Significant Increase In Cells Research Articles

Investigation of combined photodynamic and radiotherapy effects of gallium phthalocyanine chloride on MCF-7 breast cancer cells.

In this study, we evaluated the effect of gallium phthalocyanine chloride (GaPcCl) as a radio- and photosensitizer on MCF-7 breast cancer cell line. We incubated cells with GaPcCl in different concentrations (from 3.125 to 100μg/ml). Then cells in separate groups were exposed to different light doses (1.8 and 2.8J/cm2) at wavelength of 660nm and 2-Gy X-ray ionizing radiation, alone and in combination. Finally, cell survival and apoptosis were determined by MTT assay and flow cytometry, respectively. The results showed that the deactivated GaPcCl at concentration of 100µg/ml reduces the cell viability up to 15%. While, photoactivated GaPcCl (100µg/ml) at light dose of 2.8J/cm2 significantly decreases cell viability up to 55.3%. Although MTT assay demonstrated that GaPcCl is not act as a radiosensitizer, flow cytometry showed significant increase in cell apoptosis when GaPcCl was exposed to 2Gy X-ray. Using of GaPcCl-PDT (photodynamic therapy) integration with X-ray substantially increased cell death in comparison to the absence of X-ray. Furthermore, flow cytometry displayed a significant increase in apoptosis cells (especially late apoptosis) in this combination therapy. Our result proved that GaPcCl is an effective photosensitizer in MCF-7 human breast cancer cell line. The combination of GaPcCl-PDT and radiotherapy can be an efficient treatment against cancer. This approach needs further investigations on animal models for human purposes.Graphic abstract.

Acetyl-11-keto-β-boswellic acid triggers premature senescence via induction of DNA damage accompanied by impairment of DNA repair genes in hepatocellular carcinoma cells in vitro and in vivo.

Cellular senescence, a state of irreversible growth arrest, occurs in all somatic cells and causes the cells to exhaust replicative capacity. Recently, cellular senescence has been emerging as one of the principal mechanisms of tumor suppression, which can be induced by low doses of therapeutic drugs in cancer cells. Acetyl-11-keto-β-boswellic acid (AKBA), an active ingredient isolated from the plant Boswellia serrata, has been identified to induce apoptosis in hepatocellular carcinoma (HCC) cells. In this study, we found that low concentrations of AKBA treatment triggered cell growth arrest at G0/G1 phase with features of premature cellular senescence phenotype in both HCC cell lines HepG2 and SMMC7721, as observed by enlarged and flattened morphology, significant increase in cells with senescence-associated β-galactosidase staining, and decrease in cell proliferation and DNA synthesis. Furthermore, cellular senescence induced by AKBA occurred via activation of DNA damage response and impairment of DNA repair, as evidenced by strong induction of γH2AX and p53, and downregulated expressions of multiple DNA repair associated genes. Induction of p53 by AKBA caused a significant increase in p21CIP1 , which had a critical involvement in the induction of cellular senescence. Additionally, in vivo study demonstrated that induction of senescence contributed to the anticancer efficacy of AKBA. Therefore, our findings suggested that induction of premature senescence by AKBA through DNA damage response accompanied by impairment of DNA repair genes defines a novel mechanism contributing to its growth suppression in HCC cells.

PF169 LONG‐TERM INHIBITION OF CHK1/CHK2 KINASES MODIFY ACUTE LYMPHOBLASTIC LEUKEMIA CELL LINE RESPONSE TO DNA DAMAGING AGENTS

Background:Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR‐pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR‐related genes drastically affect the effectiveness of DNA‐damaging treatments in cancer cells. For this reasons selective DDR‐inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR‐inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR‐pathways in cancer cells.Aims:The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR‐related kinase, the cell cycle checkpoint kinase 1 (CHK1) and 2 (CHK2), in B‐ALL cell lines.Methods:The resistant cell lines were obtained from B‐ALL NALM‐6 cell line by prolonged incubation with a CHK1/CHK2 inhibitor, PF‐00477736. Parental cells were progressively incubated with increasing concentration of PF‐00477736 (from 100 nM to 10uM). The functional characterization of resistant cell lines was evaluated in vitro in term of response to different chemotherapy agents (doxorubicin, methotrexate and clofarabine) in term of reduction of cell viability, induction of apoptosis, cell cycle perturbation and activation of DDR pathways. The molecular characterization of resistant cell lines was evaluated in term of Copy Number Alteration (SNP‐array, Affimetrix).Results:Starting from NALM‐6 parental cell lines, we generated two resistant models, N6R‐PF4 and N6R‐PF8. N6R‐PF8 was generated form N6R‐PF4 cells after a prolong incubation with increased PF‐00477736 concentration. In term of reduction of cell viability and induction of apoptosis the resistant cell lines were not only significantly unresponsive to PF‐00477736 (increasing the IC50 up to 8‐fold) but also to different DNA damaging agents in comparison to parental NALM‐6. The cell cycle profile revealed that the resistant cell lines had a significant reduction in the percentage of cells in S phase and a significant increase of cells in G2/M phase. SNP microarray highlighted different alterations in DDR‐related genes and in particular in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN = 1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, BIRC3, CARD17, CASP1, CASP12 and CASP5) were detectable only in NALM‐6 parental cell lines and were copy number neutral in the two resistant models. Immunoblotting analysis confirmed that in the resistant cell lines the ATM/CHK2 down‐stream pathways was significant over‐expressed and activated in comparison to the parental NALM‐6. By chromosome binding analysis we identified that NALM‐6 cells were composed by a predominant clone with ATM locus CN loss and by a sub‐clone (45,X,‐Y,t(5;12)(q33;p13),t(7;19)(q11;p13)) with ATM locus in CN neutral. The treatment with PF‐00477736 progressively selected the sub‐clone, reaching 60% of clones in N6R‐PF4 and 100% of clone in N6R‐PF8.Summary/Conclusion:We showed that the higher protein expression of ATM and CHK2 affected not only the sensitivity to PF‐00477736 but also compromised the efficacy of conventional chemotherapy. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF‐00477736.

