-malignant cells and exerts additive interactions with MTX. Unfortunately, CBD produced antagonistic interaction when combined with CDDP. CBD does not cause significant cytotoxicity in HaCaT cell line. In conclusion, CBD may be considered as a part of melanoma multi-drug therapy when combined with MTX. A special attention should be paid to the combination of CBD with CDDP due to the antagonistic interaction observed in the studied malignant melanoma cell lines.

In vitro ve in silico analizi ile metforminin meme tümörü hücrelerinde protein profili üzerindeki etkinliği

Aim: This study aimed to uncover the varieties in protein profiles of Met in breast tumor (BT) cells by assessment of in vitro and in silico analysis. Materials and Methods: Here, the cells obtained from mastectomy patients were cultured, the effective Met-dose was determined as 25 mM through cell viability and BrdU tests. Protein identification in the breast tumor cells was implemented by employing LC-MS/MS technology. Results: The expression of SSR3, THAP3, FTH1, NEFM, ANP32A, ANP32B, KRT7 proteins was significantly decreased whereas the GARS protein increased in the 25 mM Met group compared to the Non-Met (0 mM) control group. In silico analysis, we analyzed the probable interactions of all these proteins with each other and other proteins, to evaluate the analysis of the larger protein network, and which metabolic pathway proteins are involved in. Conclusion: The stated proteomics analysis in our study proposes a better understanding of the prognosis of breast cancer and future studies to investigate the effect of metformin in this field on proteomic pathways in other sorts of cancer.

Circ_0000228 Promotes Cervical Cancer Progression via Regulating miR-337-3p /TGFBR1 Axis.

Objective This study aims to investigate the biological function of circular RNA (circRNA) circ_0000228 in the cervical cancer (CC). Materials and Methods In this experimental study, the GSE113696 dataset was downloaded from the Gene Expression Omnibus (GEO). GEO2R was employed to obtain differentially expressed circRNA between CC tissues and matched paracancerous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were employed to detect circ_0000228, microRNA-337-3p (miR-337-3p) and transforming growth factor, beta receptor I (TGFBR1) expression levels in the CC tissues and cells. Following gain-of-function and loss-of-function models establishment, CCK-8 and BrdU tests were conducted to examine cell proliferation. Transwell experiment was executed to examine CC cells migration and invasion. A lung metastasis model was utilized to determine the ability of circ_0000228 on the lung metastasis. Bioinformatics analysis, dual-luciferase reporter experiment and RNA immunoprecipitation (RIP) assay were applied to verify the targeting relationship among miR-337-3p, circ_0000228, and TGFBR1. Results Circ_0000228 expression in the CC tissues and cells was up-modulated. Circ_0000228 overexpression markedly enhanced cell proliferation, migration, and invasion, while knocking down circ_0000228 remarkably repressed cell proliferation, migration, and invasion. MiR-337-3p could be adsorbed by circ_0000228. TGFBR1 was identified as a target gene of miR-337-3p that indirectly and positively modulated by circ_0000228 in the CC cells. ConclusionCirc_0000228 up-modulates TGFBR1 by targeting miR-337-3p to enhance CC cell proliferation, migration and invasion. Also, Circ_0000228 is a promising therapeutic target for the CC.

Design, synthesis, molecular modeling, DFT, ADME and biological evaluation studies of some new 1,3,4-oxadiazole linked benzimidazoles as anticancer agents and aromatase inhibitors

Breast cancer is the most frequent female cancer and second cause of cancer-related deaths among women around the world. Two thirds of breast cancer patients have hormone-dependent tumors, which is very likely be treated with hormonal therapy. Aromatase is involved in the biosynthesis of estrogen thus a critical target for breast cancer. In this study, in order to identify new aromatase enzyme inhibitors, a series of benzimidazole-1,3,4-oxadiazole derivatives were synthesized and characterized by 1H NMR, 13C NMR, and MS spectra analyses. In the in vitro anticancer assay, all the compounds tested anticancer activities using MTT-based assay against five cancer cell lines (MCF-7, A549, HeLa, C6, and HepG2). Among them, compound 5a exhibited the most potent activity with IC50 values of 5.165 ± 0.211 μM and 5.995 ± 0.264 μM against MCF-7 and HepG2 cell lines. Compound 5a was included in the BrdU test to determine the DNA synthesis inhibition effects for both cell types. Furthermore, compound 5c was also found to be more effective than doxorubicin on the HeLa cell line. The selectivity of anticancer activity was evaluated in NIH3T3 cell line. In vitro, enzymatic inhibition assays of aromatase enzyme were performed for compound 5a acting on the MCF-7 cell line. For compound 5a, in silico molecular docking and dynamics simulations against aromatase enzyme was performed to determine possible protein-ligand interactions and stability. DFT study was performed to evaluate the quantum mechanical and electronic properties of compound 5a. Finally, the theoretical ADME properties of the potential aromatase inhibitor compound 5a were analyzed by calculations. Communicated by Ramaswamy H. Sarma

