Percentage Of Cells In G1 Phase Research Articles

N-acetylcysteine as a novel methacrylate-based resin cement component: effect on cell apoptosis and genotoxicity in human gingival fibroblasts

BackgroundN-acetylcysteine (NAC) reduces the cytotoxicity and genotoxicity induced by monomers leached from dental composite resins. Herein, we investigated the effects of methacrylate-based resin cement used in dental implant restoration on apoptosis and genotoxicity, as well as the antiapoptotic and antigenotoxic capabilities of its component, NAC.MethodsThe antioxidant NAC (0.1 or 1 wt.%) was experimentally incorporated into the methacrylate-based dental resin cement Premier®. The Premier® + NAC (0.1 or 1 wt.%) mixture was subsequently immersed into Dulbecco’s modified Eagle’s medium for 72 h, and used to treat human gingival fibroblasts (HGFs). The viability of HGFs was determined using the XTT assay. The formation of deoxyribonucleic acid (DNA) double-strand breaks (DNA-DSBs) was determined using a γ-H2AX assay. Reactive oxygen species (ROS), apoptosis, necrosis, and cell cycles were detected and analyzed using flow cytometry.ResultsThe eluate of Premier® significantly inhibited HGF proliferation in vitro by promoting a G1-phase cell cycle arrest, resulting in cell apoptosis. Significant ROS production and DNA-DSB induction were also found in HGFs exposed to the eluate. Incorporating NAC (1 wt.%) into Premier® was found to reduce cell cytotoxicity, the percentage of G1-phase cells, cell apoptosis, ROS production, and DNA-DSB induction.ConclusionIncorporating NAC (1 wt.%) into methacrylate-based resin cement Premier® decreases the cell cytotoxicity, ROS production, and DNA-DSBs associated with resin use, and further offers protective effects against the early stages of cell apoptosis and G1-phase cell cycle arrest in HGFs. Overall, our in vitro results indicate that the addition of NAC into methacrylate-based resin cements may have clinically beneficial effects on the cytotoxicity and genotoxicity of these materials.

Overexpression of BRG1 improves early development of porcine somatic cell nuclear transfer embryos

The epigenetic modification levels of donor cells directly affect the developmental potential of somatic cell nuclear transfer (SCNT) embryos. BRG1, as an epigenetic modifying enzyme, has not yet been studied in donor cells and SCNT embryos. In this study, BRG1 was overexpressed in porcine fetal fibroblasts (PFFs), its effect on chromatin openness and gene transcription was examined, subsequently, the development potential of porcine SCNT embryos was investigated. The results showed that compared with the control group, the percentage of G1 phase cells was significantly increased (32.3 % ± 0.87 vs 25.7 % ± 0.81, P < 0.05) in the experimental group. The qRT-PCR results showed that the expression of H3K9me3-related genes was significantly decreased (P < 0.05), HAT1 was significantly increased (P < 0.05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results revealed that SMARCA4、NANOG、SOX2、MAP2K6 and HIF1A loci had more open chromatin peaks in the experimental group. The RNA-seq results showed that the upregulated genes were mainly enriched in PI3K/AKT and WNT signaling pathways, and the downregulated genes were largely focused on disease development. Interestingly, the developmental rate of porcine SCNT embryos was improved (27.33 % ± 1.40 vs 17.83 % ± 2.02, P < 0.05), the expression of zygotic gene activation-related genes in 4-cell embryos, and embryonic development-related genes in blastocysts was significantly upregulated in the experimental group (P < 0.05). These results suggest that overexpression of BRG1 in donor cells is benefit for the developmental potential of porcine SCNT embryos.

Effect of active vitamin D on proliferation, cell cycle and apoptosis in endometriotic stromal cells

