Cell Viability Growth Research Articles

Abstract 1289: CSDE1 mediated translation reprogramming under stress

Abstract OBJECTIVE To explore the role and underlying mechanisms of CSDE1 (cold shock domain containing E1) in translation reprogramming under stress on genome-wide level. METHODS Cell 2D culture, clone formation and 3D culture were used to detect cell growth viability. Would healing assay was used to detect cell migrating ability. Flow cytometer was used to detected cell apoptosis, ROS (reactive oxygen species) and lipid-ROS. Immunofluorescence assay was taken to investigate the intracellular localization of proteins. CO-IP assay and protein spectrum were conducted to study interactive proteins. GST pull down assay was carried out to further identify the interaction between proteins. EMSA was carried out to explore the interaction between proteins and RNAs. Polysome profiling was used to isolating the actively translating mRNA. Isolated RNA was sequenced by RNA-seq to identify the mRNA regulated by CSDE1 on translational level. RESULTS Changing CSDE1 expression had little effect on cell growth, migrating, invasion in normal conditions. Then the role of CSDE1 in stress conditions was explored. For starvation stress, cells were cultured with RPMI 1640 or PBS, CSDE1 had no effect on cell vitality compared with control group. For ROS stress induced by H2O2, CSDE1 has no effect on cell grow rate and intracellular ROS level as well. For anoxic stress, the expression of CSDE1 influenced the expression of Hif-1α and cell apoptosis. For ferroptosis stress, knocking down CSDE1 increased the lipid-ROS and cell death. For DNA damage stress, cisplatin was used to construct cellular DNA damage model. CSDE1 could protect cell from DNA damage in NER (nucleotide excision repair) and DSB (double strand break) pathway and then promote cell growth. For ER (endoplasmic reticulum) stress, cells were treated with thapsigargin, and CSDE1 expression was changed over time during ER. CSDE1 can regulate the expression of CHOP, PERK and GRP78 which acted as the key proteins of unfolded protein reaction whether knocking down or overexpressing CSDE1. Besides, CSDE1 can inhibit cell autophagy and apoptosis during ER stress. Then the mechanism of CSDE1 in stress conditions was further explored. Under DNA damage stress, both CSDE1 and eIF3a regulated the translation of RPA2. CSDE1 colocalized with eIF3a and they bound with each other by CSD1 domain of CSDE1 and PCI domain of eIF3a. Besides, it is CSDE1 domain, but not eIF3a domain that can bind to RPA2 IRES. Therefore, CSDE1 acted as a bridge to connect eIF3a and RPA2 IRES and reprogram RPA2 translation. Under ER stress, knocking down CSDE1 significantly increased translation activity according to polysome profiling assay. Similarly, CSDE1 acted as a bridge to connect some proteins and RNAs to reprogram the translation. CONCLUSION CSDE1 works as a bridge to connect different protein regulators and RNAs under different stress. In this way, it can play a key role in translation reprogramming and thus reprogram the cell fate. Citation Format: Jiajia Cui, Ao-Xiang Guo, Lei-Yun Wang, Ji-Ye Yin. CSDE1 mediated translation reprogramming under stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1289.

Deciphering the Effect of Hydro-alcoholic extract of Cymodocea serrulata on the Cell Cycle Arrest and Apoptosis

Liver cancer is the most common cancer both in men and women. Hepato carcinoma is caused due to chemicals, food and also by even some drugs. Many therapies and therapeutic agents show side effects of liver cancer. A natural medicine Cymodoceae serrulata, seagrass under Cymodoceaceae family, found in the subtidal region may prove a source of benefit to avoid such side effects. Hydro ethanol extract of Cymodoceae serrulata shows high antioxidant and pharmacological activities, which may be used for the treatment of liver cancer. The earlier stage of Apoptosis can be identified by the nuclear changes such as DNA fragmentation. Propidium iodide can determine the proportion of cell cycle analysis—Annexin V conjugated to fluorochromes, including FITC. When the HepG2 test sample treated with the drug Cymodoceae serrulata, viable cell growth is significantly reduced compared to the standard camptothecin, which is an antitumor agent. Percentage of viable cell count declined significantly to 5.38%, and the percentage of the late apoptotic cell increased impressively 42.08%.