Exosomes derived from inflammatory myoblasts promote M1 polarization and break the balance of myoblast proliferation/differentiation.

BACKGROUNDAcute muscle injuries are one of the most common injuries in sports. Severely injured muscles are prone to re-injury due to fibrotic scar formation caused by prolonged inflammation. How to regulate inflammation and suppress fibrosis is the focus of promoting muscle healing. Recent studies have found that myoblasts and macrophages play important roles in the inflammatory phase following muscle injury; however, the crosstalk between these two types of cells in the inflammatory environment, particularly the exosome-related mechanisms, had not been well studied. AIMTo evaluate the effects of exosomes from inflammatory C2C12 myoblasts (IF-C2C12-Exos) on macrophage polarization and myoblast proliferation/differentiation.METHODSA model of inflammation was established in vitro by lipopolysaccharide stimulation of myoblasts. C2C12-Exos were isolated and purified from the supernatant of myoblasts by gradient centrifugation. Multiple methods were used to identify the exosomes. Gradient concentrations of IF-C2C12-Exos were added to normal macrophages and myoblasts. PKH67 fluorescence tracing was used to identify the interaction between exosomes and cells. Microscopic morphology, Giemsa stain, and immunofluorescence were carried out for histological analysis. Additionally, ELISA assays, flow cytometry, and western blot were conducted to analyze molecular changes. Moreover, myogenic proliferation was assessed by the BrdU test, scratch assay, and CCK-8 assay.RESULTSWe found that the PKH-67-marked C2C12-Exos can be endocytosed by both macrophages and myoblasts. IF-C2C12-Exos induced M1 macrophage polarization and suppressed the M2 phenotype in vitro. In addition, these exosomes also stimulated the inflammatory reactions of macrophages. Furthermore, we demonstrated that IF-C2C12-Exos disrupted the balance of myoblast proliferation/differentiation, leading to enhanced proliferation and suppressed fibrogenic/myogenic differentiation.CONCLUSIONIF-C2C12-Exos can induce M1 polarization, resulting in a sustained and aggravated inflammatory environment that impairs myoblast differentiation, and leads to enhanced myogenic proliferation. These results demonstrate why prolonged inflammation occurs after acute muscle injury and provide a new target for the regulation of muscle regeneration.

Acetylenic Synthetic Betulin Derivatives Inhibit Akt and Erk Kinases Activity, Trigger Apoptosis and Suppress Proliferation of Neuroblastoma and Rhabdomyosarcoma Cell Lines.

Neuroblastoma (NB) and rhabdomyosarcoma (RMS), the most common pediatric extracranial solid tumors, still represent an important clinical challenge since no effective treatment is available for metastatic and recurrent disease. Hence, there is an urgent need for the development of new chemotherapeutics to improve the outcome of patients. Betulin (Bet), a triterpenoid from the bark of birches, demonstrated interesting anti-cancer potential. The modification of natural phytochemicals with evidenced anti-tumor activity, including Bet, is one of the methods of receiving new compounds for potential implementation in oncological treatment. Here, we showed that two acetylenic synthetic Bet derivatives (ASBDs), EB5 and EB25/1, reduced the viability and proliferation of SK-N-AS and TE671 cells, as measured by MTT and BrdU tests, respectively. Moreover, ASBDs were also more cytotoxic than temozolomide (TMZ) and cisplatin (cis-diaminedichloroplatinum [II], CDDP) in vitro, and the combination of EB5 with CDDP enhanced anti-cancer effects. We also showed the slowdown of cell cycle progression at S/G2 phases mediated by EB5 using FACS flow cytometry. The decreased viability and proliferation of pediatric cancers cells after treatment with ASBDs was linked to the reduced activity of kinases Akt, Erk1/2 and p38 and the induction of apoptosis, as investigated using Western blotting and FACS. In addition, in silico analyses of the ADMET profile found EB5 to be a promising anti-cancer drug candidate that would benefit from further investigation.

