Normal Human Diploid Fibroblasts Research Articles

A Lombard Variety of Sweet Pepper Regulating Senescence and Proliferation: The Voghera Pepper.

Aging and its related disorders are important issues nowadays and the first cause of this physio-pathological condition is the overproduction of ROS. Ascorbic acid is an antioxidant mediator and its anti-aging proprieties are well known. Our previous data demonstrated that Voghera sweet pepper (VP), a distinctive type of pepper cultivated in Italy, is particularly rich in ascorbic acid. Based on these data, the anti-aging effect mediated by extracts of the edible part of VP was evaluated on an in vitro model of both young and old Normal Human Diploid Fibroblasts (NHDF). Using phase contrast microscopy, we observed that VP may help cells in the maintenance of physiological morphology during aging. Cytofluorimetric analyses revealed that VP extracts led to an increase in DNA synthesis and percentage of living cells, linked to a consequent increase in mitotic events. This hypothesis is supported by the enhancement of PCNA expression levels observed in old, treated fibroblasts, corroborating the idea that this extract could recover a young phenotype in adult fibroblasts, confirmed by the study of p16 and p53 expression levels and TEM analyses. Based on these results, we may suppose that VP can lead to the partial recovery of "young-like" phenotypes in old fibroblasts.

Voghera Sweet Pepper: A Potential Ally against Oxidative Stress and Aging.

In the present study, the potential functional properties of the extracts from the edible part of Capsicum annuum L. var. Peperone di Voghera (VP) were studied. The phytochemical analysis revealed a high amount of ascorbic acid, paralleled by a low carotenoid content. Normal human diploid fibroblasts (NHDF) were chosen as the in vitro model models to investigate the effects of the VP extract on oxidative stress and aging pathways. The extract of Carmagnola pepper (CP), another important Italian variety, was used as the reference vegetable. The cytotoxicity evaluation was performed firstly, using a 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, while the VP potential antioxidant and antiaging activity was investigated by immunofluorescence staining focusing on specifically selected proteins. The MTT data revealed the highest cell viability at a concentration of up to 1 mg/mL. The immunocytochemical analyses highlighted an increased expression of transcription factors and enzymes involved in redox homeostasis (Nrf2, SOD2, catalase), improved mitochondrial functionality, and the up-regulation of the longevity gene SIRT1. The present results supported the functional role of the VP pepper ecotype, suggesting a feasible use of its derived products as valuable food supplements.

Effects of oxygen on the response of mitochondria to X-irradiation and reactive oxygen species-mediated fibroblast activation

Purpose In living organisms, sensitivity to radiation increases in the presence of oxygen (O2) compared with that under anoxic or hypoxic conditions. Here, we investigated whether O2 concentration affected the response of mitochondria to X-rays radiation, which is associated with tumor microenvironment formation via fibroblast activation in radiation-related tumors. Materials and methods O2 concentrations were controlled at <5% (internal environmental oxygen condition) or anoxic levels during culture of normal human diploid lung fibroblasts TIG-3 and MRC-5. Protein expression associated with the response of mitochondria to radiation was assessed using immunostaining or western blotting. Results Induction of DNA damage (marker: γ-H2A histone family member X) and mitochondrial signaling (AMP-activated protein kinase), suppression of mitochondrial metabolic activity, and generation of reactive oxygen species occurred with radiation in cells cultured under 5% and 20% O2 conditions. However, reducing O2 concentration mitigated the effects of radiation on cell growth, mitochondrial damage (parkin), induction of antioxidant responses (nuclear factor E2-related factor 2), and fibroblast activation (α-smooth muscle actin). Radiation did not affect the markers used in this study in the absence of O2. Conclusion O2 concentration affected the response of mitochondria to radiation and reactive oxygen species-mediated fibroblast activation. Higher O2 concentrations enhanced the effects of radiation on mitochondria in human fibroblasts. In vitro studies may overestimate in vivo radiation effects due to high O2 concentrations.