Identification and Quantification of Quercetin, A Major Constituent of Artocarpus altilis by Targeting Related Genes of Apoptosis and Cell Cycle: In Vitro Cytotoxic Activity Against Human Lung Carcinoma Cell Lines

Nine phenolic compounds were identified and quantified in Artocarpus altilia fruit. One of the main compounds was quercetin, which is the major class of flavonoids has been identified and quantified in pulp part of A. altilis fruit of methanol extract. The aim of this study was to evaluate in vitro cytotoxic assay. Inhibitory concentration 50% concentration was determined using trypan blue exclusion assay. Apoptosis induction and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell cycle-related regulatory genes were assessed by RT-qPCR study of the methanol extract of pulp part on human lung carcinoma (A549) cell line. A significant increase of cells at G2/M phases was detected (P < 0.05). Furthermore, the pulp of the fruit downregulated the expression of anti-apoptosis gene BCL-2 and upregulated the expression of pro-apoptosis gene BAX. CASPASE-3 was also activated by the fruit, which started a CASPASE-3-depended mitochondrial pathway to induce apoptosis. As the results, the pulp was the most active in terms of all tests, due to high amount of quercetin in pulp part, 78% of total flavonoids. Taken together, these findings suggested that A. altilis induces apoptosis in a mitochondrial-dependent pathway by releasing and upregulating CYTOCHROME C expression and regulates the expression of downstream apoptotic components, including BCL-2 and BAX.

Long-term contractile activity and thyroid hormone supplementation produce engineered rat myocardium with adult-like structure and function

The field of cardiac tissue engineering has developed rapidly, but structural and functional immaturity of engineered heart tissues hinder their widespread use. Here, we show that a combination of low-rate (0.2 Hz) contractile activity and thyroid hormone (T3) supplementation significantly promote structural and functional maturation of engineered rat cardiac tissues (“cardiobundles”). The progressive maturation of cardiobundles during first 2 weeks of culture resulted in cell cycle exit and loss of spontaneous activity, which in longer culture yielded decreased contractile function. Maintaining a low level of contractile activity by 0.2 Hz pacing between culture weeks 3 and 5, combined with T3 treatment, yielded significant growth of cardiobundle and myocyte cross-sectional areas (by 68% and 32%, respectively), increased nuclei numbers (by 22%), improved twitch force (by 39%), shortened action potential duration (by 32%), polarized N-cadherin distribution, and switch from immature (slow skeletal) to mature (fast) cardiac troponin I isoform expression. Along with advanced functional output (conduction velocity 53.7 ± 0.8 cm/s, specific force 70.1 ± 5.8 mN/mm2), quantitative ultrastructural analyses revealed similar metrics and abundance of sarcomeres, T-tubules, M-bands, and intercalated disks compared to native age-matched (5-week) and adult (3-month) ventricular myocytes. Unlike 0.2 Hz regime, chronic 1 Hz pacing resulted in significant cardiomyocyte loss and formation of necrotic core despite the use of dynamic culture. Overall, our results demonstrate remarkable ultrastructural and functional maturation of neonatal rat cardiomyocytes in 3D culture and reveal importance of combined biophysical and hormonal inputs for in vitro engineering of adult-like myocardium. Statement of SignificanceCompared to human stem cell-derived cardiomyocytes, neonatal rat ventricular myocytes show advanced maturation state which makes them suitable for in vitro studies of postnatal cardiac development. Still, maturation process from a neonatal to an adult cardiomyocyte has not been recapitulated in rodent cell cultures. Here, we show that low-frequency pacing and thyroid hormone supplementation of 3D engineered neonatal rat cardiac tissues synergistically yield significant increase in cell and tissue volume, robust formation of T-tubules and M-lines, improved sarcomere organization, and faster and more forceful contractions. To the best of our knowledge, 5-week old engineered cardiac tissues described in this study are the first that exhibit both ultrastructural and functional characteristics approaching or matching those of adult ventricular myocardium.