LINC01123 promotes cell proliferation and migration via regulating miR-1277-5p/KLF5 axis in ox-LDL-induced vascular smooth muscle cells.

The pathophysiological mechanism of carotid atherosclerosis (CAS) involves endothelial cell dysfunction, vascular smooth muscle cells (VSMCs), and macrophage activation, which ultimately leads to fibrosis of the vessel wall. lncRNA works weightily in the formation of CAS, but the function and mechanism of lncRNA LINC01123 in stable plaque formation are still equivocal. We collected blood samples from 35 CAS patients as well as 33 healthy volunteers. VSMCs treated with oxidized low-density lipoprotein (ox-LDL) were utilized as the CAS cell models. We applied qRT-PCR for detecting LINC01123, miR-1277-5p and KLF5 mRNA expression, CCK-8 method and BrdU test for determining cell proliferation, Transwell test for measuring cell migration, as well as Western blot for assaying KLF5 protein expression. Dual-luciferase reporter experiment was adopted for assessing the interaction between LINC01123 and miR-1277-5p, as well as KLF5 and miR-1277-5p. LINC01123 and KLF5 expression were dramatically up-regulated, while miR-1277-5p expression was down-regulated in CAS patients and ox-LDL-induced CAS cell models. Overexpressed LINC01123 notedly promoted VSMCs migration and proliferation. LINC01123 knockdown repressed cell proliferation and migration. Also, LINC01123 targeted miR-1277-5p and down-regulated its expression, while miR-1277-5p could negatively regulate KLF5 expression. LINC01123 is highly expressed in CAS patients, and promotes cell proliferation and migration via regulating miR-1277-5p/KLF5 axis in ox-LDL-induced VSMCs. It might be involved in the fibrous plaque formation.

Synthetic Betulin Derivatives Inhibit Growth of Glioma Cells In Vitro.

Glioma is the most malignant tumour of the human brain still lacking effective treatment modalities. Betulin, a pentacyclic triterpene abundantly found in the birch bark, has been shown to demonstrate interesting anti-cancer activity towards many cancer cells. We determined the effects of acetylenic synthetic betulin derivatives (ASBDs) as anti-tumour agents on glioma cells in vitro. T98G and C6 glioma cell viability and proliferation were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and BrdU (bromo deoxyuridine) test, respectively. Cell-cycle progression and induction of apoptosis were investigated with flow cytometry. ASBDs significantly decreased glioma cell viability/survival and inhibited proliferation in a dose-dependent manner in vitro. Moreover, ASBDs were more cytotoxic than clinically used chemotherapeutics - temozolomide and cisplatin. ASBDs may be considered for further study as potent anti-tumour agents in glioma treatment.

Effects of BDNF and PEC Nanoparticles on Osteocytes.

Bone substitute materials loaded with mediators that stimulate fracture healing are demanded in the clinical treatment in trauma surgery and orthopedics. Brain-derived neurotrophic factor (BDNF) enhances the proliferation and differentiation of mesenchymal stem cells into osteoblast. To load the implants with BDNF, a drug delivery system that allows the release of BDNF under spatiotemporal control would improve functionality. Polyelectrolyte complex nanoparticles (PECNP) have been reported as a suitable drug delivery system. The suitability of PECNP in contact with osteocytes as the main cell type of bone is not known so far. Thus, we aimed to verify that BDNF and PECNP loaded with BDNF (PECNP+BDNF) as well as pure PECNP have no negative effects on osteocytes in vitro. Therefore, the murine osteocyte cell line MLO-Y4 was treated with BDNF and PECNP+BDNF. The effects on proliferation were analyzed by the BrdU test (n = 5). The results demonstrated a significant increase in proliferation 24 h after BDNF application, whereas PECNP+BDNF did not lead to significant changes. Thus, we conclude that BDNF is an appropriate mediator to stimulate osteocytes. Since the addition of PECNP did not affect the viability of osteocytes, we conclude that PECNP are a suitable drug delivery system for bone implants.

Adult neuronal regeneration in the telencephalon of the killifish Aphaniops hormuzensis.

