Highly diluted compounds effects on B16-F10 melanogenesis, reactive oxygen species (ROS) production and tumorigenesis.

Background: Cutaneous melanoma is a highly malignant tumor derived from pigment-producing (melanin) melanocytes of skin epidermis. Cutaneous pigmentation is described as the major physiologic defense against UV radiation. During melanin biosynthesis and other tumorigenic process, reactive oxygen species (ROS) are produced and might be critically involved in several melanomagenesis stages. ROS play key roles on regulation of many types cell proliferation, including melanoma cells. Aims: In this work we evaluated the effects of highly diluted compounds on melanogenesis and changes in reactive oxygen species after 96 hours of treatment and possible involvement in tumorigenesis. Methodology: Melanin content was measured in B16-F10 cells after 96 hours of treatment with highly diluted compounds, as well as the superoxide anion, hydrogen peroxide and nitric oxide. Furthermore, the effects of highly diluted compounds on cell proliferation were investigated by trypan blue exclusion method after 48 hours of treatment. Results: Treatment led to an increase in B16-F10 melanin content and a decrease in nitrite concentration, an intermediate product of nitric oxide. We also observed a decrease in cell proliferation after treatment. It is well recognized that nitric oxide (NO) is involved in tumor progression, including melanoma. Several articles show that NO treated B16-F10 cells exhibited higher metastatic capacity. Thereby, reduction in cell proliferation can be due to low NO levels. It is speculated that melanocytes are programmed to survive in order to preserve their photoprotective role, thus in a compensatory manner the cell may be synthesizing melanin in response to cell proliferation reduction. Conclusions: These results suggest that treatment may be reducing tumorigenic capacity via ROS reduction. However further studies are need to better understand highly diluted compounds mechanisms of action.

Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

The Effect of Epigallocatechin gallate on Cross-talk Between Autophagy and Apoptosis in NALM-6 Cell Line

Background: Acute lymphoblastic leukemia is a prevalent hematological malignancy in 2-5-year-old children. Chemotherapy, as the most common treatment for ALL, is not usually responsive. Epigallocatechin gallate (EGCG), a small molecule extracted from green tea, has significant effects on tumor cells through different mechanisms, such as DNA damage, cell cycle arrest, oxidative stress, apoptosis, and autophagy. In this study, we investigated the impact of EGCG on autophagy and apoptosis in NALM-6 cell line. Methods and results: Cell viability and apoptosis were assessed by MTT and Trypan blue exclusion assay, and flow cytometry. It was shown that EGCG remarkably inhibited proliferation, reduced cell viability, and induced apoptosis in NALM-6 cell line (P<0.05). In addition, real-time PCR and western blot analysis were used to examine autophagy. It was observed that EGCG resulted in a 4-fold increase in LC3 protein level (P<0.05) while reducing the mRNA expression level of LC3B, P62/SQSTM1, and Atg2B genes (P<0.01). It also caused around 1.3-fold increase in DRAM1 mRNA expression level (P<0.05). Finally, it was indicated that the inhibition of autophagy affects apoptosis neither in untreated nor treated cells with EGCG.Conclusion: These results show that EGCG can induce apoptosis and autophagy in NALM-6 cell line while inhibition of autophagy cannot affect apoptosis in this cell line.

Influence of oxygen availability on expression of glutaminolysis genes in human colon cancer cells

Introduction Glutaminolysis, beside glycolysis, is a key metabolic pathway of a cancer cell that provides energy and substrates for the synthesis of nucleic acids, proteins, and lipids. The pathway is mediated by both mitochondrial and cytosolic enzymes. Neither expression of glutaminolysis enzymes in colon cancer cells nor the influence of various oxygen concentrations on their expression has been studied so far. Objectives The aim of the study was to determine and compare the mRNA expression of enzymes involved in glutaminolysis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured in 1% O2 (hypoxia), 10% O2 (tissue normoxia), 21% O2 (atmospheric normoxia). Methods Cell viability was determined by Trypan Blue exclusion (TB) and Thiazolyl Blue Tetrazolium Bromide (MTT). The expression of HIF1α, GLUT1, GLS1, AST1, AST2, ACL, PC and GC1, GC2 at mRNA levelwas determined by RT-qPCR. Results. Correlation between increasing oxygen concentration and cell count was not observed. In both cell lines the number of viable cells was the lowest at 10% oxygen. The enzyme profile and expression of proteins involved in glutaminolysis varied depending on oxygen pressure and type of cell lines. In summary, our findings suggest differences in metabolic adaptation to oxygen availability in vivo between primary and metastatic colon cancer cells.