Abstract 475: Human Saphenous Vein Progenitor Cells Are Susceptible to Mechanical Stimulation. Novel Insights in Pathologic Programming of Saphenous Vein Bypass Graft Disease

Background: Implantation of saphenous vein (SV) grafts into coronary position determines structural vessel wall remodeling and intimal hyperplasia. The role of the altered wall mechanics and cell-based mechanosensing has been recently implicated in the priming of this pathologic process. We investigated the effects of cyclic uniaxial strain on human saphenous vein progenitor cells (SVPs), a cell type endowed with pericyte stem cell characteristics resident in the adventitia. Methods: CD34 + CD31 - SVPs were isolated with immuno-magnetic sorting (MACS) from SVs of patients (age 58±12.6, Mean±SD) undergoing saphenectomy. Cells were subject to uniaxial strain (10% elongation; 1Hz) for 24 and 72 hrs using the FlexCell system. Cell orientation, immunofluorescence and western analyses were performed to assess the effects of strain on cell orientation/shape, cell cycle and activity of the YAP/Hippo-dependent mechanotransduction machinery. RNA-sequencing from control vs. strained SVPs was performed at both time points using RNA from cells of 5 independent donors. Results: Results indicated an increase in the expression of the cell cycle-associated markers Ki67 and pHH3 in mechanically stimulated vs. control SVPs at 24 hrs, followed by a significant reduction at 72 hrs of stimulation. Variations in cell shape was observed as verified by a significant change in the nuclei orientation in the strain field as well as in the cell shape index/spread areas at both time points. Immunofluorescence revealed a significant increase in cells showing a nuclear localization of the YAP transcription factor at 72hrs. In line with these findings Western analyses indicated a significant decrease in the ratio between phosphorylated/total YAP and its upstream kinase LATS in mechanically stimulated vs. control SVPs, suggesting an inhibition of the HIPPO kinase pathway by mechanosensing. RNASeq gene expression analyses showed a coherent modification of gene expression pathways and the upregulation of a specific HIPPO/YAP/TEAD gene expression signature in SVPs mechanically stimulated for 72hrs. Conclusions: These findings demonstrate the direct susceptibility of human SVPs to pathologic strain and identifies this cell population as mechano-perceptors in the vein wall.

Abstract 38: Bioreactor Based Approach for Valve Tissue Engineering: Novel Application of Decellularized Porcine Pericardium

Background: Aldehyde-fixed pericardium is commonly used in valve implant manufacturing. Despite its wide employment, this tissue undergoes chronic rejection that limits implant performance and durability. In this work, we employed a method to engineer a leaflet-like tissue by seeding aortic valve interstitial cells (VIC) into fixative-free decellularized pericardium using a bioreactor based approach. Methods: Following treatment with hypotonic buffer (Tris-HCl) to induce cell lysis, porcine pericardium was incubated with TritonX-100, to remove adipose tissue and then treated with sodium dodecylsulfate to wash cellular debris. DNA was removed by incubation into a DNAse I solution. Pericardium permeability was measured on samples before and after decellularization (pressure from 735 Pa to 2200 Pa). A direct perfusion bioreactor was employed to seed (3days, 3ml/min) porcine VICs (6.5E+5 cell/scaffold) into decellularized pericardium patches (6mm diameters) and perform long-term culture (up to 14 days, 0.03ml/min). Cell seeding efficiency (Day 3) and cell proliferation (Day 7-14) were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) staining, histological analyses and fluorescence staining for quiescent/activated VIC markers (DAPI, vimentin, α-smooth muscle actin). Results: Permeability tests revealed a significant increase in decellularized samples (1-way ANOVA p &lt; 0.05), thus supporting its use in a direct perfusion bioreactor system. MTT staining revealed homogeneous cell seeding distribution, supported by DAPI staining, showing efficient cellularization through the whole patch volume. Computer-based cell nuclei counting revealed a significant cell increase from day 3 to 7 and 14 ( p &lt;0.05 1-way ANOVA). Immunofluorescence showed a marked reduction of αSMA in cells populating the inner layers. Conclusions: Our data show, for the first time, the capability to seed and culture VICs into a cell/fixative free pericardium with a direct perfusion system. Moreover, the use of pericardium treated with our decellularization procedure and recellularized under dynamic conditions supports a more physiological growth of VIC, as suggested by downregulation of αSMA in the inner pericardium layer.

Preservation of chromosomal integrity in murine spermatozoa derived from gonocytes and spermatogonial stem cells surviving prenatal and postnatal exposure to γ-rays in mice.