Research questionWhat is the effect of vitamin D3 (1,25(OH)2D3) on proliferation, cell cycle and apoptosis of endometrial stromal cells (ESC) in endometriotic patients? DesignESC isolated from 10 women with endometriosis and 10 healthy controls were treated with 1,25(OH)2D3. The proliferation of control endometrial stromal cells (CESC), eutopic endometrial stromal cells (EuESC) and ectopic endometrial stromal cells (EESC) was analysed 72 h after the treatment using methyl thiazolyl tetrazolium assay. Propidium iodide staining and flow cytometry were used to determine the cell cycle distribution in ESC. Annexin V/propidium iodide double staining was used to evaluate apoptosis in ESC. ResultsIn the presence of oestrogen, 1,25(OH)2D3 treatment inhibited the proliferation of ESC from all three origins (P = 0.009 for CESC, P = 0.005 for EuESC and P < 0.001 for EESC). The percentage of S phase cells in EESC was higher than in EuESC and CESC (P = 0.002 and P = 0.001, respectively). The percentage of S phase cells in EuESC was higher than in CESC (P = 0.005). The percentage of G1 phase cells in EESC was lower than that of EuESC and CESC (P = 0.003 and P = 0.002, respectively) and the percentage of G1 phase cells in EuESC was lower than that of CESC (P = 0.007). Moreover, 1,25(OH)2D3 inhibited cell cycle regardless of cell type (P = 0.002 in EESC, P = 0.001 in EuESC and P = 0.014 in CESC), but in the absence of oestrogen, inhibited cell cycle only in EuESC (P = 0.012). ConclusionsAlthough 1,25(OH)2D3 increased apoptotic and necrotic cells and decreased live cells in the EuESC and EESC, it did not affect apoptosis in CESC and only increased necrotic cells. These findings indicate that 1,25(OH)2D3 potentially has a growth-inhibiting and pro-apoptotic effect on ESC from endometriotic patients.

SPATA33 affects the formation of cell adhesion complex by interacting with CTNNA3 in TM4 cells

Communication between Sertoli cell is essential during spermatogenesis and testicular development in mice, and the dynamic balance of this communication is regulated by some adhesion proteins. In this study, we found that SPATA33 and CTNNA3 were involved in this process. Quantitative real-time PCR and western blotting showed similar trend of expression of two proteins in the testis of mice of different ages. Subsequently, CRISPR-Cas9 technique was used to prepare Spata33 knockout cell lines with TM4 cells, cell wound scratch assay showed that Spata33 gene knockout affected cell migration, and flow cytometry assay showed that Spata33 knockout resulted in a decreased percentage of G1 phase cells in TM4 cell line. In addition, phalloidin staining assay showed that Spata33 gene knockout disrupted the formation of F-actin. Moreover, the protein immunoprecipitation experiment showed the interaction between SPATA33 and CTNNA3, which affected the interaction between CTNNA3 and CTNNB1. SPATA33 inhibits the formation of CDH1-CTNNB1-CTNNA3 complex through its interaction with CTNNA3, thus weakening adhesion between Sertoli cell and promoting cell migration.

Predict the role of lncRNA in kidney aging based on RNA sequencing

BackgroundLong noncoding RNAs (lncRNAs) are involved in physiological and pathological processes. However, no studies have been conducted on the relationship between lncRNAs and renal aging.ResultsFirst, we evaluated the histopathology of young (3-month-old) and old (24-month-old) C57BL/6J mouse kidneys. Masson trichrome staining and PAS staining showed interstitial collagen deposition and fibrosis, mesangial matrix expansion, a thicker basement membrane and renal interstitial fibrosis in old mouse kidneys. Senescence-associated β-galactosidase (SA-β-gal)-positive areas in the kidneys of old mice were significantly elevated compared to those of young mice. Then, we analyzed the differential expression of lncRNAs and mRNAs in the kidneys of young and old mouse kidneys by RNA-seq analysis. 42 known and 179 novel differentially expressed lncRNAs and 702 differential mRNAs were detected in the mouse kidney. Next, we focused on the differentially expressed mRNAs and lncRNAs by RNA-seq. GO and KEGG analyses were performed based on differentially expressed mRNAs between young and old mouse kidneys. Transregulation based on RIsearch and the correlation coefficient of mRNA-lncRNA were also calculated. The mRNA-lncRNA network was constructed by choosing a Spearman correlation coefficient > 0.9 or <-0.9. GO and KEGG pathway enrichment analyses revealed that differentially expressed mRNAs participated in aging-related pathways. A total of 10 lncRNAs and trans-regulated mRNAs were constructed. Finally, we validated the role of lncRNA Gm43360 by CCK-8, flow cytometry, western blot and SA-β-gal staining. The expression level of Adra1a was positively correlated and Csnk1a1 was negatively correlated with lncRNA Gm43360. The cell counting kit-8 (CCK-8) results showed that lncRNA Gm43360 promoted cell viability. LncRNA Gm43360 increased the percentage of S phase cells and decreased the percentage of G1 phase cells compared with the negative control. LncRNA Gm43360 decreased the expression of p53, p21 and SA-β-gal.ConclusionsLncRNA Gm43360 may play a protective role in kidney aging.