Cytotoxic triterpene and steroidal glycosides from the seeds of Digitalis purpurea and the synergistic cytotoxicity of steroidal glycosides and etoposide in SBC-3 cells

The phytochemical investigations of the seeds of Digitalis purpurea have revealed their richness in cardenolide and pregnane glycosides exhibiting potent cytotoxicity; further chemical examinations of the D. purpurea seeds have achieved the isolation of six triterpene glycosides (1–6), six spirostanol glycosides (7–12), and three furostanol glycosides (13–15), including seven previously unidentified compounds (1–3, 10–12, and 14). Here, the structures of 1–3, 10–12, and 14 were determined via extensive spectroscopic analyses, including two-dimensional (2D) NMR; hydrolysis, followed by chromatographic and spectroscopic analyses; and X-ray crystallographic analysis. The cytotoxic activities of the isolated compounds (1–15) against SBC-3 small cell lung carcinoma and TIG-3 normal human diploid fibroblast cells were evaluated. Triterpene glycoside 3 and spirostanol glycoside 9 exhibited considerable cytotoxicity with IC50 values of 1.0 and 1.7 µM, respectively; they induced apoptotic cell death, which was accompanied by the activation of caspase-3 in SBC-3 cells. Spirostanol glycoside 7 exhibited cytotoxicity toward the SBC-3 cells (IC50 1.3 μM). Additionally, 7 at 0.1 and 1.0 µM synergistically enhanced the cytotoxicity of etoposide against SBC-3 cells; compound 7 induced the release of DAMPs; the release of HMGB1, the secretion of ATP, and the exposure of CALR in the SBC-3 cells. Furthermore, the combination of 7 and etoposide resulted in increasing the extracellular release of DAMPs. These data indicated that 7, as well as its combination with etoposide, might potentially cause immunogenic cell death.

Radiation affects glutathione redox reaction by reduced glutathione peroxidase activity in human fibroblasts.

The glutathione (GSH) redox control is critical to maintain redox balance in the body’s internal environment, and its perturbation leads to a dramatic increase in reactive oxygen species (ROS) levels and oxidative stress which have negative impacts on human health. Although ionizing radiation increases mitochondrial ROS generation, the mechanisms underlying radiation-induced late ROS accumulation are not fully understood. Here we investigated the radiation effect on GSH redox reactions in normal human diploid lung fibroblasts TIG-3 and MRC-5. Superoxide anion probe MitoSOX-red staining and measurement of GSH peroxidase (GPx) activity revealed that high dose single-radiation (SR) exposure (10 Gy) increased mitochondrial ROS generation and overall oxidative stress in parallel with decrease in GSH peroxidase (GPx) activity, while GSH redox control was effective after exposure to moderate doses under standard serum conditions. We used different serum conditions to elucidate the role of serum on GSH redox reaction. Serum starvation, serum deprivation and DNA damage response (DDR) inhibitors-treatment reduced the GPx activity and increased mitochondrial ROS generation regardless of radiation exposure. Fractionated-radiation was used to evaluate the radiation effect on GSH reactions. Repeated fractionated-radiation induced prolonged oxidative stress by down-regulation of GPx activity. In conclusion, radiation affects GSH usage according to radiation dose, irradiation methods and serum concentration. Radiation affected the GPx activity to disrupt fibroblast redox homeostasis.

Clinically relevant concentration of propofol and benzodiazepines did not affect in vitro angiogenesis.