Pre- and postnatal male mice were acutely (659-690 mGy/min) and continuously (0.303 mGy/min) exposed to 2 Gy γ-rays to evaluate spermatogenic potential and chromosome damage in their germ cells as adults. Acute irradiation on Days 15.5, 16.5, and 17.5 post-coitus affected testicular development, as a result of massive quiescent gonocyte loss; the majority of the seminiferous tubules in these testes were devoid of germ cells. Acute irradiation on Days 18.5 and 19.5 post-coitus had less effect on testicular development and spermatogenesis, even though germ cells were quiescent gonocytes on these days. Adverse effects on testicular development and spermatogenesis were observed following continuous irradiation between Days 14.5 and 19.5 post-coitus. Exposure to acute and continuous postnatal irradiation after the differentiation of spermatogonial stem cells and spermatogonia resulted in nearly all of the seminiferous tubules exhibiting spermatogenesis. Neither acute nor continuous irradiation was responsible for the increased number of multivalent chromosomes in primary-spermatocyte descendents of the exposed gonocytes. In contrast, a significant increase in cells with multivalent chromosomes was observed following acute irradiation on Days 4 and 11 post-partum. No significant increases in unstable structural chromosomal aberrations or aneuploidy in spermatozoa were observed, regardless of cell stage at irradiation or the radiation dose-rate. Thus, murine germ cells that survive prenatal and postnatal irradiation can restore spermatogenesis and produce viable spermatozoa without chromosome damage. These findings may provide a better understanding of reproductive potential following accidental, environmental, or therapeutic irradiation during the prenatal and postnatal periods in humans.

Protective effects of melatonin and omega-3 on the hippocampus and the cerebellum of adult Wistar albino rats exposed to electromagnetic fields

The purpose of the study was to investigate the effects of pulsed digital electromagnetic radiation emitted by mobile phones on the central nervous system of the adult Wistar albino rats. The study evaluated structural and functional impacts of four treatment arms: electromagnetic field (EMF) exposed; EMF exposed+melatonin treated group (EMF+Mel); EMF exposed+omega-3 (ω3) treated group (EMF+ω3); and control group (Cont). The 12-weeks-old rats were exposed to 900MHz EMF for 60min/day (4:00–5:00p.m.) for 15days. Stereological, biochemical and electrophysiological techniques were applied to evaluate protective effects of Mel and ω3. Significant cell loss in the CA1 and CA2 regions of hippocampus were observed in the EMF compared to other groups (p<0.01). In the CA3 region of the EMF+ω3, a significant cell increase was found compared to other groups (p<0.01). Granular cell loss was observed in the dentate gyrus of the EMF compared to the Cont (p<0.01). EMF+ω3 has more granular cells in the cerebellum than the Cont, EMF+Mel (p<0.01). Significant Purkinje cell loss was found in the cerebellum of EMF group compared to the other (p<0.01). EMF+Mel and EMF+ω3 showed the same protection compared to the Cont (p>0.05). The passive avoidance test showed that entrance latency into the dark compartment was significantly shorter in the EMF (p<0.05). Additionally, EMF had a higher serum enzyme activity than the other groups (p<0.01). In conclusion, our analyses confirm that EMF may lead to cellular damage in the hippocampus and the cerebellum, and that Mel and ω3 may have neuroprotective effects.

The E3 Ubiquitin Ligase HUWE1 Is a Potential Therapeutic Target for Multiple Myeloma

BackgroundThe use of proteasome inhibitors to disrupt the ubiquitin proteasome system (UPS) has provided a significant therapeutic advance and highlights the importance of this pathway in Multiple Myeloma (MM). Selective inhibition of other enzymes that regulate the UPS may enable more specific targeting of aberrant signalling pathways with reduced side-effects. Using UPS-specific microarrays we found higher expression of the E3 ligase HUWE1 in MM cell lines compared to normal counterparts. HUWE1 is involved in the regulation of key proteins such as ARF, p53 and MYC. It has previously been reported to be deregulated in tumorigenesis and small molecule inhibitors of HUWE1 have recently shown efficacy in in vitro models of colorectal cancer. The aim of this study was to elucidate the role and therapeutic potential of HUWE1 in MM.MethodsTo validate our UPS microarray studies, HUWE1 expression levels were analysed in publically available datasets (GSE6477, GSE6691, GSE2113), CD138+ cells from healthy bone marrow (n=3) and MM patients (n=5) and MM cell lines [U266 (p53 mutant), OPM-2 (p53 mutant), KMS-18 (p53 WT/-), JJN3(p53 WT/-), MM.1S (p53 WT)]. shRNA-mediated gene knockdown (Source Biosciences) was used to investigate the therapeutic potential of targeting HUWE1. HUWE1 knockdown cells were analysed for viability and cell cycle status; effects on known downstream substrates were analysed by Western blotting. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1.ResultsElevated HUWE1 expression (≥ 2-fold) was confirmed in MM cell lines and primary CD138+ MM cells compared to healthy CD138+ cells at both the gene and protein level. Analysis of publically available datasets (GSE6477, GSE6691) also demonstrated that HUWE1 was significantly increased in MM patients compared with healthy donors. Moreover, further analysis of datasets GSE6477 and GSE2113 revealed that HUWE1 expression increased significantly (p≤0.05) through a spectrum of plasma cell diseases (MGUS, smoldering MM, newly diagnosed MM, plasma cell leukemia). shRNA knockdown of HUWE1 resulted in a significant increase of cells in G2/M, an accumulation of p53 and MIZ1 and a decrease in MYC protein levels (p≤0.05). HUWE1 depletion significantly decreased (p≤0.01) the proliferation of all MM cell lines, cultured alone or in co-culture with MM patient-derived bone marrow stromal cells, regardless of p53 status. Conversely, we show that MM cell lines that carry mutant p53 were largely resistant to the MDM2 inhibitor Nutlin-3. Finally, proteomic profiling identified more than 80 putative substrates of HUWE1, confirming its known roles as an RNA binding protein and in core histone regulation. Furthermore, this analysis revealed previously unidentified involvement of HUWE1 in regulation of cell cycle (3 proteins) and MAPK signalling (4 proteins).ConclusionsThe proteasome is an established target in MM and there are opportunities for more specific targeting of enzymes within the UPS with reduced side-effects. Here we demonstrate that the E3 ligase HUWE1 is elevated in MM and inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines, decreased expression of MYC and increased levels of p53. These effects were independent of p53 status, suggesting that inhibition of HUWE1 offers a more valuable approach than MDM2 inhibitors which show efficacy only in p53 wild type myeloma. Future studies will validate novel substrates of HUWE1 identified in this study and investigate recently identified small molecule inhibitors of HUWE1 in MM. This study demonstrates that HUWE1 is important in the growth and survival of MM cells and may provide a novel therapeutic target in MM. DisclosuresNo relevant conflicts of interest to declare.