Total Phenols, Identification of Active Compounds and Anticancer Activity of Salvia judaica Boiss against the breast Cancer Cell MDA-231

Salvia judaica is an annual herb from genus Salvia L.; the largest genera of Lamiaceae. It’s a medicinal plant prominent in pharmaceutical applications in many countries around the world. This study aimed to explore bioactive compounds likely to be responsible for the plant anticancer activity, and evaluate anticancer effects, after determining the total content of phenols in the ethanol extract and essential oil in this species. Ethanol extract (EE) and essential oil (EO) were prepared from dried aerial parts (leaves and the flower). GC-MS analysis of EO showed the presence of/43/ effective compounds in varying proportions, the major compounds were sesquiterpenes like delta-cadinene, alpha-Gurjunene, beta-humulene, and alpha-caryophyllene. This is the first study revealed that S.judaica is so rich in phenols which proceeded S.officinalis, noting the superiority of the EE over the EO samples in the total phenols. Anticancer properties of EE and EO of S. judaica against MDA-231 breast cancer cell line were studied -for the first time - by cell cycle analysis and Annexin V/PI apoptosis assay using Flow cytometry technique. Cells were treated with EE (0.001, 0.01, 0.02, 0.1mg/ml) and EO (0.005, 0.01, 0.02, 0.03, 0.04 mg/ml) at various concentrations for48 h. The results revealed that both EE and EO induced cell cycle arrest at G1-phase. Cells treated with EE and EO for 48h showed increasing the percentage of cells in G1-phase and decreasing the percentage of cells in S-phase with increasing concentration compared with untreated cells (control). Annexin V-FITC/PI assay confirmed that EO and EE were able to induce apoptosis. Cells treated with EOat (0.04 mg/ml) for 48h resulted in apoptotic cells at 96.68%, and necrotic cells at 0.12%, compared with untreated cells. On the other hand, Cells treated with EE at (0.1 mg/ml) for 48h resulted in apoptotic cells at 94.43%, and necrotic cells at 0.47%, compared with control. Results revealed that EO is better than EE as anticancer; treatment with EO resulted in more apoptotic cells and less necrotic cells, and there were significant differences between them. This confirmed that EO contains specific anticancer compounds as showed by GC-MS analysis. However, more studies should be performed to explore antioxidants present in S.judaica and determine the underlying mechanism of their anti-breast cancer properties.

Dioscorea deltoidea Leaf Extract (DDLE) Targets PI3K/AKT/mTOR Pathway and Inhibits Ovarian Cancer Cell Growth.

Ovarian cancer is the malignant tumour of the female reproductive organ with highest mortality rate among all the types of gynaecological tumours. This study investigated the effect of Dioscorea deltoidea leaf extract (DDLE) on OV-90 and CAOV4 ovarian cancer cells. The results demonstrated that DDLE suppresses OV-90 and CAOV3 cell viability significantly in dose dependent manner. The OV-90 and CAOV3 cell viability were reduced to 24 and 27% respectively with 20 mg/mL DDLE treatment. Five mg/mL DDLE treatment of OV-90 and CAOV4 cells raised percentage of cells in G2-phase to 55.9 and 51.2%, respectively. In 5mg/mL DDLE -treated OV-90 and CAOV4 cells a prominent suppression in cyclin-D1 and cyclin B1 proteins was observed in 48 h. The DDLE treatment promoted OV-90 and CAOV3 cell apoptosis to 34.65 and 29.89%, respectively. The Fas, FasL, cleaved caspase-3, and Bax levels were up-regulated markedly in the cells after DDLE treatment. Moreover, DDLE treatment suppressed p-mTOR, p-AKT and p-PI3K expression in OV-90 and CAOV3 cells. Thus, DDLE suppressed ovary cancer cell viability and elevated cell apoptosis. Inhibitory effect of DDLE on ovarian cancer cells is associated with targeting PI3K/AKT/mTOR pathway.

Study of thermal injury effects on human HaCaT cells under simulated microgravity environment