Cell investigation into the biocompatibility of adult human dermal fibroblasts with PCL nanofibers/TiO2 nanotubes on the surface of Ti-30Ta alloy for biomedical applications.

The polymer poly(ε-caprolactone) (PCL) has been used in the biomaterial field for its relatively inexpensive price and suitability for modification. Also, its chemical and biological properties are desirable for biomedical applications. The electrospinning process has been used for producing polymer fibers of PCL due in large part to an increased interest in nanoscale properties and technologies. Moreover, the use of biocompatible polymers for the viability of cell growth is a promising alternative to improve osseointegration. Characterization techniques such as scanning electron microscopy and contact angle were used for analyses of samples. Adult human dermal fibroblasts (neonatal) were utilized to evaluate the biocompatibility of the association of the electrospinning process of the biocompatible polymer (PCL) with TiO2 nanotubes on the Ti-30Ta alloy surface. The results of this study showed a favorable response for adhesion on the surface. This promising material is due to the modulation of the biological response.

Effect of siRNA interference of ubiquitin-specific protease 9X on apoptosis and growth of diffuse large B cell lymphoma cell line

Ubiquitin-specific protease 9X (USP9X) is crucial in the diagnosis and treatment of many tumor types, but its role in Diffuse Large B Cell Lymphoma (DLBCL) has not been determined. The current study aimed to examine the effects of RNA interference on USP9X expression, and subsequently on the bioactivity of DLBCL Farage and Pfeiffer cells. There were two groups in the study: USP9X-siRNA and NC. USP9X siRNA was transiently transferred into DLBCL cells by Cationic liposome. The total RNA was extracted using Fe2O3 and was retrieved into the DNA using the MagBeads Total RNA Extraction Kit. The protein expression of USP9X in Farage, Pfeiffer, and normal human B cell line at the cellular level was observed by Western blot. The Farage and Pfeiffer cells were infected with USP9X-siRNA. Cell apoptosis and cell growth viability were analyzed by flow cytometry and CCK8, Mcl-1 protein, a potential target of USP9X, and apoptosis factor proteins (such as Bak, Cytochrome C, Caspase 3, Caspase 8, PARP) were detected by Western blot after siRNA interference. The results showed that the protein expression of USP9X in malignant B cells was four times higher than that of the normal B cells. Inhibition of USP9X reduced the Mcl-1 activity, and increased the caspase-3, Bak and Cytochrome C activity. In the malignant B cells, Mcl-1 and Bak were binding in vivo; Bak was a new partner of Mcl-1. Inhibition of USP9X reduced cell proliferation and increased apoptosis. The expression of USP9X is upregulated in Diffuse large B cell lymphoma cells, Farage, and Pfeiffer. Inhibition expression of USP9X may induce cell apoptosis, inhibit cell growth, and downregulate Mcl-1 protein expression in Diffuse large B cell lymphoma cells, Farage, and Pfeiffer. USP9X has the ability in regulating cell apoptosis.

RGFP966 Suppresses Tumor Growth and Migration Through Inhibition of EGFR Expression in Hepatocellular Carcinoma Cells in vitro.