The potential of central nervous system regeneration was evaluated for the first time in the injured brain of the old world killifish Aphaniops hormuzensis. The histomorphological organization in the regeneration procedure was evaluated using the hematoxylin and eosin (H&E) staining and the bromodeoxyuridine (BrdU) immunohistochemistry technique. The histological tissue sections were sampled daily for 10 days. Based on the H&E staining, a large gliosis reaction was detected along with vacuolization and telencephalon deformation on 1-day post-lesion (dpl). The vacuolated zone declined fast and the telencephalon hemisphere recovered on 3 dpl. The symptoms of injured telencephalon nervous tissue were resolved within 7 dpl in both genders. In the BrdU test of the control group, BrdU-labeled cells were observed in the ventricular zone (VZ), pallium (Pa), and lateral pallium (LPa). On 1 dpl, the BrdU+ cells accumulated in the VZ, Pa, and LPa (located near the injury area). From 3 dpl onwards, the BrdU+ cells were reduced in the telencephalic VZ, Pa, and LPa. Based on the BrdU+ results, the adult brain in A. hormuzensis possesses a remarkable capacity for neuronal regeneration. By taking into account the high neural regeneration potency of A. hormuzensis and its relatively short lifespan, it could be concluded that besides the currently known models, the members of aphaniid fishes could probably be valuable animals to study the regeneration phenomenon in the vertebrates.

Cell Proliferation to Evaluate Preliminarily the Presence of Enduring Self-Regenerative Antioxidant Activity in Cerium Doped Bioactive Glasses.

(1) Background: a cell evaluation focused to verify the self-regenerative antioxidant activity is performed on cerium doped bioactive glasses. (2) Methods: the glasses based on 45S5 Bioglass®, are doped with 1.2 mol%, 3.6 mol% and 5.3 mol% of CeO2 and possess a polyhedral shape (~500 µm2). Glasses with this composition inhibit oxidative stress by mimicking catalase enzyme (CAT) and superoxide dismutase (SOD) activities; moreover, our previous cytocompatibility tests (neutral red (NR), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Bromo-2-deoxyUridine (BrdU)) reveal that the presence of cerium promotes the absorption and vitality of the cells. The same cytocompatibility tests were performed and repeated, in two different periods (named first and second use), separated from each other by four months. (3) Results: in the first and second use, NR tests indicate that the presence of cerium promotes once again cell uptake and viability, especially after 72 h. A decrease in cell proliferation it is observed after MTT and BrdU tests only in the second use. These findings are supported by statistically significant results (4) Conclusions: these glasses show enhanced proliferation, both in the short and in the long term, and for the first time such large dimensions are studied for this kind of study. A future prospective is the implantation of these bioactive glasses as bone substitute in animal models.

β2AR-dependent signaling contributes to in-vivo reendothelialization capacity of endothelial progenitor cells by shear stress

Endothelial progenitor cells (EPCs) play a crucial role in the endothelial repair after arterial injury. Shear stress has a beneficial effect on modulating EPC functions. The molecular mechanism underlying the influence of EPCs on the endothelial integrity and shear stress effects on EPC regulation remained unclear. Herein, we investigated the influence of β2 adrenergic receptor (β2AR)-dependent signaling on in-vitro shear stress-mediated function and in-vivo reendothelialization capacity of human EPCs. The human EPCs from healthy population were exposed to in-vitro 5, 10, and 20 dyn/cm shear stress for 15 h, and 10 dyn/cm for 5, 10, and 15 h, respectively. The in-vitro proliferation was assessed by CCK8 and BrDU tests. The migration and adhesion were evaluated by Transwell system and human umbilical vein endothelial cells (HUVECs) incorporation assays. Meanwhile, the angiogenic cytokine stromal derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) concentration of supernatant were tested by ELISA. Phosphorylated β2AR, Akt, and eNOS were detected by western blot. In an in-vivo study, mice carotid injury models were produced through denuding the endothelium with a curved flexible wire, and thereafter CM-Dil-labeled EPCs were injected intravenously. After 3 days, cells recruited to the injury sites were detected by fluorescent microscopy, and the in-vivo reendothelialization capacity was assessed by Evans blue dye. Shear stress improved in-vitro functions and in-vivo reendothelialization capacity of human EPCs. In parallel, shear stress up-regulated the phosphorylation of β2AR, Akt, and eNOS, and promoted vascular endothelial growth factor (VEGF) secretion of human EPCs. With ICI118,551 (a β2AR inhibitor) treatment, shear stress-induced Akt and eNOS phosphorylation as well as VEGF secretion were suppressed. After β2AR/PI3K/Akt/eNOS pathway of EPCs was blocked, the effects of shear stress on in-vitro functions and in-vivo reendothelialization capacity of EPCs were inhibited. The present study provided the novel data that shear stress-induced β2AR/Akt/eNOS pathway enhanced reendothelialization capacity of EPCs. Shear stress-induced β2AR-dependent pathway may be a novel and important therapeutic target for endothelial repair.