Formation of extracellular traps by circulating neutrophils and monocytes in rheumatoid arthritis patients: a study of new citrullinated autoantigen

Detection of subcellular structures containing typical citrullinated rheumatoid autoantigens in a single compartment presents a special interest, due to importance of anticitrulline autoantibodies for the autoimmune response in RA. Neutrophil and monocyte extracellular traps (NETs and ETs, respectively) may be considered such candidate structures. Our objective was to assess ability of blood neutrophils and monocytes from RA patients to generate NETs and ETs spontaneously and after in vitro induction.32 patients with verified RA and 30 healthy volunteers as controls were included into the study. Circulating neutrophils and monocytes were isolated with one-step density gradient centrifugation using three layers of ficoll-amidotrizoate gradient. Composition of isolated cellular fractions, their viability, and non-specific activation were evaluated microscopically using Trypan Blue exclusion test, as well as Nitro-Blue Tetrazolium test. The NETs were induced by phorbol-12-myristate-13-acetate, and ETs by bacterial LPS. Spontaneous and induced formation of extracellular traps was assessed using fluorescence microscopy. Neutrophil and monocyte fractions contained minute percentages of impurities and low extents of activated and dead cells. Spontaneous NET and ET formation in RA patients was significantly increased comparing to healthy controls. Neutrophils from ACPA-positive RA patients were found to have higher frequency of NET formation, compared to ACPA-negative RA patients. The monocytes did not demonstrate such differences between these subgroups. There were no substantial morphological differences in NETs and ETs patterns between the individuals from both groups. Induced extracellular trap production in RA was significantly higher compared to healthy controls. The level of myeloperoxidase-specific fluorescence in ETs was considerably lower than in NETs. NETs could probably be considered as a source of citrulline autoantigen participating in autoantibody production and stimulation of inflammatory autoimmune responses in RA, whereas ETs may play less important role in this process.

Effective Treatment against ESBL-Producing Klebsiella pneumoniae through Synergism of the Photodynamic Activity of Re (I) Compounds with Beta-Lactams

Background: Extended-spectrum beta-lactamase (ESBL) and carbapenemase (KPC+) producing Klebsiella pneumoniae are multidrug-resistant bacteria (MDR) with the highest risk to human health. The significant reduction of new antibiotics development can be overcome by complementing with alternative therapies, such as antimicrobial photodynamic therapy (aPDI). Through photosensitizer (PS) compounds, aPDI produces local oxidative stress-activated by light (photooxidative stress), nonspecifically killing bacteria. Methodology: Bimetallic Re(I)-based compounds, PSRe-µL1 and PSRe-µL2, were tested in aPDI and compared with a Ru(II)-based PS positive control. The ability of PSRe-µL1 and PSRe-µL2 to inhibit K. pneumoniae was evaluated under a photon flux of 17 µW/cm2. In addition, an improved aPDI effect with imipenem on KPC+ bacteria and a synergistic effect with cefotaxime on ESBL producers of a collection of 118 clinical isolates of K. pneumoniae was determined. Furthermore, trypan blue exclusion assays determined the PS cytotoxicity on mammalian cells. Results: At a minimum dose of 4 µg/mL, both the PSRe-µL1 and PSRe-µL2 significantly inhibited in 3log10 (>99.9%) the bacterial growth and showed a lethality of 60 and 30 min of light exposure, respectively. Furthermore, they were active on clinical isolates of K. pneumoniae at 3–6 log10. Additionally, a remarkably increased effectiveness of aPDI was observed over KPC+ bacteria when mixed with imipenem, and a synergistic effect from 3 to 6log10 over ESBL producers of K. pneumoniae clinic isolates when mixed with cefotaxime was determined for both PSs. Furthermore, the compounds show no dark toxicity and low light-dependent toxicity in vitro to mammalian HEp-2 and HEK293 cells. Conclusion: Compounds PSRe-µL1 and PSRe-µL2 produce an effective and synergistic aPDI effect on KPC+, ESBL, and clinical isolates of K. pneumoniae and have low cytotoxicity in mammalian cells.