Involvement of Cold Inducible RNA-Binding Protein in Severe Hypoxia-Induced Growth Arrest of Neural Stem Cells In Vitro

Neonatal hypoxia is the leading cause of brain damage with birth complications. Many studies have reported proliferation-promoting effect of mild hypoxia on neural stem cells (NSCs). However, how severe hypoxia influences the behavior of NSCs has been poorly explored. In the present study, we investigated the effects of 5, 3, and 1 % oxygen exposure on NSCs in vitro. MTT, neurosphere assay, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation revealed a quick growth arrest of C17.2 cells and primary NSCs induced by 1 % oxygen exposure. Cell cycle analysis showed that this hypoxia exposure caused a significant increase of cells in G0/G1 phase and decrease of cells in S phase that is associated with decrease of Cyclin D1. Interestingly, the expression of cold inducible RNA-binding protein (CIRBP), a cold responsive gene reacting to multiple cellular stresses, was decreased in parallel with the 1 % oxygen-induced proliferation inhibition. Forced expression of CIRBP under hypoxia could restore the proliferation of NSCs, as showed by EdU incorporation and cell cycle analysis. Furthermore, the expression of Cyclin D1 under hypoxia was also restored by CIRBP overexpression. Taken together, these data suggested a growth-suppressing effect of severe hypoxia on NSCs and, for the first time, revealed a novel role of CIRBP in hypoxia-induced cell cycle arrest, suggesting that modulating CIRBP may be utilized for preventing hypoxia-induced neonatal brain injury.

A novel, clinically relevant use of a piglet model to study the effects of anesthetics on the developing brain.

BackgroundAnesthesia-induced neurotoxicity research in the developing brain must rely upon an unimpeachable animal model and a standardized treatment approach. In this manner, identification of mechanisms of action may be undertaken. The goal of this study was to develop a novel, clinically relevant, translational way to use a piglet model to investigate anesthesia effects on the developing brain.Methods29 newborn piglets were assigned to either: (1) control (no intervention, n = 10); (2) lipopolysaccharide (LPS; positive inflammatory control, n = 9); or (3) isoflurane anesthesia (n = 10). Positive inflammatory control animals were given 100 mcg/kg LPS from Escherichia coli intraperitoneally (IP) on the same day as those receiving isoflurane. Isoflurane was administered for 3 h while care was taken to ensure human perioperative conditions. To establish a clinical scenario, each animal was intubated and monitored with pulse oximetry, invasive and non-invasive blood pressure, electrocardiogram, temperature, end-tidal CO2, anesthetic concentration, and iSTAT blood analysis. All animals were sacrificed after 48 h using transcardiac perfusion of ice-cold, heparinized phosphate buffered saline (PBS) followed by 4 % paraformaldehyde (PFA). Brains were collected and histopathological analysis focused on the entorhinal cortex looking for degenerative changes due to its critical role in learning and memory. Reliable identification of entorhinal cortex was achieved by using colored ink on the surface of the brains, which was then cross-referenced with microscopic anatomy. Hematoxylin & eosin-stained high-power fields was used to quantify cells. ImageJ™ (National Institutes of Health, Bethesda, MD, USA) was used to count absolute number of progenitor glial cells (PGC) and number of PGCs per cluster. Immunohistochemistry was also utilized to ensure positive identification of cellular structures.ResultsHistopathological sections of 28 brains were analyzed. One animal in the LPS group died shortly after administration, presumably from inadvertent intravascular injection. There was an acute basal ganglia ischemic infarct in one isoflurane-treated animal. A large number of small, round nucleated cells were seen throughout layer II of the entorhinal cortex in all animals. These cells were identified as PGCs using immunohistochemistry and light microscopy. Although there was no difference in the absolute number of PGCs between the groups, animals given isoflurane or LPS demonstrated a significant increase in cells forming ‘clusters’ in the entorhinal cortex. An apparent change in the pattern of doublecortin labeling also suggests changes in neuronal precursors and undifferentiated neurons.ConclusionsThis study represents the first novel use of a clinically relevant neonatal piglet model to study anesthesia effects on the developing brain. LPS induces neuroinflammation, and this is a potential mechanism for LPS and perhaps isoflurane in causing a change in progenitor cell distribution. We postulate that the isoflurane-induced change in glial progenitor cell distribution could have important implications for cell differentiation, maturation and neural circuit behavior in the rapidly developing brain.