Objective: To investigate the thermal injury effects on human HaCaT cells under simulated microgravity environment. Methods: The human HaCaT cells were collected and divided into simulated microgravity thermal injury (SMGTI) group, normal gravity thermal injury (NGTI) group, and normal gravity false injury (NGFI) group according to the random number table. Cells in NGTI and NGFI groups were cultured routinely in culture bottle, and cells in SMGTI group were cultured in the rotary cell culture system to simulate microgravity environment. Cells in SMGTI and NGTI groups were bathed in hot water of 45 ℃ for 10 minutes to make thermal injury model, and cells in NGFI group were bathed in warm water of 37 ℃ for 10 minutes to simulate thermal injury. At post injury hour (PIH) 12, cell morphology of 3 groups was observed under inverted phase contrast electron microscope. At PIH 2, 6, and 12, single cell suspension in the 3 groups was collected to detect the cell cycle by flow cytometer and the mRNA expressions of heat shock protein 70 (HSP70), matrix metalloproteinase 9 (MMP-9), and cysteine-aspartic protease 3 (caspase-3) by real time fluorescence quantitative reverse transcription polymerase chain reaction, and the experiments were repeated for 3 times. At PIH 2, 6, and 12, cell culture supernatant in the 3 groups was collected to detect the concentration of heparin-binding epidermal growth factor (HB-EGF) by enzyme linked immunosorbent assay method, the experiment was repeated for 3 times. The sample in each group and each time point was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference test, Kruskal-Wallis H test, and Mann-Whitney U test. Results: (1) At PIH 12, cells in NGFI group showed regular shape and regular arrangement, with no cell debris. The cell shape in NGTI group was generally regular, with fewer cell debris and closer arrangement than that in NGFI group. The cells in SMGTI group showed more irregular shapes, different sizes, and dead cell debris. (2) The percentage of G1 phase cells in NGTI group was significantly higher than that in NGFI group and SMGTI group at PIH 2, respectively (P<0.05), and the percentage of G1 phase cells in NGTI group was significantly lower than that in NGFI group and SMGTI group at PIH 6 and 12, respectively (P<0.05). The percentage of G2/M phase cells in NGTI group was significantly lower than that in SMGTI group at PIH 2 (P<0.05), and the percentage of G2/M phase cells in NGTI group was significantly higher than that in NGFI group and SMGTI group at PIH 6 and 12, respectively (P<0.05). The percentage of S phase cells in NGTI group at PIH 2, 6, and 12 was significantly higher than that in SMGTI group (P<0.05), and the percentage of S phase cells in NGTI group at PIH 2 and 6 was significantly lower than that in NGFI group (P<0.05). (3) The HSP70 mRNA expressions of cells in NGTI group were 2.50±0.30 and 3.99±0.35 at PIH 2 and 6, which were significantly higher than 1.14±0.15 and 0.82±0.27 in NGFI group (P<0.05), and 1.17±0.53 and 1.65±0.59 in SMGTI group (P<0.05). The MMP-9 mRNA expression of cells in SMGTI group was significantly higher than that in NGTI group at PIH 2, 6, and 12, respectively (Z=-2.319, -2.882, -2.908, P<0.05). At each time point after injury, the mRNA expression of caspase-3 of cells in NGTI group was similar to that in NGFI group and SMGTI group, respectively (P>0.05). (4) The concentration of HB-EGF in cell culture supernatant of NGTI group was significantly lower than that in NGFI group at PIH 2, 6 and 12 (P<0.05), and the concentration of HB-EGF in cell culture supernatant of SMGTI group was significantly higher than that in NGTI group at PIH 2 and 6 (P<0.05). Conclusions: The proliferation and secretion functions and expression of wound repair related protein of human HaCaT cells inflicted with thermal injury in simulated microgravity environment showed complex and diversified changes, which provide theoretical basis for further research on damage repair under weightlessness.

Allicin Inhibits Proliferation and Promotes Apoptosis of Human Epidural Scar Fibroblasts

Allicin can suppress liver and cardiac fibrosis; thus, we hypothesized that it might prevent scar tissue from extensive epidural fibrosis after laminectomy. Human epidural scar fibroblasts were isolated from surgical specimens and treated with allicin at a gradient of concentrations. Morphology, viability, migration rate, cell cycle, and apoptosis rate were measured by fluorescence microscope, Cell Counting Kit-8 assay, scratch assay, and flow cytometry. Western blot was used to measure the expression level of proliferation-related proteins. After treatment by allicin, cell viability (P=0.042) and migration rate (P= 0.010 in scratch assay and P= 0.025 in transwell assay) decreased significantly in a dose-dependent manner. The percentage of G1 phase cells significantly decreased (P= 0.017), whereas the percentage of S phase cells (P= 0.096) and G2 phase cells (P= 0.038) significantly increased in a dose-dependent manner. Similarly, the percentage of apoptotic cells increased significantly in a dose-dependent manner (P=0.036). Compared with the control group, the expression level of proliferating cell nuclear antigen protein (P= 0.081) and Bcl-2 protein (P= 0.029) significantly decreased, whereas the BAX protein level significantly increased (P= 0.017). Allicin can suppress human epidural scar fibroblast migration, induce cell apoptosis, and block cell proliferation at S phase and G2 phase.