Emodin Ameliorate High Glucose-induced Podocyte Apoptosis via Regulating AMPK/mTOR- mediated Autophagy Signaling Pathway

Background: Podocyte apoptosis and autophagy dysfunction have been considered to be one of the important causes of diabetic nephropathy (DN). Emodin has the function of regulating autophagy. The present study was performed to investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to the induction of AMPK/mTOR-mediated autophagy in MPC5 cells in vitro.Methods: The viability and apoptosis of podocytes (MPC5 cells) were detected using CCK-8 assay, trypan blue exclusion assay and flow cytometry analysis, respectively. The expression levels of Cleaved caspase-3, autophagy maker LC3 I/II, and AMPK/mTOR signaling pathway-related proteins were evaluated with western blot analysis. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.Results: HG (20-160 mmol/L) dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 μmol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage. Emodin (4 μmol/L) significantly increased LC3-II levels and induced RFP-LC3-containing punctate structures in MPC5 cells. Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 μmol/L) abolished emodin-induced autophagy activation.Conclusion: Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for DN.

The effect of Epitranscriptomic writer (METTL3) silencing on Hepatocellular carcinoma cell line

Worldwide, HCC is the sixth most common malignancy and the third most common cause of cancer-related death. RNA epigenetics becomes a hot topic in recent years. Among more than a hundred different RNA modifications, m6A is the most abundant modification. m6A is involved in regulating mRNA stability, splicing, and translation. However, the implications of m6A modification in human carcinogenesis remain poorly understood. METTL3 is a major RNA methyltransferase implicated in mRNA biogenesis, decay, and translation control through m6A modification. We aimed to target METTL3 in HepG2 cell lines by siRNA (small interfering RNA), and then evaluated the effect of this interference on viability and proliferative activity of HepG2 cells. Material and methods Using HepG2 cell lines, METTL3 was targeted using siRNA. The viability of HepG2 was conducted by Trypan blue exclusion test. The cell proliferation was tested by CellTiter 96® AQueous One Solution Cell Proliferation Assay. Results viable cell number and viability percent were significantly reduced in HepG2 cells transfected with siMETTL3 compared to mock cell lines (treated with transfection reagent only) (p&lt;0.05). The active proliferative cell count was lower in cells transfected with siMETTL3 than mock cells (p&lt;0.05). Conclusions Knockdown of METTL3 in HepG2 cell lines successively reduced cell viability and active proliferative cell count. METTL3 may be involved in liver tumorigenesis and its targeting may be of therapeutic benefit.

Viability and DNA Damage of Buccal Mucosa Cells in Patients Exposed to Panoramic X-ray

Panoramic X-ray is well known to cause DNA damage and induces cellular death. The aim of the present study was to evaluate the cytotoxicity of radiation exposure from panoramic radiography on human buccal mucosa cells by assessing the cell viability using the simple-trypan blue exclusion test. The genotoxicity effect was evaluated by assessing comet assay score. This research included a total of 20 healthy patients who had panoramic radiography for a routine dental examination. Buccal mucosa cells were collected from all participants before X-ray exposure and at 30 min or 24 h after exposure in Groups 1 and 2, respectively, and subjected to a comet assay and trypan blue exclusion test to assess cell viability and DNA damage. Cell viability was calculated as the ratio of live (translucent) to total counted cells. Comet assay output images were analysed using OpenComet software and a visual score by measuring the percentages of tail DNA and summing the visual score, respectively. A statistically significant (p < 0.05) reduce in cell viability was observed at 30 min after exposure, furthermore there is no more reduction after 24 h. Both comet assay measurements showed a significant (p < 0.05) increase in the percentage of tail DNA and visual score at 30 min after exposure, then tend to decrease after 24 h of exposure, although it was not significant (p > 0.05). The results showed that panoramic radiography interfered cell viability and induced DNA damage in buccal mucosa cells within 30 min after exposure, but these effects were ceased after 24 h.