Angiogenesis, one of regenerative medicine, is essential in the process of wound healing. The detailed effects of intravenous anesthetics and sedatives used during perioperative period have not yet been clarified. We investigated the effects of benzodiazepines and propofol on in vitro capillary tube formation. The effects of midazolam, diazepam and propofol (1, 10, 50µM each) on proliferation of human umbilical vein endothelial cells (HUVEC) and normal human diploid fibroblasts (NHDF) were determined. Quantitation of migration was achieved by measuring the fluorescence of migrating HUVEC using angiogenesis system. The effects of midazolam, diazepam and propofol on in vitro angiogenesis were investigated in co-cultured HUVEC and NHDF incubated. The effects of midazolam on activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases were examined by Western blot analysis using phospho-specific antibodies. Parametric data were analyzed with one-way repeated measures analysis of variance followed by the Scheffé test. A value of P < 0.05 was considered statistically significant. Fifty µM of midazolam significantly impaired endothelial cell proliferation, migration, and in vitro capillary tube formation. Propofol, diazepam or lower dose midazolam did not show any enhancing or suppressive effects on in vitro angiogenesis. Fifty µM of midazolam remarkably activated ERK, but not p38 MAPK in HUVEC. Propofol and benzodiazepines except high-dose midazolam did not affect in vitro angiogenesis. High-dose midazolam may impair in vitro capillary tube formation due to by suppressing proliferation and migration of endothelial cells via activation of ERK.

Silencing of long non-coding RNA KCNQ1OT1 alleviates LPS-induced lung injury by regulating the miR-370-3p/FOXM1 axis in childhood pneumonia

PurposeLong non-coding RNAs (lncRNAs) play important roles in the development of pneumonia. We aimed to explore the role of the lncRNA KCNQ1OT1 in pneumonia and its underlying mechanisms.MethodsThe expression of KCNQ1OT1, FOXM1, and miR-370-3p was detected in the serum of 24 children with pneumonia and in 24 healthy controls. Normal human embryonic lung-derived diploid fibroblasts (WI-38 cells) were stimulated with LPS (10 μg/mL) to simulate the cellular model of pneumonia, and cell viability, apoptosis, and inflammation were analysed. Dual luciferase reporter and/or RNA binding protein immunoprecipitation assays were performed to test the relationship between miR-370-3p and KCNQ1OT1/FOXM1. Mice were intratracheally administered LPS (5 mg/kg) to induce an in vivo model of pneumonia, and pathological injury and inflammation were analysed.ResultsThe expression of KCNQ1OT1 and FOXM1 was up-regulated, and miR-370-3p was down-regulated in the serum of children with pneumonia, LPS-treated WI-38 cells, and in lung tissues of LPS-treated mice. Silencing of KCNQ1OT1 or overexpression of miR-370-3p suppressed cell apoptosis and inflammation and facilitated cell viability in LPS-treated WI-38 cells. KCNQ1OT1 directly targets miR-370-3p and negatively regulates its expression. FOXM1 was targeted by miR-370-3p and negatively modulated by miR-370-3p. In addition, silencing of KCNQ1OT1 mitigated LPS-induced lung injury and inflammation in mice. The protective effects of KCNQ1OT1 silencing in LPS-treated WI-38 cells and mice were reversed by silencing of miR-370-3p or overexpression of FOXM1.ConclusionSilencing of KCNQ1OT1 alleviates LPS-induced lung injury by regulating the miR-370-3p/FOXM1 axis in pneumonia.

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.

Lactate preconditioning promotes a HIF-1\u03b1-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts

Recent evidence has emerged that cancer cells can use various metabolites as fuel sources. Restricting cultured cancer cells to sole metabolite fuel sources can promote metabolic changes leading to enhanced glycolysis or mitochondrial OXPHOS. However, the effect of metabolite-restriction on non-transformed cells remains largely unexplored. Here we examined the effect of restricting media fuel sources, including glucose, pyruvate or lactate, on the metabolic state of cultured human dermal fibroblasts. Fibroblasts cultured in lactate-only medium exhibited reduced PDH phosphorylation, indicative of OXPHOS, and a concurrent elevation of ROS. Lactate exposure primed fibroblasts to switch to glycolysis by increasing transcript abundance of genes encoding glycolytic enzymes and, upon exposure to glucose, increasing glycolytic enzyme levels. Furthermore, lactate treatment stabilized HIF-1α, a master regulator of glycolysis, in a manner attenuated by antioxidant exposure. Our findings indicate that lactate preconditioning primes fibroblasts to switch from OXPHOS to glycolysis metabolism, in part, through ROS-mediated HIF-1α stabilization. Interestingly, we found that lactate preconditioning results in increased transcript abundance of MYC and SNAI1, key facilitators of early somatic cell reprogramming. Defined metabolite treatment may represent a novel approach to increasing somatic cell reprogramming efficiency by amplifying a critical metabolic switch that occurs during iPSC generation.