MiR-133b inhibits cell proliferation, migration and invasion of esophageal squamous cell carcinoma by targeting EGFR

BackgroundEsophageal squamous cell carcinoma (ESCC) is an aggressive tumor entity characterized by early metastasis and late diagnosis. MicroRNA-133b (miR-133b) has been considered as a tumor suppressor in many human cancers by regulating epidermal growth factor receptor (EGFR). However, the specific effects of miR-133b and EGFR on ESCC remain unclear. MethodsqRT-PCR and western blotting were applied for measuring expression of mRNA and protein. Flow cytometry was used for detecting cell cycle and apoptosis. Cell proliferation, migration and invasion were detected by colony formation and transwell assays. Luciferase reporter assay was used to confirm the interaction between miR-133b and EGFR. ResultsLow expression of miR-133b and high expression of EGFR were identified in ESCC cells and tissues. Overexpression of miR-133b or knockdown of EGFR suppressed the cell proliferation, migration, and invasion of ESCC cells, and raised the percentage of G1 phase cells. The apoptosis of ESCC cells were promoted by increasing miR-133b and decreasing EGFR expression. Luciferase reporter assay confirmed EGFR as the target of miR-133b in ESCC cells. Overexpression of miR-133b significantly decreased the phosphorylation of PI3K, ERK and AKT by directly down-regulating EGFR. Higher expression of E-cadherin and CK-18 and lower expression of Vimentin and N-cadherin were observed after the transfection of miR-133b mimics or shEGFR. ConclusionOverexpression of miR-133b could suppress proliferation, migration and invasion of ESCC cells by inhibiting MAPK/ERK and PI3K/AKT signaling pathways through targeting EGFR, indicating that miR-133b might be a potential therapeutic target for the treatment of ESCC.

Self-complementary and tyrosine-mutant rAAV vectors enhance transduction in cystic fibrosis bronchial epithelial cells

Recombinant adeno-associated virus (rAAV) vector platforms have shown considerable therapeutic success in gene therapy for inherited disorders. In cystic fibrosis (CF), administration of first-generation rAAV2 was safe, but clinical benefits were not clearly demonstrated. Therefore, next-generation vectors that overcome rate-limiting steps in rAAV transduction are needed to obtain successful gene therapy for this devastating disease. In this study, we evaluated the effects of single-strand or self-complementary (sc) rAAV vectors containing single or multiple tyrosine-to-phenylalanine (Y-F) mutations in capsid surface-exposed residues on serotypes 2, 8 or 9. For this purpose, CF bronchial epithelial (CFBE) cells were transduced with rAAV vectors, and the transgene expression of enhanced green fluorescence protein (eGFP) was analyzed at different time points. The effects of vectors on the cell viability, host cell cycle and in association with co-adjuvant drugs that modulate intracellular vector trafficking were also investigated. Six rAAV vectors demonstrated greater percentage of eGFP+ cells compared to their counterparts at days 4, 7 and 10 post-transduction: rAAV2 Y(272,444,500,730)F, with 1.95-, 3.5- and 3.06-fold increases; rAAV2 Y(252,272,444,500,704,730)F, with 1.65-, 2.12-, and 2-fold increases; scrAAV2 WT, with 1.69-, 2.68-, and 2.32-fold increases; scrAAV8 Y773F, with 57-, 6.06-, and 7-fold increases; scrAAV9 WT, with 7.47-, 4.64-, and 3.66-fold increases; and scrAAV9 Y446F, with 8.39-, 4.62-, and 4.4-fold increases. At days 15, 20, and 30 post-transduction, these vectors still demonstrated higher transgene expression than transfected cells. Although the percentage of eGFP+ cells reduced during the time-course analysis, the delta mean fluorescence intensity increased. These vectors also led to increased percentage of cells in G1-phase without eliciting any cytotoxicity. Prior administration of bortezomib or genistein did not increase eGFP expression in cells transduced with either rAAV2 Y(272,444,500,730)F or rAAV2 Y(252,272,444,500,704,730)F. In conclusion, self-complementary and tyrosine capsid mutations on rAAV serotypes 2, 8, and 9 led to more efficient transduction than their counterparts in CFBE cells by overcoming the intracellular trafficking and second-strand DNA synthesis limitations.

Effects of Quercetin on Proliferation and H₂O₂-Induced Apoptosis of Intestinal Porcine Enterocyte Cells.