PurposeHistone deacetylase 3 (HDAC3) has been suggested to play a role in hepatocellular carcinoma (HCC). In the present report, we aimed to identify the effects of RGFP966, a specific HDAC3 inhibitor, on the cell proliferation and migration of HCC cell lines.MethodsHuman HCC cell lines, which were identified using short tandem repeat (STR) DNA profiling analysis, were used in this report. Cell proliferation assay was used to identify the growth viability of cells. Wound healing and transwell assay were used to identify the migration ability of cells. Further, a human phospho-receptor tyrosine kinases array kit was used to screen out RGFP966 effects on key receptor tyrosine kinases. Then, the mRNA expression was quantified by real-time PCR, and protein expression was identified by Western blot immunoassay.ResultsWe found that RGFP966 inhibited both proliferation and migration of HCC cells. Further, RGFP966 represses the expression and phosphorylation levels of epidermal growth factor receptor (EGFR) in HCC cells. Moreover, HDAC3 is involved in the inhibition of EGFR by RGFP966. Overall, we elucidated an inhibitive function of RGFP966 in HCC progression.ConclusionRGFP966 inhibits EGFR signaling pathway and suppresses proliferation and migration of HCC cells.

Antifungal activity of ZnO thin films prepared by glancing angle deposition

Thin films of zinc oxide (ZnO) were produced by reactive DC magnetron sputtering with incidence angles of α = 0°, 40°, 60° and 80° (glancing angle deposition configuration), aiming to be tested against the opportunist pathogenic fungus Candida albicans. The results showed the formation of stoichiometric ZnO thin films, with inclined columns for incidence angles equal to or >40°. All thin films presented high transparency in the visible range above the bandgap region (near 380 nm). The deposition conditions gave rise to a three-fold increase of the surface porosity with the increment of the incidence angle. Noteworthy, is the formation of different types of pores distributions, from micro-, through meso-, to macropores. Regarding the biological effect, the thin films produced with inclined columns presented a significant antifungal activity, with the inhibition of viable cell growth by 68%. Moreover, the formation of mesoporous films enhanced the antifungal properties of ZnO thin films against Candida albicans. The overall behaviour indicates that these thin films are promising candidates to be applied in antimicrobial surfaces, as well as to be used in further studies to determine the molecular mechanisms involved in the antimicrobial action of ZnO.

Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppressestumor growth invivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.

Transient gene expression using valproic acid in combination with co-transfection of SV40 large T antigen and human p21CIP /p27KIP.

Transient gene expression (TGE) in HEK293 cells was optimized by Vink et al. by co-expression of human cell cycle inhibitors p21CIP /p27KIP and Simian virus 40 large T antigen (SVLT). In this study, we investigated the effect of this enhancer protein complex on the TGE experiments in a cell-cycle arrested condition of HEK293F cells induced by valproic acid. Growth profiles, consumptions of nutrients, formations of waste products, and product titers of recombinant human antibodies (huAb) were monitored during the 7-day cultivation time. Our results showed that the use of enhancer proteins increased the product yields in a growth arrest condition as well. During the growth phase, no differences were detected regarding viable cell densities (VCDs), viabilities, growth rates, and cell diameters between the TGE experiments with and without enhancer proteins. However, during the declining phase VCD and viability showed slightly higher values at day 6 and 7 in the presence of enhancers. Furthermore, we could not detect any differences in glucose and glutamine metabolism during batch cultivations with co-expression of enhancer proteins. Taken together, the special complex of enhancer proteins did not contribute to further enhancement of growth arrest and shift in the main cell metabolisms, but resulted in higher cell viability during the decline phase. Our observations suggest that the human cell cycle inhibitors p21CIP /p27KIP together with very low amount of SVLT antigen may induce alternative functional activities than growth arrest to further improve the yield of recombinant proteins.

How the competitive exclusion principle can be validated using optical density measurements collected on artificially reconstituted soil ecosystems

A mathematical model, validated on experimental data aiming at describing and predicting soil bacteria growth on an essential limited substrate in batch pure cultures is proposed as an extension of the Monod’s one in revisiting the way where the optical density is modelled. This model takes into account viable cell growth, substrate consumption, cell mortality, non-viable cell accumulation in the culture medium and partial dead cell recycling into substrate. The least squares method is used to identify model parameters. The model is extended and validated for mixed cultures proving, for artificially reconstituted soil ecosystems, that there is only competition for the substrate. Mathematics Subject Classification: 34D23, 35N25, 37B25, 49K40, 00A71.