Apoptosis: A target for anticancer therapy with novel cyanopyridines.

One of the many methods of treating cancer is to terminate the uncontrolled growth of cancer cells. So, aiming the apoptotic pathway is an exciting approach to finding new anticancer agents. A novel series of cyanopyridines was designed and synthesized for antiproliferative evaluation. 2-Amino-6-(4-(benzyloxy)phenyl)-4-(4-(dimethylamino)phenyl) nicotinonitrile 10f was the most potent inhibitor against the growth of PC-3, and HepG-2 cancer cell lines with IC50 values of 2.04 uM (selectivity index, SI=78.63, 43, respectively). Also, 10f was safe against the growth of normal human diploid lung fibroblasts cell line (WI-38) with an IC50 value of 160.04 uM. Its analogs, 10b, 10d, 10g, and 11b, were also active against the growth of PC-3, and HepG-2 while against MCF-7 cell line, they displayed good cytotoxic activity compared to the reference standard 5-FU. Remarkably, mechanistic studies indicated that compounds 10b, 10d, 10f, 10g, and 11b stimulated the level of active caspase 3 and boosted the BAX/BCL2 ratio 20-95 folds in comparison to the control. Our results have also indicated that 10b, 10d, 10f, 10g, and 11b exhibited a very potent inhibitory activity against PIM-1 kinase enzyme, where the IC50 values unraveled very potent molecules in the micromolar range (0.47-1.27μM). Further investigations have shown that 10f, the most potent PIM-1 kinase inhibitor, induced a cell cycle arrest at the G2/M phase. Moreover, in silico evaluation of ADME properties indicated that all the cyanopyridine compounds are orally bioavailable with no permeation to the blood brain barrier.

Telomere analysis using 3D fluorescence microscopy suggests mammalian telomere clustering in hTERT-immortalized Hs68 fibroblasts

Telomere length and dynamics are central to understanding cell aging, genomic instability and cancer. Currently, there are limited guidelines for analyzing telomeric features in 3D using different cellular models. Image processing for telomere analysis is of increasing interest in many fields, however a lack of standardization can make comparisons and reproducibility an issue. Here we provide a user's guide for quantitative immunofluorescence microscopy of telomeres in interphase cells that covers image acquisition, processing and analysis. Strategies for determining telomere size and number are identified using normal human diploid Hs68 fibroblasts. We demonstrate how to accurately determine telomere number, length, volume, and degree of clustering using quantitative immunofluorescence. Using this workflow, we make the unexpected observation that hTERT-immortalized Hs68 cells with longer telomeres have fewer resolvable telomeres in interphase. Rigorous quantification indicates that this is due to telomeric clustering, leading to systematic underestimation of telomere number and overestimation of telomere size.

Osmanicin, a Polyketide Alkaloid Isolated from Streptomyces osmaniensis CA-244599 Inhibits Elastase in Human Fibroblasts.

The strain Streptomyces osmaniensis CA-244599 isolated from the Comoros islands was submitted to liquid-state fermentation coupled to in situ solid-phase extraction with amberlite XAD-16 resin. Elution of the trapped compounds on the resin beads by ethyl acetate afforded seven metabolites, osmanicin (1), streptazolin (2), streptazone C (3), streptazone B1 (4), streptenol C (5), nocardamine (6) and desmethylenylnocardamine (7). Osmanicin (1) is a newly reported unusual scaffold combining streptazolin (2) and streptazone C (3) through a Diels-Alder type reaction. Experimental evidence excluded the spontaneous formation of 1 from 2 and 3. The isolated compounds were evaluated for their ability to inhibit elastase using normal human diploid fibroblasts. Compound 1 exhibited the most potent activity with an IC50 of 3.7 μM.