Weanling stress and toxicosis, which are harmful to the health of pigs’ intestines, are associated with oxidative stress. Quercetin (Que) is a polyphenolic compound that shows good anti-cancer, anti-inflammation and anti-oxidation effects. This study aimed to elaborate whether or not Que promotes IPEC-J2 (intestinal porcine enterocyte cells) proliferation and protects IPEC-J2 from oxidative damage. Thus, we examined the effects of Que on proliferation and H2O2-induced apoptosis in IPEC-J2. The results showed that Que increased IPEC-J2 viabililty, propelled cells from G1 phase into S phase and down-regulated gene levels of P27 and P21, respectively. Besides, H2O2-induced cell damage was alleviated by Que after different exposure times, and Que depressed apoptosis rate, reactive oxygen species (ROS) level and percentage of G1 phase cells and elevated the percentage of cells in G2 phase and S phase and mitochondrial membrane potential (Δψm) after IPEC-J2 exposure to H2O2. Meanwhile, Que reduced the value of Bax/Bcl-2 in H2O2 exposed cells. In low-degree oxidative damage cells, lipid peroxidation product malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were increased. In turn, Que could reverse the change of MDA content and SOD activity in low-degree damage cells. Nevertheless, catalase (CAT) activity was not changed in IPEC-J2 incubated with Que under low-degree damage conditions. Interestingly, relative expressive levels of the proteins claudin-1 and occludin were not altered under low-degree damage conditions, but Que could improve claudin-1 and occludin levels, slightly. This research indicates that Que can be greatly beneficial for intestinal porcine enterocyte cell proliferation and it protects intestinal porcine enterocyte cells from oxidation-induced apoptosis, and could be used as a potential feed additive for porcine intestinal health against pathogenic factor-induced oxidative damages and apoptosis.

Effect of Threonine on the Proliferation and Viability of Porcine Granulosa Cells

The proliferation and viability of granulosa cells directly affects the development and function of follicles. The current study examined the effect of threonine on cell morphology and cell cycle of porcine granulosa cells. It would help to understand hormone secretion, reproductive health, and disease prevention and treatment in females. Granulosa cells were isolated from pig ovaries, and different passages of cells were stained and identified by FSHR antibody. Threonine was added to the culture medium, and cells were stained with HE to examine the changes in granulosa cell morphology. Cell viability was measured using the CCK-8 assay, and cell cycle phase was determined by flow cytometry. Immunofluorescence staining showed &gt;98% FSHR-positive cells demonstrating that the isolated granulosa cells were &gt;95% pure. HE-staining of different passages of granulosa cells showed that cells from the second to fourth passage have stable morphology, whereas those from the fifth and subsequent passages began to degenerate and degrade. CCK-8 results showed that =0.5 mM threonine can significantly enhance granulosa cell viability after 0.5, 1 and 2 h of treatment, and cells treated with 1 mM threonine for 4 h had the highest cell viability. In contrast, 5 and 10 mM threonine significantly reduced cell viability, indicating that only a certain amount of threonine can enhance granulosa cell viability. Flow cytometry results demonstrated that the percentage of G1 phase cells was higher in the 0.1, 0.5 and 1 mM groups than in the control group, but lower in the 10 mM group than in control group. While the 5 mM group had the highest percentage of S phase cells, the 10 mM group had no S phase cells. In contrast, the percentage of G2 phase cells was much higher in the 10 mM group than in the control group and other dose groups, indicating that an appropriate amount of threonine can promote granulosa cell proliferation, but excessive threonine can inhibit cell proliferation.

Effect of etoposide-induced alteration of the Mdm2-Rb signaling pathway on cellular senescence in A549 lung adenocarcinoma cells.

The present study aimed to investigate the effect of various concentrations of etoposide (VP-16) on the E3 ubiquitin-protein ligase Mdm2 (Mdm2)-retinoblastoma (Rb) signaling pathway in the cellular senescence of A549 lung adenocarcinoma cells. A549 cells were randomly divided into the following four groups: Control group (no treatment), group 1 (1 µmol/l VP-16), group 2 (5 µmol/l VP-16) and group 3 (25 µmol/l VP-16). Each group was cultured for 48 h after treatment prior to observation of the alterations to cellular morphology. The cell cycle distribution of each group was also detected by flow cytometry. In addition, the activity of cellular senescence-associated β-galactosidase, and the expression of Mdm2 and phosphorylated (p-) Rb protein, was measured. The percentage of senescent cells was significantly higher following VP-16 treatment compared with the control group. The percentage of G1 phase cells, and p-Rb protein and Mdm2 protein expression were also significantly different following VP-16 treatment compared with the control group. VP-16 increased the activity of β-galactosidase in the A459 cells. VP-16 also decreased the expression level of Mdm2 and p-Rb protein and inhibited cell cycle progression in G1. These results indicate that VP-16 induces the cellular senescence of A549 cells via the Mdm2-Rb signaling pathway. However, further investigations are required to validate the mechanisms underlying these effects of VP-16.