Cytoprotective Activity of Neichitti (Vernonia cinerea) in Human Embryonic Kidney (HEK293) Normal Cells and Human Cervix Epitheloid Carcinoma (HeLa) Cells against Cisplatin Induced Toxicity: A Comparative Study

Introduction: Traditional Siddha Medicine literatures suggest to use the decoction of Vernonia cinerea (VC) to alleviate toxic effects caused by metals. Cisplatin, a metal used in cancer treatment is known to cause nephrotoxicity.Aim: To evaluate and compare protective activity of aqueous extract and fractions of VC in Human Embryonic Kidney (HEK293) cells and Human Cervix Epitheloid Carcinoma (HELA) cell lines against cisplatin induced cytotoxicity.Materials and Methods: The Crude Aqueous Extract (CAE) was obtained from whole plant of VC. CAE was fractioned using nonpolar to polar solvents, and Butanol Fraction (BF) as well as Aqueous Fractions (AF) were obtained. The cell lines were exposed with the IC50 dose of cisplatin, then treated with different doses of the extract or fractions. After 48 hours of incubation, viable cell growth was determined by MTT reduction assay in all the treatments by observing at absorbance of 540 nm. Dose response (% cell viability) curve was drawn and the values were statistically compared using One-Way ANOVA, followed by Dunnett’s (2-sided) post-hoc test.Results: Aqueous fraction showed 2.8 times higher cytoprotection (improvement in cell viability by 54%) than CAE against cisplatin induced toxicity in HEK293 cells. BF did not show protective activity in HEK293 cells. In HELA cells, AF showed 1.73 times higher cytoprotection than CAE and 1.55 times higher cytoprotection than BF against cisplatin, as evidenced by improvement in cell viability by 30%, 11% and 13% in AF, CAE and BF respectively. AF exhibited 80% higher cytoprotection against cisplatin cytotoxicity in normal HEK293 than cancer HELA cells. Whereas, CAE and BF showed lesser cytoprotective activity than AF.Conclusion: This study created scientific basis for using Vernonia cinerea against metal toxicities in Siddha system and suggests that the AF is suitable for further discovery of drug that selectively protect normal renal cell during cisplatin treatment in cancer patients.

MiR-126-3p inhibits ovarian cancer proliferation and invasion via targeting PLXNB2

Ovarian cancer is one of the leading malignancies in women and the 5-year survival rate of ovarian cancer still remains poor. In the present study, we aimed to investigate the interaction between the miR-126-3p and PLXNB2 in the progression of ovarian cancer. The qRT-PCR data revealed a reduction of miR-126-3p level in ovarian cancer tissues comparing to the adjacent normal tissues. Over-expression of miR-126-3p in ovarian cancer cells suppressed cell proliferation and invasion and the phosphorylation of AKT and ERK1/2. The cell cycle assay results showed that the over-expression of miR-126-3p induced cells in G1-phase and reduced cells in S-phase. We further performed bioinformatics analysis and luciferase assay to investigate the relationship between miR-126-3p and PLXNB2 in ovarian cancer cells. The results of TargetScan suggested that PLXNB2 is a direct target of miR-126-3p in ovarian cancer cells, and luciferase assay confirmed bioinformatics prediction. Knocking down of PLXNB2 with PLXNB2 siRNA results in repressed ovarian cancer cell proliferation and invasion, and decreased phosphorylation of AKT and ERK1/2, which is similar to the effect of over-expression of miR-126-3p in OC cells. The synergistic effect of combination of miR-126-3p over-expression and PLXNB2 down-regulation on the cell growth viability, cell colony, and cell invasion was also identified. All these findings indicated that miR-126-3p is involved in the progression of ovarian cancer via direct regulating PLXNB2.