MicroRNA-124 (MiR-124) Inhibits Cell Proliferation, Metastasis and Invasion in Colorectal Cancer by Downregulating Rho-Associated Protein Kinase 1(ROCK1)

Background/Aims: MiR-124 inhibits neoplastic transformation, cell proliferation, and metastasis and downregulates Rho-associated protein kinase (ROCK1) in Colorectal Cancer (CRC). The aim of this study was to further investigate the roles and interactions of ROCK1 and miR-124 and the effects of knockdown of ROCK1and MiR-124 in human Colorectal Cancer (CRC). Methods: Three Colorectal cancer cell lines (HCT116, HT29 and SW620) and one Human Colonic Mucosa Epithelial cell line (NCM460) were studied. The protein expression of ROCK1 was examined by Western-blot and qRT-PCR were performed to examine the expression levels of ROCK1 mRNA and miR-124. Furthermore, We performed transfection of cancer cell line (SW620) with pre-miR-124(mimics), anti-miR-124(inhibitor), ROCK1 siRNA and the control, then observed the affects of ROCK1 protein expression by westen-blot, cell proliferation by EDU (5-ethynyl-2'deoxyuridine assay) and expression levels of ROCK1mRNA by qRT-PCR . A soft agar formation assay, Migration and invasion assays were used to determine the effect of regulation of miR-124 and ROCK1, and survivin on the transformation and invasion capability of colorectal cancer cell. Results: MiR-124 expression was significantly downregulated in CRC cell lines compare to normal (P < 0.05). In contrast, ROCK1 protein expression was significantly increased in CRC cell lines compared to the normal (P < 0.05), whereas the gene (ROCK1mRNA) expression remained unaltered (P > 0.05). ROCK1 mRNA was unaltered in cells transfected with miR-124 mimic and miR-124 inhibitor, compared to normal controls. There was a significant reduction in ROCK1 protein in cells transfected with miR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P < 0.05). Cell proliferation, transformation and invasion of cells transfected with miR-124 inhibitor were significantly increased compared to those in normal controls (P<0.05). However, cell proliferation, transformation and invasion of cells transfected with ROCK1 siRNA were significantly decreased compared to control (P < 0.05). Conclusions: In conclusion, our results demonstrated that miR-124 not only promoted cancer cell hyperplasia and significantly associated with CRC metastasis and progression, but also downregulated ROCK1 protein expression. More importantly, increased ROCK1 expression or inhibited miR-124 expression may constitute effective new therapeutic strategies for the treatment of renal cancer in the future.

ERK1/2 Phosphorylation in the Rat Supraoptic Nucleus, Dorsal Raphe Nucleus, and Locus Coeruleus Neurons Following Noxious Stimulation to the Hind Paw

Phospho-ERK1/2 (pERK1/2) fluorescence-immunohistochemistry is specifically well suited to mirror neuronal activity in the pain pathway at the cellular level. This study employed this method to visualize neuronal activity in 3 rat CNS nuclei following an acute noxious stimulation. The rat hind paw was stimulated either by heat or by a sequence of mustard oil and heat. Two min after the thermal stimulation or after the combined mustard oil and thermal stimulation, there was a significant increase in cells showing pERK1/2 immunoreactivity in the supraoptic nucleus (SON), in the dorsal raphe nucleus (DRN), and in the locus coeruleus (LC). Pretreatment with the opioid analgesic morphine or the N-methyl-<smlcap>D</smlcap>-aspartate antagonist MK-801 markedly attenuated ERK1/2 phosphorylation. These findings support the concept that the SON, the DRN, and the LC are integrated into pain processing at the hypothalamic and brain stem level.