Hesperetin-etoposide combinations induce cytotoxicity in U2OS cells: Implications on therapeutic developments for osteosarcoma

Osteosarcoma chemotherapy has improved survival rates, however, chemoresistance and drug toxicity still limit therapy. Drug combinations may overcome these limitations by allowing fewer chemoresistant cells to survive. The aim of this study was to evaluate the cytotoxic potential of hesperetin to osteosarcoma and to analyze the cell cycle effects of combinations of hesperetin with chemotherapeutic agents. For this, the U2OS human osteosarcoma cell line was exposed to hesperetin or hesperetin combined with etoposide or doxorubicin in defined proportions. Hesperetin was less cytotoxic compared to chemotherapeutic agents, as shown by cell growth, viability and clonogenic assays. Notwithstanding, hesperetin combined with etoposide showed additive effects on the inhibition of cell growth. Furthermore, hesperetin induced G2-phase arrest, associated with decreased gene expression of cyclins B1 and E1 and cyclin-dependent kinases 1 and 2. The combination with higher additive effect resulted in higher percentage of cells in G2-phase, showing that G2-phase arrest is associated with cytotoxicity. Moreover, hesperetin induced cytostatic effects. In conclusion, our results suggest that G2-phase arrest is an important step for hesperetin-induced cytotoxicity in U2OS cells. Hesperetin shows potential cytotoxicity when combined with etoposide, which may have implications on therapeutic developments for osteosarcoma.

The effects of up-regulating the expression of microRNA-1224-5p on hepatocellular carcinoma tumor endothelial cells

Objective To investigate the effect of microRNA-1224-5p (miR-1224-5p) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). Methods Oligonucleotides were chemically synthesized and transfected into TECs using Lipofectamine 2000. TECs were divided into 3 groups: a control (CON) group consisted of TECs without transfection, a negative control (NC) group consisted of TECs transfected with negative control oligonucleotides and Green Fluorescent Protein (GFP), and a micro-up (MU) group consisted of TECs transfected with miR-1224-5p mimic and GFP. The expression of miR-1224-5p was quantified via quantitative reverse transcription polymerase chain reaction (RT-qPCR). The proliferation of TECs was detected using MTT (Thiazolyl Blue Tetrazolium Bromide) assay. Apoptosis and cell cycle were detected via flow cytometry. The migration and invasion of TECs were detected using Ttranswell assay. The ability of tube fromation of TECs was detected using tube formation assay. RESULTS Oligonucleotides were successfully transduced into TECs, and specifically up-regulated the expression of miR-1224-5p. Results The results of RT-qPCR showed that the expression of miR-1224-5p was significantly increased in the MU group (2-ΔΔCt=3.27±0.15) compared with the CON group (2-ΔΔCt=1.00) and the NC group (2-ΔΔCt=1.08±0.11) (P<0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MU group for the 4 time points compared with the CON group and the NC group (P<0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MU group (19.29±0.95)% compared with the CON group (8.73±0.64)% and the NC group (9.51±0.56)% (P<0.01). The results of cell cycle detection showed that percentage of G1 phase cells in the MU group (73.3±2.0)% was significantly higher than the CON group (50.7±1.3)% and the NC group (51.7±1.9)% and the percentage of S phase cells in the MU group (23.0±3.0)% was significantly decreased compared with the CON group (31.4±2.7)%, and the NC group (29.1±1.6)%. (P<0.05 or P<0.01). The ability of migration was significantly inhibited in the MU group (51.0±3.6) compared with the CON group (77.7±2.5), and the NC group (79.2±3.5) (P<0.01). And the ability of invasion of TECs was also significantly inhibited in the MU group (9.8±1.3) compared with the CON group (15.8±0.8) and the NC group (15.4±0.9) (P<0.01). The ability of tube formation of TECs in the MU group was inhibited absolutely, but TECs in the CON group and the NC group were not affected. Conclusion Up-regulation of miR-1224-5p could decrease proliferation, induce apoptosis, inhibit migration and invasion, and supress tube formation of TECs of human HCC. Key words: Hepatocellular carcinoma; Vascular endothelial cells of human hepatocellular carcinoma; MicroRNA-1224-5p

UCN enhances TGF-beta-mediated mitoinhibition of VSMCs via counteracting TGF-beta-induced cPLA2 expression and activation