Induction of immune responses by protein vaccines formulated with adjuvants against Leishmania major in vivo

We used TSA as a principal antigen of Leishmania major with adjuvants. The aim of this research was to assess the efficacy of protein vaccine in the presence of Montanide vis-a-vis chitosan nanoparticle. The expression of recombinant protein was confirmed with SDS (sodium dodecyl sulfate) page and Western blotting. A total of 36 BALB/c mice were divided into six groups (TSA/high chitosan, TSA/low chitosan, TSA/Montanide, high chitosan, low chitosan, and PBS groups) and subcutaneously immunized with 20 mg of vaccine at three time intervals on days 0, 14, and 28. The mice were challenged with parasites 21 days after the final immunization. For assaying lymphocyte proliferation, the Brdu test and for evaluation of IgG subclasses and cytokines, the ELISA method was carried out. No statistically noteworthy variation was found between the vaccinated groups before and after the infectious challenge or intervention in terms of IgG and IFN-γ values. Statistically, significant difference was seen among the vaccinated and control groups in terms of IgG and IFN-γ. In terms of IL-4 before and after intervention, no statistically substantial difference was noted in the vaccinated and control groups, whereas, IgG isotypes rose to a great extent prior to and following intervention in all vaccine-immunized groups as compared with control groups. The formulated recombinant TSA protein vaccine with adjuvants induced lymphocytes proliferation and increased cytokines and antibodies as compared with the control groups. Given their potency, the formulated candidate vaccines may be recommended for further studies.

Evaluation of citrinin-induced toxic effects on mouse Sertoli cells

Penicillium citrinum-derived mycotoxin citrinin (CTN) is known to be a toxic agent for humans and animals. Previous studies have shown that CTN leads to toxicity in many biological systems; however, a limited number of studies have been performed to demonstrate the harmful effects of CTN on the male reproductive system. In the present study, the effects of CTN on cytotoxicity and apoptosis were examined in Sertoli cells as a model. Sertoli cells were treated with eight different CTN concentrations (from 0 up to 200 µM, for 6–72 h). Toxic potential of CTN was estimated by measuring metabolic activity (MTT test), DNA synthesis (BrdU test), and cell membrane damage (LDH test) as well as apoptosis and necrosis (via staining with propidium iodide and Hoechst 33342). The results showed that CTN significantly decreased the cell viability and cell proliferation, increased cytotoxicity, apoptosis, and necrosis in a concentration-dependent manner. Furthermore, CTN showed cytotoxicity in Sertoli cells with an IC50 value of 116.5 µM for 24 h. In conclusion, these findings clearly showed that, CTN affects Sertoli cells even at low concentrations. Thus, as a result of the damage of CTN shown in Sertoli cells, it can be deduced that CTN may also have detrimental effects on the testes.

The Effects of Dental Pulp Stem Cell Conditioned Media on the Proliferation of Peripheral Blood Mononuclear Cells

Background: Dental Pulp Stem Cells (DPSCs) are multipotent mesenchymal stem cells. DPSCs can renew themselves and differentiate into various cell types such as adipocytes, osteocytes, neurons, etc. DPSCs possess immunomodulatory properties and can inhibit peripheral blood mononuclear cell (PBMC) proliferation. Recent studies showed that conditioned-medium mesenchymal stem cells also had immunosuppressive activity. The ability of DPSC conditioned medium to suppress proliferation of allogeneic PBMC determined using BrdU (5-bromo-2’-deoxyuridine) proliferation assay. Materials and Methods: Dental pulp stem cells were extracted from a wisdom tooth. These cells are characterized for differentiation potential to adipogenic and osteogenic lineage and expression of mesenchymal stem cells markers, including CD105, CD73, CD90, CD14, CD-34, and CD45. The characterized DPSCs were cultured, and the conditioned medium (CM) got isolated. Stimulated and non-stimulated PBMCs from the allogeneic donor were cultured with DPSC-CM for 24, 48, and 72 hours. The proliferation of PBMCs was measured with BrdU assay. Results: The BrdU test results showed that DPSC-CM reduced allogeneic PBMC proliferation at different time points. DPSC-CM could inhibit stimulated and non-stimulated PBMC in 48 and 72 hours after incubation.Conclusion: This study demonstrated that DPSC-CM had an immunomodulatory effect on the proliferation of allogeneic cells.