Lamin B receptor (LBR) is involved in the induction of cellular senescence in human cells

Cellular senescence is a phenomenon of irreversible growth arrest in mammalian somatic cells in culture. Various stresses induce cellular senescence and indeed, we have found that excess thymidine effectively induces cellular senescence in human cells. Further, many reports indicate the implication of chromatin proteins in cellular senescence. Here we analysed the role of lamin B receptor (LBR), a nuclear envelope protein that regulates heterochromatin organization, in cellular senescence induced by excess thymidine. We then found that the LBR protein was down-regulated and showed aberrant localization in cells upon induction of cellular senescence by excess thymidine. Additionally, we also found that knock-down of LBR facilitated the induction of cellular senescence by excess thymidine in cancerous HeLa cells, and importantly, it induced cellular senescence in normal human diploid fibroblast TIG-7 cells. These results suggested that decreased LBR function is involved in the induction of cellular senescence in human cells.

Cell Kinetic Approaches to the Search for Anti-Aging Drugs: Thirty Years After

This is a brief overview of the ideas of the possibility of using the cell kinetic model developed by the author in the 1980s to test, in experiments on cell cultures, potential geroprotectors and geropromoters that slow down or accelerate, respectively, the aging process in animals and humans. The history of the evolution of this model—from estimation of only the cell reproduction rate and saturation density in a non-subcultured cell culture to constructing survival curves in the stationary phase of growth and to a further analysis of the possible interrelation between all parts of the curve of cells’ growth and subsequent dying out—is considered. Possible approaches to mathematical and statistical analysis of the data obtained within the framework of this model system are analyzed. It is emphasized that such studies can be carried out on cells of a very different nature (normal and transformed human and animal cells, plant cells, yeast, mycoplasmas, bacteria, etc.), which makes possible an evolutionary approach to the interpretation of the results obtained. At the same time, in the author’s opinion, the most promising experiments are those carried out on immortalized cells of humans and animals, since they are not cancerous on the one hand and have an unlimited mitotic potential on the other hand and, therefore, do not “age” in the process of numerous divisions, as, for example, normal human diploid fibroblasts do. It is assumed that the appropriate mathematical analysis of the entire growth and dying out curve of a non-subcultured cell culture (from seeding into a culture flask to the complete death of all cells) may allow the clarification of certain relationships between the development and aging of a multicellular organism and to increase the reliability of identifying promising geroprotectors.

A dual-type L2 11–88 peptide from HPV types 16/18 induced strong immune responses and broad spectrum cross-reactive antibodies in mice

The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell−cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells.

PIM1-catalyzed CBX8 phosphorylation promotes the oncogene-induced senescence of human diploid fibroblast

The proto-oncogene PIM1 encodes Ser/Thr kinase and regulates cell growth, differentiation and apoptosis. However, more and more studies including ours have found that PIM1 can induce senescence in normal human diploid fibroblasts and behave as a tumor suppressor. But the relevant molecular mechanism of this process is not yet clear. It has been reported that Chromobox homolog 8 (CBX8) binds directly to INK4A as a transcriptional repressor, thereby suppressing stress-induced senescence. Here we report that PIM1 can phosphorylate CBX8 to promote its degradation, thereby up-regulating p16, during PIM1-induced cell senescence. Overexpression of CBX8 can inhibit PIM1-induced cell senescence. These data suggest that to promote CBX8 degradation may be an important molecular mechanism of PIM1-induced cell senescence.

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118cells, previously plated 3 days before at 1.04, 5.21, and 26.1×103cells/cm2. Hepatocytes plated onto lower TIG-118cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118cell density, which helps to design better engineered hepatocytes for future applications such as invitro cell-based assays and transplantable hepatocyte tissues.

Evaluation of the Antioxidant Capacity, Antimicrobial and Antiproliferative Potential of Fir (Abies alba Mill.) Honeydew Honey Collected from Gorski kotar (Croatia).