Expression and function of SLAM family molecule SLAMF3 (CD229) in myeloma

e242 [U266 (p53 mutant), OPM-2 (p53 mutant), KMS-18 (p53 WT/-), JJN3(p53 WT/-), MM.1S (p53 WT)]. Effects on downstream substrates were analysed by Western blotting. Elevated HUWE1 expression was confirmed in MM cell lines and primary CD138+ MM cells compared to healthy CD138+ cells at both the gene and protein level ( 2-fold). shRNA knockdown of HUWE1 significantly decreased (p 0.01) the proliferation of all MM cell lines, regardless of p53 status; this effect was maintained when cells were cultured in the presence of bone marrow stromal cells. Conversely, we show that MM cell lines that carry mutant p53 were largely resistant to the MDM2 inhibitor Nutlin-3. Finally, we have demonstrated that HUWE1 depletion is associated with a significant increase of cells in G2/M, an accumulation of p53 and MIZ1 and a decrease in MYC protein levels (p 0.05). The proteasome is an established target in MM and there are opportunities for more specific targeting of enzymes within the UPS. Here we demonstrate that the E3 ligase HUWE1 is important in the growth and survival of MM cells and offers a more valuable therapeutic target than MDM2. Future work will investigate the effect of small molecule inhibitors of HUWE1 in MM.

Downregulation of rho-associated protein kinase 1 by miR-124 in colorectal cancer.

To investigate the roles and interactions of rho-associated protein kinase (ROCK)1 and miR-124 in human colorectal cancer (CRC). Expression of ROCK1 protein was examined by Western blotting, and quantitative reverse transcriptase PCR was performed to measure expression of ROCK1 mRNA and miR-124. Two cancer cell lines were transfected with pre-miR-124 (mimic) and anti-miR-124 (inhibitor) and the effects on ROCK1 protein and mRNA expression were observed. In addition, cell proliferation was assessed via a 5-ethynyl-2' deoxyuridine assay. Soft agar formation assay, and cell migration and invasion assays were used to determine the effect of survivin on the transformation and invasion activity of CRC cells. miR-124 was significantly downregulated in CRC compared to normal specimens (0.603 ± 0.092 vs 1.147 ± 0.286, P = 0.016) and in metastatic compared to nonmetastatic CRC specimens (0.416 ± 0.047 vs 0.696 ± 0.089, P = 0.020). Expression of miR-124 was significantly associated with CRC metastasis, tumor T and N stages, and tumor grade (all P < 0.05). ROCK1 protein was significantly increased in CRC compared to normal tissues (1.896 ± 0.258 vs 0.866 ± 0.136, P = 0.026), whereas ROCK1 mRNA expression was unaltered (2.613 ± 0.251 vs 2.325 ± 0.246). miR-124 and ROCK1 were inversely expressed in CRC tissues and cell lines. ROCK1 mRNA was unaltered in cells transfected with miR-124 mimic and miR-124 inhibitor, compared to normal controls. There was a significant reduction in ROCK1 protein in cells transfected with miR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (Ps < 0.05). Transformation and invasion of cells transfected with miR-124 inhibitor were significantly increased compared to those in normal controls (P < 0.05). Cells transfected with miR-124 inhibitor showed increased cell proliferation. miR-124 promotes hyperplasia and contributes to invasion of CRC cells, but downregulates ROCK1. ROCK1 and miR-124 may play important roles in CRC.

Detection of aneugenicity and clastogenicity in buccal epithelial cells of pan masala and gutkha users by pan-centromeric FISH analysis.

Chewing of betel quid, smoking and alcohol consumption are all associated with higher incidences of oral cancer. Genetic damage can be detected by fluorescent in situ hybridization (FISH) using human centromeric probes. In the present study FISH was performed on buccal epithelial cells of pan masala and gutkha chewers alone with and without additional tobacco smoking and/or alcohol consumption. The study comprised of 1500 male individuals. The present study found the highest frequency of micronuclei without a centromeric region (MN(-)) among gutkha users who also smoked and drank (P < 0.05). A significant increase in cells having micronuclei with a centromeric region (MN(+)) was observed among pan masala users who also smoked (P < 0.05). The study reveals that the clastogenic effects of pan masala/gutkha increase with smoking and alcohol consumption, but aneugenic effects were also observed among the pan masala chewers who smoked.

Antineoplastic effects of Chlorella pyrenoidosa in the breast cancer model

ObjectivesThere has been considerable interest in both clinical and preclinical research about the role of phytochemicals in the reduction of risk for cancer in humans. The aim of this study was to determine the antineoplastic effects of Chlorella pyrenoidosa in experimental breast cancer in vivo and in vitro. MethodsIn this experiment, the antineoplastic effects of C. pyrenoidosa in the chemoprevention of N-methyl-N-nitrosourea–induced mammary carcinogenesis in female rats were evaluated. Chlorella powder was administered through diet at concentrations of 0.3% and 3%. The experiment was terminated 14 wk after carcinogen administration. At autopsy, mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. In vitro cytotoxicity assay, parameters of apoptosis, and proliferation after chlorella treatment in human breast adenocarcinoma (MCF-7) cells were carried out. ResultsBasic parameters of experimental carcinogenesis, mechanism of action (biomarkers of apoptosis, proliferation, and angiogenesis), chosen metabolic variables, and side effects after long-term chlorella treatment in animals were assessed. Chlorella at higher concentration suppressed tumor frequency by 61% (P < 0.02) and lengthened tumor latency by 12.5 d (P < 0.02) in comparison with the controls. Immunohistochemical analysis of rat tumor cells showed caspase-7 expression increase by 73.5% (P < 0.001) and vascular endothelial growth factor receptor-2 expression decrease by 19% (P = 0.07) after chlorella treatment. In a parallel in vitro study, chlorella significantly decreased survival of MCF-7 cells in a dose-dependent manner. In chlorella-treated MCF-7 cells, a significant increase in cells having sub-G0/G1 DNA content and significant increase of early apoptotic and late apoptotic/necrotic cells after annexin V/PI staining assay were found. Decreases in mitochondrial membrane potential and increasing reactive oxygen species generation were observed in the chlorella-treated MCF-7 cells. ConclusionsThis study is the first report on the antineoplastic effects of C. pyrenoidosa in experimental breast cancer in vivo and in vitro.