Chemopreventive properties of young green barley extracts in in vitro model of colon cancer

Young green barley is the most valuable source of nutrients and bioactive substances. It has a broad spectrum of health-promoting properties such as antioxidant, anti-inflammatory, hypoglycaemic, anti-depressant, anti-atherosclerotic and anticancer. The presented study is an attempt to extend this knowledge with particular emphasis on the possibility of using green barley in colon cancer prevention. Extracts were prepared on the basis of two commercial products: ground dried barley grass (YGB INT) and powder of young green barley juice (YGB GW). Their influence on colon epithelial cells (CCD841 CoN) viability and proliferation were analyzed by LDH and MTT assays. Anticancer properties of extracts were screened on colon cancer cell lines (LS180, HT-29) by MTT and BrdU assays. Changes in cells morphology induced by extracts were investigated after May-Grünwald-Giemsa staining. Tested extracts were not toxic against CCD841 CoN and did not affected their proliferation or morphology (LDH test, MTT test, microscopy observation). The MTT revealed that extracts significantly inhibited proliferation of colon cancer cells in a dose-dependent manner. Results of BrdU test confirmed antiproliferative properties of extracts, but opposite to MTT test, indicated YGB GW as a better anticancer agent. Light microscopy observation proved the data obtained from both MTT and BrdU tests and additionally suggested the ability of the extracts to induce necrosis in LS180 and HT-29 cells. The study demonstrated that YGB extracts specifically inhibit proliferation of colon cancer cells without any undesirable effect on colon epithelial cells. Obtained results will provide a rationele for the future development of dietary supplements which could be beneficial in colon cancer chemoprevention.

Lycopene, sulforaphane, quercetin, and curcumin applied together show improved antiproliferative potential in colon cancer cells in vitro.

Lycopene, sulforaphane, quercetin, and curcumin, ingredients of daily diet, show significant anticancer and chemopreventive potential; however, no data are available showing thorough evaluation of jointly used phytochemicals on cancer cell proliferation. Here, we compare anticancer potential of mentioned substances applied separately or in combination (as MIX) by measuring mitochondrial activity (MTT test), DNA synthesis (BrdU test) and lactate dehydrogenase release (LDH test) in colon epithelial (CCD841 CoTr), and colon cancer (HT-29, LS174T) cells. Additive inhibitory effect of simultaneously used phytochemicals on cancer cells proliferation has been shown. In epithelial cells, tested combination effectively inhibited mitochondrial activity, but not DNA synthesis. LDH test revealed cytotoxicity of tested mixture against cancer cells without negative effect on normal cells. Furthermore, we demonstrated that MIX enhances antiproliferative effect of common cytostatics: 5-fluorouracil and cisplatin. Presented data suggest chemopreventive potential of the proposed combination of natural substances and their usefulness as adjuvant strategy during chemotherapy. PRACTICAL APPLICATIONS: Colorectal cancer is one of the most common causes of cancer death worldwide. Since its development and progression is strongly correlated with dietary habits, healthy diet as well as supplementation with proved anticancer agents seems to be reasonable strategy of colon cancer prevention and treatment. In the present study, we have focused on four natural compounds abundantly found in daily diet i.e., lycopene, sulforaphane, quercetin, and curcumin, with well established anticancer potential. Their individual and collective impact both on normal colon epithelium cells and colon cancer cells viability, growth, and proliferation was examined. Furthermore, activity of the substances combined as MIX to influence antiproliferative potential of commonly used in colon cancer treatment cytostatics, 5-fluorouracil, and cisplatin was verified. Proposed in the study combination of phytochemicals with experimentally proven antiproliferative activity may propose an effective strategy for prevention and treatment of colon cancer.