Cell and Bacteria-Baterial Interactions on the Ti10Mo8Nb Alloy After Surface Modification

The purpose of this study is the surface modification of Ti10Mo8Nb experimental alloy by using TiO2 nanostructure growth. Ingots of the Ti10Mo8Nb experimental alloy were produced by fusion from sheets of molybdenum, niobium, and titanium commercially pure in an arc melting furnace under argon atmosphere. The potentiostatic anodic oxidation was performed using an electrolyte formed of glycerol and H2O (1: 1 by vol.) with the addition of 2.7% (w/v) NH4F under 20 V for 3 h at room temperature. After the surface treatment, the samples were annealing. The surface of the alloy was analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), contact angle, surface energy measurements. In vitro studies, such as cell and bacterial adhesion, were performed to evaluate the response of this treatment. The Ti10Mo8Nb alloy exhibited beta phase after the processing. After the surface treatment, a nanoporous layer of TiO was obtained. The anatase phase was found in the annealed samples (450 oC for 3 hours). In vitro analyzes showed cell growth viability and decreased bacterial proliferation due to the larger specific surface area.

Effect of Electrospun Phb and Hap-Phb Composite Scaffolds Characteristics on Mesenchymal Stem Cell Growth Viability

Effect of Electrospun Phb and Hap-Phb Composite Scaffolds Characteristics on Mesenchymal Stem Cell Growth Viability

Isocitrate Dehydrogenase 1 Mutant Cancers Are Metabolically Vulnerable to Inhibition of Acetyl CoA Carboxylase Via a 2-Hydroxyglutarate Independent Mechanism

Introduction: Mutations substituting arginine 132 of isocitrate dehydrogenase 1 (IDH1) are recurrent in acute myeloid leukemia (AML) and several other cancers, resulting in the aberrant production of the onco-metabolite, R-2-hydroxyglutarate (2-HG), as well as an inability to convert cytoplasmic alpha-ketoglutarate to isocitrate via reductive carboxylation. Currently, small molecules that effectively inhibit the neomorphic enzyme and abrogate the production of 2-HG, such as AG-120, are in clinical trials with promising results. However, these inhibitors have not proven to be curative in most AML cases, indicating a need for additional targeted therapies. We have previously investigated synthetic lethal vulnerabilities in IDH1-mutated AML and identified an interaction with BCL2 leading to increased susceptibility to ABT-199 (Chan et al, 2015). Synthetic lethal approaches targeting 2-HG independent metabolic vulnerabilities conferred by mutant IDH1 may complement IDH1 mutant inhibitors. Using a novel computational method (MiSL) based on Boolean implication (if-then rules) mining of pan-cancer data, we identified acetyl CoA carboxylase (ACACA) as a potential druggable target in IDH1-mutated AML. ACACA is the rate-limiting step in the de novo synthesis of fatty acids, and mutant IDH1 leads to a reduction in malonyl-CoA, a key building block for fatty acids, in a 2-HG independent manner. This finding