SUMMARYThe paper examines the antiproliferative, antimicrobial and antioxidative effects of fir (Abies alba Mill.) honeydew honey from mountain region of Croatia (Gorski kotar) as a potential replacement for standard antibiotics and chemotherapeutic agents. Cell viability, annexin V assay and flow cytometry analysis served to analyse the antiproliferative effect on, apoptosis induction in and cell death of cancer cell lines: HeLa, MCF-7, SW620, CFPAC-1, MIA PaCa-2 and normal diploid human fibroblasts (BJ). Antimicrobial activity was tested against Staphylococcus and Acinetobacter strains by agar well diffusion and microdilution assays. The DPPH˙ assay determined the radical scavenging activity, while mathematical models helped to evaluate the kinetic data of DPPH˙ inhibition. Antiproliferative effect on all tested cell lines and the prominent effect on normal diploid human fibroblasts (BJ), colorectal adenocarcinoma (SW620, metastatic) and breast epithelial adenocarcinoma (MCF-7, metastatic) was observable. The mechanisms of antiproliferative effect included accumulation of cells in the sub-G1 phase in all tested cells and induction of apoptosis in SW620 and MCF-7 cells predominantly. The antibacterial assays showed that antibiotic-resistant strains of both bacteria, including multi-resistant strain A. baumannii ATCC® BAA-1605™, were sensitive to all tested honey samples. Radical scavenging assay suggests that antioxidants present in the honey possess different radical suppressing abilities and that they react at different rates with radicals, thereby causing two steps of reaction. The results of the study indicate that Croatian fir honeydew honey has a therapeutic potential due to the strong biological activity and can serve to protect human health.

Synthesis and biological evaluation of 4β-(thiazol-2-yl)amino-4′-O-demethyl-4-deoxypodophyllotoxins as topoisomerase-II inhibitors

A series of 4β-(thiazol-2-yl)amino-4′-O-demethyl-4-deoxypodophyllotoxins were synthesized, and their cytotoxicities were evaluated against four human cancer cell lines (A549, HepG2, HeLa, and LOVO cells) and normal human diploid fibroblast line WI-38. Some of the compounds exhibited promising antitumor activity and less toxicity than the anticancer drug etoposide. Among them, compounds 15 and 17 were found to be the most potent synthetic derivatives as topo-II inhibitors, and induced DNA double-strand breaks via the p73/ATM pathway as well as the H2AX phosphorylation in A549 cells. These compounds also arrested A549 cells cycle in G2/M phase by regulating cyclinB1/cdc2(p34). Taken together, these results show that a series of compounds are potential anticancer agents.

GNG11 (G-protein subunit γ 11) suppresses cell growth with induction of reactive oxygen species and abnormal nuclear morphology in human SUSM-1 cells

Enforced expression of GNG11, G-protein subunit γ 11, induces cellular senescence in normal human diploid fibroblasts. We here examined the effect of the expression of GNG11 on the growth of immortalized human cell lines, and found that it suppressed the growth of SUSM-1 cells, but not of HeLa cells. We then compared these two cell lines to understand the molecular basis for the action of GNG11. We found that expression of GNG11 induced the generation of reactive oxygen species (ROS) and abnormal nuclear morphology in SUSM-1 cells but not in HeLa cells. Increased ROS generation by GNG11 would likely be caused by the down-regulation of the antioxidant enzymes in SUSM-1 cells. We also found that SUSM-1 cells, even under normal culture conditions, showed higher levels of ROS and higher incidence of abnormal nuclear morphology than HeLa cells, and that abnormal nuclear morphology was relevant to the increased ROS generation in SUSM-1 cells. Thus, SUSM-1 and HeLa cells showed differences in the regulation of ROS and nuclear morphology, which might account for their different responses to the expression of GNG11. Thus, SUSM-1 cells may provide a unique system to study the regulatory relationship between ROS generation, nuclear morphology, and G-protein signaling.