Urocortins (UCNs) and transforming growth factor-beta (TGF-beta) have been demonstrated to participate in various cardiovascular diseases, many of which involve vascular smooth muscle cells (VSMCs) proliferation. Cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release is an important cause of VSMCs proliferation. The work was to investigate the regulation of VSMCs proliferation by UCN/TGF-beta and whether cPLA2 was a link between their signaling pathways. VSMCs proliferation was measured by colorimetric assay and immunofluorescence microscopy. Using cell flow cytometry, the changes in the cell cycle phases were investigated. Lentiviral Vector Particle was performed to overexpress cPLA2 gene. Both UCN and TGF-beta inhibited VSMCs proliferation and an additive effect was observed when the cells were treated with UCN plus TGF-beta. TGF-beta increased the percentage of cells in G1-phase while UCN increased the cell percentage in G2-phase with a concomitant decrease in S-phase. Furthermore, cPLA2 expression was increased by TGF-beta but decreased by UCN and UCN attenuated TGF-beta-induced cPLA2 expression. In primary VSMCs, TGF-beta induced cPLA2 phosphorylation, and this effect was also attenuated by UCN. Similar to UCN, the cPLA2 inhibitor, pyrrophenone (PYR), also played a role in enhancing TGF-beta-mediated mitoinhibition. Inversely, overexpression of cPLA2 eliminated the effect of UCN on the mitoinhibition. The pretreatment with UCN counteracted TGF-beta-mediated cPLA2 expression and activation, thereby contributing to TGF-beta-mediated mitoinhibition of VSMCs.

Transcription factor LSF (TFCP2) inhibits melanoma growth.

Late SV40 factor 3 (LSF), a transcription factor, contributes to human hepatocellular carcinoma (HCC). However, decreased expression level of LSF in skin melanoma compared to that in benign melanocytic tumors and nevi in mice and humans was found in this study. Anchorage-dependent and -independent growth of melanoma cells was suppressed by LSF overexpression through an increased percentage of G1 phase cells and an increased p21CIP1 expression level in vitro and in vivo. Anchorage-dependent growth in LSF-overexpressed melanoma cells was promoted by depletion of LSF in the LSF-overexpressed cells. Integrated results of our EMSA and chromatin immunoprecipitation assays showed binding of LSF within a 150-bp upstream region of the transcription start site of p21CIP1 in melanoma cells. Taken together, our results suggest potential roles of LSF as a growth regulator through control of the transcription of p21CIP1 in melanocytes and melanoma cells as well as a biomarker for nevus.

Evaluation of rapid cell division in non-uniform cell cycles.

To better understand the mechanisms of development of harmful algal blooms (HABs), accurate estimates of species-specific in situ growth rates are needed. HABs are caused by rapid cell division by the causative microorganisms. To accurately estimate the in situ growth rates of harmful algae having non-uniform and/or irregular cell cycles, we modified a standard equation based on the cell cycle, and calculated the in situ growth rate to describe the process of bloom development in nature. Sampling of a developing bloom of Heterosigma akashiwo in Pohang Bay, Korea, was conducted every 3 h from 15:00 on August 2 to 07:00 on August 4, 2006. The amount of H. akashiwo DNA was measured using flow cytometry following tyramide signal amplification-fluorescence in situ hybridization. On August 2, the percentage of G1 phase cells decreased from 15:00 to 19:00 then increased until 22:00; it then decreased until 07:00 on August 3, followed by an increase to 10:00. This indicates the ability of the cells in nature to undergo more than one round of division per day. During the following night two rounds of division did not occur. The in situ growth rates estimated using the modified equation ranged from 0.31 to 0.53 d(-1) . We conclude that the use of this equation enables more accurate estimates of bloom formation by rapidly dividing cells.

Effect of trypsin on the growth of a skin squamous cell carcinoma cell line A431

Objective To evaluate the effect of trypsin on the proliferation, migration and adhesion of a skin squamous cell carcinoma cell line A431. Methods Cultured A431 cells were divided into several experimental groups treated with trypsin at concentrations of 0.1, 1, 10 and 100 nmol/L for 24, 48 and 72 hours respectively, and a control group treated with DMEM complete medium only. Cell counting kit-8 (CCK8) assay was conducted to evaluate cellular proliferative activity to select the optimal concentration of trypsin. Then, some A431 cells treated with trypsin at the selected concentration for 24, 48 and 72 hours respectively (or 48 hours only) served as the experimental groups (or group), and other A431 cells treated with DMEM complete medium served as the control group. Flow cytometry was performed to assess cell cycle distribution and proliferation index, fibronectin-based adhesion assay to estimate cell adhesive capacity, and wound healing assay and Transwell assay were conducted to evaluate the migratory capacity of cells in two- and three-dimensional space. Statistical analysis was carried out by using analysis of variance, paired samples t test and chi-square test. Results The proliferative activity of A431 cells increased along with the increase of trypsin concentrations, with the strongest increasing effect observed at 100 nmol/L. After treatment with 100 nmol/L trypsin, the experimental group showed a decrease in the percentage of G1-phase cells, but an increase in the percentage of S-phase cells, proliferation index, migratory and adhesive capacity compared with the control group (all P < 0.05). Conclusion Trypsin can promote the proliferation, migration and adhesion of A431 cells. Key words: Carcinoma, squamous cell; Trypsin; Cell growth processes; Cell cycle; Cell movement; Cell line, tumor