led us to investigate a potential synthetic lethal interaction between mutant IDH1 and ACACA based on the hypothesis that the combination causes marked inhibition of fatty acid synthesis required for cell growth.Methods: Boolean implications (MiSL) were used to identify candidate synthetic lethal interactions with mutant IDH1 by isolating genes deleted only in the absence of the mutation and with differential gene expression within pan-cancer TCGA data. Validation was performed using THP-1 cells transduced with doxycycline-inducible wildtype and R132H mutant IDH1 lentiviral vectors, and primary patient IDH1-mutant and wildtype AML samples, using both shRNAs and a targeted pharmacologic inhibitor of ACACA. Metabolomics was performed using semi-targeted mass spectrometry and liquid chromatography. Finally, primary AML samples and IDH1-mutant and wildtype cancer cell lines (HT-1080, U118, U87) were transduced with validated shRNA and engrafted into NSG mice.Results: Our computational method found that IDH1 mutation and ACACA deletions were mutually exclusive in pan-cancer TCGA data, ACACA deletions resulted in lowered expression of ACACA, and ACACA was differentially over-expressed in IDH1-mutant AML compared to IDH1-wildtypeAML. Pharmacologic inhibition of ACACA with 2 uM TOFA caused a marked reduction in cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox; p = 0.0001). Similarly, knockdown of ACACA with independent shRNAs caused a defect in viable cell growth in the presence of IDH1 R132H (+ dox), but not in its absence (- dox) or with scrambled shRNA (p=0.009, shRNA #1 vs. scrambled; p=0.01, shRNA #2 vs. scrambled). Primary IDH1 R132 mutated purified AML blasts were selectively sensitive to TOFA treatment compared to IDH1 wildtype normal karyotype blasts (IC50 0.6 uM vs 6 uM, p=0.009) in viable growth assays. Furthermore, when transduced with lentivirus encoding shRNA to ACACA, primary IDH1-mutant AML cells exhibited markedly reduced engraftment of RFP-positive human CD45+CD33+ leukemic cells compared to scrambled non-targeting shRNA (p < 0.05, Mann-Whitney U). As predicted, IDH1-mutant AML blasts pre-treated with 10uM AG-120 (sufficient to inhibit production of detectable 2-HG) remained susceptible to ACACA inhibition in vitro. Strikingly, in vivo models of IDH1 R132C mutated, but not wildtype, sarcoma cell lines exhibited a dramatic decrease in cell growth after ACACA inhibition that was not reversible by treatment with AG-120. Finally, metabolomic profiling revealed a major perturbation in multiple phospholipid fatty acid species and decreased malonyl-CoA conferred by IDH1 R132H, consistent with our proposed mechanism.Conclusion: We have identified de novo lipogenesis through ACACA as a critical metabolic vulnerability linked to IDH1 mutation in AML and provide evidence that therapeutic inhibition of ACACA with small molecules may be beneficial in AML, as well as in other cancers with IDH1 mutations. DisclosuresMajeti:Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