O1.05 * AXITINIB, A SELECTIVE VEGFR-1,-2,-3 TYROSINE KINASE INHIBITOR, INHIBITS TUMOR GROWTH AND INCREASES INFILTRATION OF IMMUNE CELLS IN AN INTRACRANIAL MELANOMA MOUSE MODEL

VEGF is a key mediator of pathological cancer associated neoangiogenesis and immunosuppression. VEGFR targeted therapy holds promise to counteract cancer associated neoangiogenesis but also to alleviate cancer associated immune suppression. Melanoma patients are at high risk for developing brain metastases. These lesions are strongly vascularized and have a high risk for spontaneous bleeding. Melanoma is susceptible to immunotherapy and patients affected by brain metastases might benefit from combining immunotherapy with anti-VEGFR therapy. The aim of this study was to investigate the effect of axitinib, a small molecule tyrosine kinase inhibitor of VEGFRs, on MO4 melanoma cell growth in vitro and on the composition of tumor-infiltrating immune cells in a subcutaneous and intracranial melanoma syngeneic mouse model. We first treated MO4 melanoma cells in vitro with increasing concentrations of axitinib and found no induction of apoptosis. Furthermore, increasing amounts of axitinib had no effect on T-cell proliferation or DC maturation, thus excluding a potential suppressive effect of axitinib on antigen presenting and T-cell function. When subcutaneous tumor-bearing mice were treated with axitinib (oral gavage, 25 mg/kg, bid), tumor growth was strongly inhibited and survival was significantly improved as compared to the control group. Next, subcutaneous and intracranial tumor bearing mice were treated with axitinib. After 7 days of treatment a significant increase in the number of total immune cells (CD45 + cells) within the spleen, subcutaneous and intracranial tumor was observed. Whereas the increase in immune cells within the spleen was most marked for CD3 + CD8 + T cells, a significant increase in the number of CD11b + cells was noted within the tumors. Upon further characterizing of this CD11b+ population, we found that there was a significant increase in cells with a monocytic myeloid derived suppressor cell phenotype (moMDSCs,CD11b + Ly6ChighLy6G-). However, in vitro proliferation assays using highly purified splenic MDSCs showed that moMDSCs isolated from axitinib-treated mice to have a reduced suppressive capacity on CD4+ and CD8+ T cells than those isolated from vehicle-treated mice on a per cell basis. When highly purified tumor MDSCs were used in in vitro proliferation assays we found that moMDSCs isolated from axitinib-treated mice almost lost their suppressive capacity on CD4+ and CD8+ T cells. Based on these observations we conclude that axitinib delays tumor growth, increases survival and is associated with an increased infiltration of the tumor micro-environment with immune cells both in subcutaneous as well as intracranial localizations. Axitinib seems to specifically increase moMDSCs within the CD11b+ cell population but to reduce their suppressive capacity. Further investigation of combination therapies with axitinib and immunotherapy seems warranted.

The effect of substrate elasticity on the adhesion and elasticity of vascular smooth muscle cells (1077.8)

In previous studies, we found vasoactive agonists induce coordinated changes in elasticity and integrin‐mediated adhesion to extracellular matrix (ECM) in primary vascular smooth muscle cells (VSMC) that were cultured on plastic/glass dishes. In this study, we hypothesized that the mechanical properties/stiffness of the substrate may play an important role in determining cellular elasticity and adhesion in VSMC. To simulate the in vivo mechanical environment of the ECM for VSMCs, a flexible polyacrylamide (PA) gel 2D substrate with tunable elasticity was used for cell attachment. A comparison of two substrate elasticities (7 and 30 kPa) were tested and collagen‐I was coated on the surface of PA gel to provide a biologically functional substrate to promote adhesion of VSMCs. An atomic force microscope (AFM) was used to measure VSMC elasticity and VSMC adhesion to FN coated AFM probes. Quantitative characterization of changes in cell morphology, adhesion, and elasticity were determined by custom‐developed software. Initial results showed a significant increase in cell elasticity/stiffness (~ +120%), adhesion force (~ +30%), and adhesion probability (~ +100%) of the VSMCs cultured on the stiffer (30 kPa) substrates compared with the cells cultured on softer substrates (7 kPa). These findings support the hypothesis that elasticity of substrate exerts an effect on the mechanical and adhesive properties of VSMCs.Grant Funding Source: NIH P01HL095486