The Suppression of CCL2 Expression Decrease the Proliferation of HL-60 Cells through Downregulation of Cyclin d1 Via Arresting Cells at the G1 Phase

Background and purpose: Chemokine (C-C motif) ligand 2(CCL2) is a member of the CC subfamily which displays chemotactic activity for monocytes and basophils. It has played a very important role in many solid tumors and changes in bone marrow microenvironment. However, its role in acute myeloid leukemia (AML) has not yet been clear. In this point, we established a cell line with CCL2 down-expression to explore the effect of CCL2 gene on leukemogenesis.Methods: Lentivirus with CCL2-knockdown was successfully constructed after screening effective CCL2 shRNA sequence and tranfected into HL-60 cells which was validated on the level of mRNA and protein by real-time PCR and Western blot. The cells coming from parental, sh-Vectors and shCCL2 were detected for cell growth viability by CCK-8 assay, cell cycles and apoptosis by Flow cytometry. We applied exon sequencing technology to identify the gene profiling between the CCL2 knockdown and the control, of which, Cyclin d1 was selected for further experiments as its expression level was significantly downregulated. Then we successfully down regulated cyclin d1 expression in HL-60 by means of RNA interference to detect the cell proliferation through CCK-8 assay, cell cycles and apoptosis through Flow cytometry.Results: HL-60 cell line expressed the highest level of CCL2 among acute leukemia cell lines (Figure 1). Among 4 pairs of CCL2 interference sequences, only pair 2 had the most efficient potential in knockdown CCL2 expression which was constructed into sh-Vector, GV248, and validated by real-time RT-PCR and Western blot(Figure 2). Low expression of CCL2 significantly decreased HL-60 cell growth. Meanwhile, the CCL2-shRNA-mediated HL-60 cells showed about 12% more cells arrested in G1 phase compared with controls (Table 1, Figure 3). The results of expression profiling showed that there were total 159 genes differentially expressed (Figure 4), of which, ten top pathways were illustrated in Table 2. Cyclin D1 was related to cell cycle, NOD-like receptor signaling pathway, TNF signaling pathway and NF-kappa B signaling pathway which was the lowest expression among cell cycle gene related in HL-60 cells transfect with shCCL2(Table 2, highlighted raw) and further validated by real-time RT-PCR and Western blot (Figure 5). After Cyclin D1 was decreased on the level of mRNA and protein of HL-60, the cell proliferation was evidently slow and cell cycle analysis also indicated a similar pattern of CCL2 (Figure 6).Conclusion:CCL2 involved in cell proliferation which was mediated by cyclin D1 via blocking more cells at G1 phase.Figure 3. Knockdown of CCL2 inhibits cell proliferation via G1 phase arrested. ADown regulation of CCL2 influenced cell proliferation. From day 2 to 5, the proliferation rate of HL-60 cells transfected by shCCL2 grew significantly slower than controls. B: CCL2 played a role in cell cycle process. More cells transfected shCCL2 were arrested in G1 phase compared with controls. *Indicate significant differences with P-values <0.05Figure 5. Cylin D1 was the most influenced gene among cell proliferation related genes profile. Aquantitative RT-PCR analysis showed that mRNA levels of preliferation related genes: PCNA, cyclin D1, c-jun, surviving, erk1, and erk2. Among them, Cyclin D1 was expressed lowest. B: Western blot analysis confirmed that the protein expression of total cyclin D1 was much lower compared with controls.Figure 6. The effect of Cyclin D1 on cell proliferation. ACyclin D1 was successfully knockdowned in HL-60. mRNA levels were determined by quantitative RT-PCR and revealed that Cyclin D1 expression was knockdowned by the vector of shccnd. B: Knockdown of cyclin D1 inhibits cell proliferation. Compared with control, HL-60 cells with low level of Cyclin D grew significant slowly. C: Down regulation of Cyclin D1 influences more cells arrested in G1 phase compared with control.*P-values <0.05 [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vitro biocompatibility study of keratin/agar scaffold for tissue engineering

The porous scaffold was fabricated from a binary blend of keratin/agar for tissue engineering. The miscibility of keratin and agar polymers into their blend was confirmed by Fourier transform infrared spectroscopy and X-ray diffractometer study. The scaffold fabricated from freeze extraction method resulted in a porous interconnected structure with apparent porosity 94.40±2.34%. Scanning electron microscopy study reveals the presence of interconnected pores with a pore size ranges from 50 to 300μm. The hydrophilic nature of the scaffold was confirmed by water retention capacity studies, which was observed 160±7.89%. The scaffold's tensile strength of 0.154±0.031MPa and percent of elongation at break with 16.33±2.52% justify its mechanical capability. The positive antimicrobial property and in vitro degradation was recorded for the fabricated scaffold. The in vitro biocompatibility study of the scaffolds confirms the attachment and proliferation of the cultured mammalian myofibroblast cell line. Negative cytotoxicity and a viable cell growth ensure that the fabricated scaffold can serve a potential source of biomaterial for tissue engineering and can be applicable for wound healing and skin regeneration.

An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair.

AimTo compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair.MethodsHuman BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively.ResultsA greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro-Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source.ConclusionChondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue.

HBx protein-induced upregulation of microRNA-221 promotes aberrant proliferation in HBV‑related hepatocellular carcinoma by targeting estrogen receptor-α.

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). Emerging evidence has shown the association between aberrantly expressed miR-221 and cancer development; however, little is known concerning its potential role in hepatitisB virus (HBV)-related HCC. In the present study, functional studies demonstrated that HBx leads to the promotion of cell proliferation and cell growth viability. Obviously overexpressed miR-221 was found in HBx-transfected cells compared with the mock counterparts. Suppression of miR-221 significantly inhibited HCC cell proliferation. Western blot analysis indicated that estrogen receptor-α (ERα) was downregulated in HCC tissues and cell lines. Bioinformatic analysis combined with validation experiments identified ERα as a direct target of miR-221. The present study suggests that miR-221 modulates HCC cancer cell proliferation by suppressing ERα, functioning as a tumor promoter. Moreover, our data imply that miR-221 has potential as an miRNA-based therapeutic target for HBV-related HCC.

MiR-145 is downregulated in human ovarian cancer and modulates cell growth and invasion by targeting p70S6K1 and MUC1

MicroRNAs (miRNAs) are a family of small non-coding RNA molecules that regulate gene expression at post-transcriptional levels. Previous studies have shown that miR-145 is downregulated in human ovarian cancer; however, the roles of miR-145 in ovarian cancer growth and invasion have not been fully demonstrated. In the present study, Northern blot and qRT-PCR analysis indicate that miR-145 is downregulated in ovarian cancer tissues and cell lines, as well as in serum samples of ovarian cancer, compared to healthy ovarian tissues, cell lines and serum samples. Functional studies suggest that miR-145 overexpression leads to the inhibition of colony formation, cell proliferation, cell growth viability and invasion, and the induction of cell apoptosis. In accordance with the effect of miR-145 on cell growth, miR-145 suppresses tumor growth in vivo. MiR-145 is found to negatively regulate P70S6K1 and MUC1 protein levels by directly targeting their 3′UTRs. Importantly, the overexpression of p70S6K1 and MUC1 can restore the cell colony formation and invasion abilities that are reduced by miR-145, respectively. MiR-145 expression is increased after 5-aza-CdR treatment, and 5-aza-CdR treatment results in the same phenotype as the effect of miR-145 overexpression. Our study suggests that miR-145 modulates ovarian cancer growth and invasion by suppressing p70S6K1 and MUC1, functioning as a tumor suppressor. Moreover, our data imply that miR-145 has potential as a miRNA-based therapeutic target for ovarian cancer.

Effect of hOGG1 Over-Expression on Cisplatin Resistance in Esophageal Squamous Carcinoma Cells

Human 8-oxoguanine DNA glycosylase (hOGG1) is an ubiquitous protein. It initiates the DNA base excision repair (BER) pathway to repair the 8-oxoguanine lesion. This may be associated with chemotherapeutics. In this article, the effect of hOGG1 over-expression on cisplatin resistance in esophageal squamous cell carcinoma (ESCC) EC9706 and ET13 cells was investigated. Recombinant adenovirus pAd/CMV/V5-DEST-hogg1 and control adenovirus pAd/CMV/5-GW/lacZ were constructed and transferred into EC9706 and ET13 cells, respectively. The protein expression and localization were determined by Western blot and by immunofluorescence assay. The cell growth viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5 diphe-nyltetrazolium bromide (MTT) assay and clonogenic survival assay. The apoptotic cells were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. The oxidative DNA damage (8-Hydroxyguanine [8-oxoG] DNA level) was semi-quantified by immunohistochemistry assay. The over-expression of hOGG1 protein was mainly in the nucleus in hOGG1 cells. After exposure to a common chemotherapeutic agent cisplatin, hOGG1 over-expression cells exhibited longer survival ability, lower cell apoptosis, and less 8-oxoG oxidative damage, compared with vector-treated cells and no-treated cells (p<0.05). BER pathway to repair 8-oxoG lesion may be associated with ESCC sensitivity to cisplatin, and over-expression of hOGG1 in the nucleus can repair more 8-oxoG oxidative damage. The findings implied that over-expression of hOGG1 can protect ESCC cells from cisplatin-induced apoptosis and prolong cancer cell survival time. Modulation of DNA damage repair activity in the nucleus or in the mitochondria may lead to a different approach regarding cisplatin-induced resistance to chemotherapy.